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Genetische Modifikation humaner mesenchymaler Stammzellen zur Stimulation der Knochenheilung / Synergistic effect of Indian hedgehog and BMP-2 gene transfer to increase the osteogenic potential of human mesenchymal stem cellsSchmalzl, Jonas Georg January 2016 (has links) (PDF)
Fragestellung:
Die Therapie von Knochendefekten kritischer Größe mit kompromittiertem Regenerationspotential, stellt ein schwerwiegendes Problem dar. Die Forschung auf dem Gebiet der Knochenheilung hat sich in jüngster Vergangenheit daher auf die Anwendung mesenchymaler Vorläuferzellen (MSZ) zur Stimulierung des Knochenwachstums konzentriert. In der vorliegenden Studie wurde in humanen MSZ eine Überexpression spezifischer Wachstumsfaktoren induziert mit dem Ziel, deren osteogenes Potential zu steigern.
Methodik:
MSZ wurden nach etablierten Protokollen expandiert. Durch adenovirale Transfektion wurde eine überexpression von grün fluoreszierendem Protein (GFP, Kontrolle), indian hedgehog (IHH), bone morphogenetic protein 2 (BMP-2) und IHH in Kombination mit BMP-2 induziert. Die MSZ wurden für 28 Tage mit osteogenem Differenzierungs- und Kontrollmedium kultiviert. Als weitere Kontrolle dienten native MSZ. Es wurden die Auswirkungen der jeweiligen genetischen Veränderungen auf die metabolische Aktivität (Alamar Blau), die Proliferation (Qubit dsDNA BR), die Aktivität des Enzyms alkalische Phosphatase (ALP)(p-Nitrophenylphosphat), die Mineralisierung (Alizarinrot S, Calcium O-Cresolphthalein) sowie auf die Expression charakteristischer Markergene untersucht (qRT-PCR).
Ergebnis:
In den ersten 72h nach Transfektion konnte eine leichte, im Vergleich zu nativen Zellen nicht signifikante Abnahme der metabolischen Aktivität in allen Gruppen beobachtet werden. Das Proliferationsverhalten transfizierter und nativer MSZ unterschied sich während des Untersuchungszeitraums nicht signifikant. Bei der Analyse der ALP-Aktivität zeigte sich ein typisches Rise-and-Fall Muster. Alle ost Gruppen wiesen sowohl im Assay als auch in der PCR eine signifikant höhere ALP-Aktivität auf. Die Überexpression von BMP-2 und IHH+BMP-2 bewirkte eine signifikant stärkere Mineralisierung an Tag 28. In der PCR zeigte sich für BMP-2 ost und IHH+BMP2 ost ein signifikanter Anstieg der Osteopontin und BMP-2 Expression über die Zeit. Zudem stieg bei allen ost Gruppen die Runx2 Expression bis Tag 21 an.
Schlussfolgerung:
Die virale Transfektion hatte keinen negativen Einfluss auf die metabolische Aktivität der Zellen oder deren Proliferationsverhalten. Die Überexpression von BMP-2 ohne oder in Kombination mit IHH führte zu einer vermehrten Produktion extrazellulärer Matrix und zu einer gesteigerten Genexpression osteogener Marker. Die virale Transfektion stellt daher eine vielversprechende Möglichkeit dar, das osteogene Potential von MSZ zu steigern. / Introduction:
To stimulate healing of large bone defects research has concentrated on the application of mesenchymal stem cells (MSCs).
Methods:
In the present study, we induced the overexpression of the growth factors bone morphogenetic protein 2 (BMP-2) and/or indian hedgehog (IHH) in human MSCs by adenoviral transduction to increase their osteogenic potential. GFP and non-transduced MSCc served as controls. The influence of the respective genetic modification on cell metabolic activity, proliferation, alkaline phosphatase activity (ALP), mineralization in cell culture, and osteogenic marker gene expression was investigated.
Results:
Transduction had no negative influence on cell metabolic activity or proliferation. ALP activity showed a typical rise-and-fall pattern with a maximal activity at day 14 and 21 after osteogenic induction. Enzyme activity was significantly higher in groups cultured with osteogenic media. The
overexpression of BMP-2 and especially IHH+BMP-2 resulted in a significantly higher mineralization after 28 days. This was in line with obtained qRT-PCR analyses, which showed a significant increase in osteopontin and osteocalcin expression for osteogenicly induced BMP-2 and IHH+BMP-2 transduced cells when compared to the other groups. Moreover, an increase in runx2 expression was observed in all osteogenic groups toward day 21. It was again more pronounced for BMP-2 and IHH+BMP-2 transduced cells cultured in osteogenic media.
Conclusion:
In summary, viral transduction did not negatively influence cell metabolic activity and proliferation. The overexpression of BMP-2 in combination with or without IHH resulted in an increased deposition of mineralized extracellular matrix, and expression of osteogenic marker genes. Viral transduction therefore represents a promising means to increase the osteogenic potential of MSCs and the combination of different transgenes may result in synergistic effects.
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Identification of microRNAs involved in osteoblast differentiation of murine embryonic stem cellsKaniowska, Dorota 09 August 2012 (has links) (PDF)
Skeletal development requires stringent control of programs for gene activation and suppression in response to physiological cues. There has been a principal focus on the identification of the mechanisms by which a particular cell phenotype is activated. MicroRNAs (miRNAs, miRs) have emerged as key negative regulators of diverse biological and pathological processes, including developmental timing, organogenesis, apoptosis, cell proliferation and differentiation; how they regulate osteoblast specific gene expression, is poorly understood. miRNAs are small 22 nucleotides (nt) endogenous non-coding RNAs (ncRNAs) that anneal to 3’ untranslated region (3’UTR) of target messenger RNA (mRNA) to mediate inhibition of translation and lower protein level. It remains to be established how specific miRNAs contribute to regulate the onset of a tissue-specific phenotype.
One previously identified important player in the activation of skeletal-related genes that control formation of bone tissue is Wnt (wingless) signaling. The Wnts are regulating the differentiation of multiple cell types but also are driving embryonic stem cells (ESCs) into specific lineages, for example they support osteoblastogenesis. By attaching to the membrane, Wnts direct a signaling cascade for accumulation of β-catenin (CatnB), which in turn activates osteoblast-essential genes. The contribution of global mechanisms is equally important for understanding tissue development and diseases.
The aim of this study was to identify miRNAs that are differentially expressed in osteogenically differentiated ESCs. In addition, functional characterization of these miRNAs was performed to further unravel the molecular mechanisms underlying osteogenesis. Finally, an important goal was to identify the mRNA targets of these miRNAs, which are required for differentiation of ESCs into osteoblasts with a primary focus on mRNAs associated with the Wnt signaling pathway.
miRNA expression profiling reveals an overall down-regulation of miRNAs during osteogenic differentiation of ESCs
To identify miRNAs that are potentially involved in osteogenesis ESCs were differentiated into osteoblasts and compared to undifferentiated ESCs using a miRNA microarray. miRNA profiling during the initial stages of osteoblast differentiation showed 25 miRNAs significantly differentially expressed. Differential expression of 4 miRNAs tested was confirmed using quantitative real-time PCR (RT-qPCR). Many miRNAs were expressed at low levels in differentiated ESCs. Indeed, down-regulation of miRNAs appeared to be common during differentiation. Furthermore, related miRNAs encoded on the same chromosome showed similar expression profiles. In summary, though several miRNAs were identified that can significantly distinguish between undifferentiated and osteogenically differentiated ESCs, 11 were chosen for further functional analysis.
Functional studies show that miR-127, miR-183, miR-291b-5p, miR-293, miR-361, miR-467b and miR-665 affect osteogenesis of ESCs
Undifferentiated and differentiated ESCs were used for functional studies of 11 miRNAs (miR-22, miR-127, miR-130a, miR-183, miR-291b-5p, miR-293, miR-300, miR-361, miR-467b, miR-665 and miR-690), which were down-regulated during osteogenic differentiation. To asses the function of these miRNAs, gain- and loss-of-function experiments were performed. Overexpressing and knocking down these miRNAs caused changes in cell survival, cell morphology, and osteogenic differentiation capacity as measured with calcium deposition, ALP activity and expression of osteogenic markers. Particularly, overexpression of miR-361 and knockdown of miR-665 significantly enhanced mineralization and expression levels of osteogenic markers. Thus, both miRNAs might regulate osteogenic differentiation in the early stages of lineage specification and commitment.
miRNAs are modulators of osteogenic differentiation
To identify miRNA target candidates that may account for the observed effects on cell survival and osteogenic differentiation of ESCs, a combined approach of bioinformatic predictions, mRNA expression analysis, and TurboGFP reduction upon miRNA overexpression coupled with the search of known literature was performed to identify cellular events that the identified miRNAs might be involved in.
Target identification suggested that the candidate miRNAs may interfere with the Wnt pathway as many target candidates were detected that were known to be Wnt signaling-associated. To confirm that miR-183, miR-293, miR-361, miR-665 and miR-690 regulated osteoblast differentiation, target mRNA/miRNA interaction was studied using RT-qPCR. Overexpression of these miRNAs reduced the levels of the key factors involved in Wnt signaling; particularly Wnt inhibitor factor 1 (WIF-1) levels were decreased by miR-293, nuclear factor of activated T cells 3 (NFATc-3) and Prickle-1 by miR-361, Dishevelled 1 (Dvl-1) by miR-665 and for forkhead box O 3 (FoxO-3), Ras homolog gene family, member A (RhoA) and CatnB-1 by miR-690.
Thus, to address the hypothesis that miR-361 activates osteoblast differentiation by targeting Prickle-1 and NFATc-3, the p2FP-RNAi vector system was applied. It was shown that expression of miR-361 down-regulates Prickle-1 levels, which to our knowledge have not been described so far. As it was found previously, Prickle-1 reduced Dvl-3 levels by promoting its ubiquitination, resulting in inhibition of Wnt canonical signaling in liver cancer. Since Dvls are positive regulators of osteogenesis by elevating CatnB levels and stimulating lymphoid enhancer factor/T cell factor proteins (LEF/TCF) -dependent transcription in the canonical Wnt pathway, Prickle-1 might be a negative regulator of osteogenic differentiation by eliminating Dvls from the complex. This interaction offers a novel mechanism of Wnt signaling activation in osteogenesis and can be explored to identify key components in the Wnt signaling pathway.
In summary, we suggested that miR-361 acts as an activator in osteogenic differentiation of ESCs. / Die Embryonalentwicklung des Skelettsystems ist in Bezug auf programmierte Genaktivierung in Antwort auf physiologische Schlüsselreize strikten Kontrollen unterworfen. Studien zur Untersuchung solcher Kontrollelemente haben sich dabei vor allem auf die Identifikation von Mechanismen fokussiert, die einen bestimmten zellulären Phänotyp aktivieren. Zum Vorschein kamen microRNAs (miRNAs), die als negative Schlüsselregulatoren diverser biologischer und pathologischer Prozesse wirken, wie zum Beispiel der zeitlichen Regulation von Entwicklung, der Organogenese, Apoptose, zellulärer Proliferation und Differenzierung. Wie sie allerdings die Osteogenese, den Prozess der Knochenbildung, regulieren ist weitestgehend unbekannt.
MiRNAs sind kurze 22 Nukleotid lange endogene nicht-kodierende RNAs (ncRNAs), die an die 3\' nicht translatierte Region (3\'UTR) einer Ziel mRNA binden und somit die Inhibition der Translation vermitteln, was letzten Endes zu einer Erniedrigung des Proteinlevels führt. Es bleibt allerdings zu etablieren, wie spezifische miRNAs zur Spezifikation in einen bestimmten Zell- oder Gewebephänotyps beitragen.
Einer der bisher identifizierten Akteure, der die Aktivierung von skelettalen Genen kontrolliert, ist der Wnt (wingless) Signalweg. Wnt Moleküle regulieren die Differenzierung vieler unterschiedlicher Zelltypen, aber lenken auch die Differenzierung von embryonalen Stammzellen (ESCs) in spezifische Richtungen, so z.B. in die Richtung von Knochenzellen, den Osteoblasten. Indem sie an die Zellmembran andocken, dirigieren Wnts eine Signalkaskade, die die Akkumulation von beta-catenin (CatnB) im Zellkern nach sich zieht, wodurch knochenspezifische Gene aktiviert werden. Obwohl die Wnt Signalkaskade weitestgehend beschrieben ist, ist der Beitrag globalerer Regulationsmechanismen, wie die der miRNAs, an der Osteogenese jedoch gleichfalls für das Verständnis von Gewebeentwicklung und -fehlfunktion von Bedeutung.
Das Ziel dieser Arbeit war es deshalb bestimmte miRNAs zu identifizieren, die differentiell in ESCs exprimiert werden, die zu Knochenzellen ausdifferenzieren. Desweiteren sollten diese miRNAs funktionell charakterisiert werden, um die molekularen Mechanismen, die der Osteogenese unterliegen, aufzudecken. Letztendlich war es ein weiteres wichtiges Ziel die Ziel mRNAs der knochenspezifischen miRNAs zu identifizieren und deren Bezug zum Wnt Signalweg zu charakterisieren.
miRNA Expression ist während der osteogenen Differenzierung herunter reguliert
Um solche miRNAs zu identifizieren, die potentiell in die Osteogenese eingreifen, wurden ESCs zu Osteoblasten differenziert und mit undifferenzierten ESCs mit Hilfe eines miRNA Microarrays verglichen. Das so durchgeführte miRNA Profiling zeigte, dass 25 miRNAs während der initialen Phase der osteogenen Differenzierung signifikant unterschiedlich exprimiert wurden. Die differentielle Expression von 4 getesteten miRNAs wurde in einem nächsten Schritt über quantitative real-time PCR (RT-qPCR) beispielhaft bestätigt. Generell zeigte sich, dass differenzierende ESCs viele miRNAs auf geringem Niveau exprimieren. Tatsächlich schien die Herunterregulation der miRNA Expression mit der Differenzierung der Zellen einherzugehen. Desweiteren zeigten miRNAs, die auf dem gleichen Chromosom kodiert sind, ähnliche Expressionsmuster. Zusammenfassend fanden sich etliche miRNAs, die in undifferenzierten Zellen im Vergleich zu differenzierenden Zellen unterschiedlich exprimiert werden, von denen schlussendlich 11 für weitere Analysen ausgewählt wurden (miR-22, miR-127, miR-130a, miR-183, miR-291b-5p, miR-293, miR-300, miR-361, miR-467b, miR-665 and miR-690).
miR-127, miR-183, miR-291b-5p, miR-293, miR-361, miR-467b und miR-665 beeinflussen die Osteogenese
In einem nächsten Schritt wurden undifferenzierte und differenzierende ESCs für funktionelle Studien dieser 11 herrunterregulierten miRNAs herangezogen. Um die Funktion dieser miRNAs aufzudecken, wurden sogenannte Gain-of-function und Loss-of-function Studien durchgeführt. Die experimentelle Überexpression und der Knock-down dieser miRNAs führten zu Änderungen in der zellulären Morphologie, der Viabilität und der osteogenen Differenzierungskapazität wie durch einen Kalziumdepositionsassay, einen ALP Aktivitätsassay und die Expression knochenspezifischer Markergene gezeigt werden konnte. Im Besonderen erhöhte die Überexpression der miR-361 und der Knock-down der miR-665 den Mineralisierungsgrad der Zellen und die Expressionniveaus knochenspezifischer Gene. Daher ist zu schließen, dass beide miRNAs das Potential besitzen, die Osteogenese - besonders in den frühen Stadien der Keimbahnspezifikation - zu regulieren.
miRNAs als Modulatoren der Osteogenese
Um miRNA Zielkandidaten zu identifizieren, die die beobachteten Effekte auf die Zellviabilität und auf die osteogene Differenzierungen bedingen könnten, wurde ein kombinierter Ansatz aus Bioinformatischer Sequenz- und Prädiktionsanalyse, mRNA Expressionsanalyse und TurboGFP Reduktion nach miRNA Überexpression gewählt. Gepaart mit einer Literatursuche deutete diese Zielkandidatenanalyse darauf hin, dass die identifizierten miRNAs tatsächlich den Aktivierungsstatus des Wnt Signalwegs manipulieren könnten, da viele der prädiktierten Target mRNAs bekannt dafür sind, mit dem Wnt Signalweg zu interagieren.
Um zu bestätigen, dass miR-183, miR-293, miR-361, miR-665 und miR-690 die Osteogenese regulieren, wurde die mRNA/miRNA Interaktion indirekt mittels RT-qPCR studiert. Die Überexpression dieser miRNAs führte zu einer Erniedrigung des mRNA Expressionsspiegels von WIF-1 (Wnt inhibitory factor 1) durch miR-293, NFATc-3 (nuclear factor of activated T cells 3) und Prickle-1 durch miR-361, Dishevelled 1 (Dvl-1) durch miR-665, sowie forkhead box O3 (FoxO-3), Ras homolog gene family, member A (RhoA) und CatnB durch miR-690.
In einem nächsten Schritt konnte durch Nutzung eines speziellen Reportersystems (TurboGFP) eine direkte Interaktion zwischen miR-361 und Prickle-1 nachgewiesen werden. Wie bereits in anderen Studien gezeigt, ist Prickle-1 in der Lage, die Spiegel an Dvl-3 durch Ubiquitinierung des Proteins zu reduzieren, was zur Inhibierung des kanonischen Wnt Signalweges führt. Da Dvls als positive Regulatoren der Osteogenese bekannt sind, indem sie den CatnB Spiegel erhöhen und die lymphoid enhancer factor/T cell factor protein (LEF/TCF) abhängige Transkription stimulieren, könnte Prickle-1 als negativer Regulator fungieren, indem es Dvls von diesem Transkriptionskomplex entfernt.
Abschließend lässt sich zusammenfassen, dass miR-361 in dieser Arbeit als neuartiger Aktivator der osteogenen Differenzierung vorgeschlagen wird. Die molekulare Interaktion zwischen miR-361, Prickle-1 und Dvls bietet einen neuartigen Mechanismus der Wnt Signalaktivierung während der Osteogenese und kann für weitere Untersuchungen zur Identifizierung von Schlüsselkomponenten des Wnt Signalweges herangezogen werden.
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Der Einfluss des Transkriptionsfaktors Runx2 auf osteogene und adipogene Differenzierungsmarker, insbesondere auf PPARγ / The influence of the transcription factor Runx2 on osteogenic and adipogenic differentiation markers, particularly on PPARγDeuschl, Jana Daniela 11 December 2013 (has links)
Mesenchymale Stammzellen können sich durch den Einfluss verschiedener Transkriptionsfaktor zu Osteoblasten, Adipozyten, Chondrozyten oder Myoblasten differenzieren. Während sie sich unter Runx2-Einfluss entlang der osteoblastären Linie differenzieren, entwickeln sie sich bei vorliegendem PPARγ entlang des adipogenen Differenzierungswegs. Das Gleichgewicht zwischen beiden Faktoren und ihr Zusammenspiel stellen einen wichtigen Bereich in der Osteoporoseforschung dar. In dieser Dissertation wurde durch Runx2-Suppression bzw. Runx2-Überexpression die Rolle dieses Faktors in pHOB und SCP1-Zellen erfasst und die Interaktion zwischen Runx2 und PPARγ untersucht.
Der Runx2-Knockdown’ erfolgte mittels RNA-Interferenz, die Runx2-Überexpression durch ein Runx2 exprimierendes Plasmid. In RT-PCRs wurden mRNA-Messungen durchgeführt. Die Proteinbestimmung erfolgte im ‚Westernblot’. Der funktionelle Einfluss der Runx2-Überexpression auf die PPARγ-Transkription wurde durch Kotransfektion des an Luziferase gekoppelten PPARg-Promotorgens erfasst. Die funktionelle Aktivität des PPARg-Proteins wurde durch die Transfektion des an Luziferase gekoppelten PPRE-Gens gemessen. Promotoraktivität und Funktionalität der Proteine wurden in Luziferase-Reportergenassays erfasst.
Unter basalen Kulturbedingungen differenzierten sich pHOB osteogen. Durch zweimalige siRunx2-Transfektion gelang auf mRNA-Ebene eine suffiziente Runx2-Suppression über 29 Tage auf durchschnittlich 10,1%. Neben einer Steigerung der PPARγ-mRNA nach sieben Tagen konnte darunter auch eine Suppression der osteogenen Differenzierungsmarker OC und AP beobachtet werden. Ein ‚Rescue’ der supprimierten Runx2-Genexpression konnte durch osteogene Stimulation nicht erreicht werden.
In den Runx2-/PPARγ-Interaktionsversuchen wurden SCP1-Zellen adipogen stimuliert, um die PPARγ2-mRNA und PPARγ-Promotoraktivität zu erhöhen. Darunter konnte ebenfalls eine gesteigerte Funktionalität des PPARγ-Proteins beobachtet werden. Durch Runx2-Überexpression wurde in SCP1-Zellen die PPARγ-Promotoraktivität und somit der Beginn der mRNA-Synthese gehemmt. Die PPARγ2-mRNA hingegen blieb unbeeinflusst.
Die zentrale Rolle des Runx2 in der osteogenen Differenzierung scheint durch den Einfluss auf die osteogenen Marker OC und AP in pHOB bestätigt zu werden. Auch der Einfluss auf die adipogene Differenzierung erfolgt über Runx2. Im Rahmen dieser Dissertation konnte erstmalig die Hemmung des PPARγ-Promotors durch Runx2 beschrieben werden. Hierdurch werden die PPARγ-Transkription und somit voraussichtlich die Interaktion zwischen Adipogenese und Osteogenese beeinflusst.
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Identification of microRNAs involved in osteoblast differentiation of murine embryonic stem cellsKaniowska, Dorota 29 May 2012 (has links)
Skeletal development requires stringent control of programs for gene activation and suppression in response to physiological cues. There has been a principal focus on the identification of the mechanisms by which a particular cell phenotype is activated. MicroRNAs (miRNAs, miRs) have emerged as key negative regulators of diverse biological and pathological processes, including developmental timing, organogenesis, apoptosis, cell proliferation and differentiation; how they regulate osteoblast specific gene expression, is poorly understood. miRNAs are small 22 nucleotides (nt) endogenous non-coding RNAs (ncRNAs) that anneal to 3’ untranslated region (3’UTR) of target messenger RNA (mRNA) to mediate inhibition of translation and lower protein level. It remains to be established how specific miRNAs contribute to regulate the onset of a tissue-specific phenotype.
One previously identified important player in the activation of skeletal-related genes that control formation of bone tissue is Wnt (wingless) signaling. The Wnts are regulating the differentiation of multiple cell types but also are driving embryonic stem cells (ESCs) into specific lineages, for example they support osteoblastogenesis. By attaching to the membrane, Wnts direct a signaling cascade for accumulation of β-catenin (CatnB), which in turn activates osteoblast-essential genes. The contribution of global mechanisms is equally important for understanding tissue development and diseases.
The aim of this study was to identify miRNAs that are differentially expressed in osteogenically differentiated ESCs. In addition, functional characterization of these miRNAs was performed to further unravel the molecular mechanisms underlying osteogenesis. Finally, an important goal was to identify the mRNA targets of these miRNAs, which are required for differentiation of ESCs into osteoblasts with a primary focus on mRNAs associated with the Wnt signaling pathway.
miRNA expression profiling reveals an overall down-regulation of miRNAs during osteogenic differentiation of ESCs
To identify miRNAs that are potentially involved in osteogenesis ESCs were differentiated into osteoblasts and compared to undifferentiated ESCs using a miRNA microarray. miRNA profiling during the initial stages of osteoblast differentiation showed 25 miRNAs significantly differentially expressed. Differential expression of 4 miRNAs tested was confirmed using quantitative real-time PCR (RT-qPCR). Many miRNAs were expressed at low levels in differentiated ESCs. Indeed, down-regulation of miRNAs appeared to be common during differentiation. Furthermore, related miRNAs encoded on the same chromosome showed similar expression profiles. In summary, though several miRNAs were identified that can significantly distinguish between undifferentiated and osteogenically differentiated ESCs, 11 were chosen for further functional analysis.
Functional studies show that miR-127, miR-183, miR-291b-5p, miR-293, miR-361, miR-467b and miR-665 affect osteogenesis of ESCs
Undifferentiated and differentiated ESCs were used for functional studies of 11 miRNAs (miR-22, miR-127, miR-130a, miR-183, miR-291b-5p, miR-293, miR-300, miR-361, miR-467b, miR-665 and miR-690), which were down-regulated during osteogenic differentiation. To asses the function of these miRNAs, gain- and loss-of-function experiments were performed. Overexpressing and knocking down these miRNAs caused changes in cell survival, cell morphology, and osteogenic differentiation capacity as measured with calcium deposition, ALP activity and expression of osteogenic markers. Particularly, overexpression of miR-361 and knockdown of miR-665 significantly enhanced mineralization and expression levels of osteogenic markers. Thus, both miRNAs might regulate osteogenic differentiation in the early stages of lineage specification and commitment.
miRNAs are modulators of osteogenic differentiation
To identify miRNA target candidates that may account for the observed effects on cell survival and osteogenic differentiation of ESCs, a combined approach of bioinformatic predictions, mRNA expression analysis, and TurboGFP reduction upon miRNA overexpression coupled with the search of known literature was performed to identify cellular events that the identified miRNAs might be involved in.
Target identification suggested that the candidate miRNAs may interfere with the Wnt pathway as many target candidates were detected that were known to be Wnt signaling-associated. To confirm that miR-183, miR-293, miR-361, miR-665 and miR-690 regulated osteoblast differentiation, target mRNA/miRNA interaction was studied using RT-qPCR. Overexpression of these miRNAs reduced the levels of the key factors involved in Wnt signaling; particularly Wnt inhibitor factor 1 (WIF-1) levels were decreased by miR-293, nuclear factor of activated T cells 3 (NFATc-3) and Prickle-1 by miR-361, Dishevelled 1 (Dvl-1) by miR-665 and for forkhead box O 3 (FoxO-3), Ras homolog gene family, member A (RhoA) and CatnB-1 by miR-690.
Thus, to address the hypothesis that miR-361 activates osteoblast differentiation by targeting Prickle-1 and NFATc-3, the p2FP-RNAi vector system was applied. It was shown that expression of miR-361 down-regulates Prickle-1 levels, which to our knowledge have not been described so far. As it was found previously, Prickle-1 reduced Dvl-3 levels by promoting its ubiquitination, resulting in inhibition of Wnt canonical signaling in liver cancer. Since Dvls are positive regulators of osteogenesis by elevating CatnB levels and stimulating lymphoid enhancer factor/T cell factor proteins (LEF/TCF) -dependent transcription in the canonical Wnt pathway, Prickle-1 might be a negative regulator of osteogenic differentiation by eliminating Dvls from the complex. This interaction offers a novel mechanism of Wnt signaling activation in osteogenesis and can be explored to identify key components in the Wnt signaling pathway.
In summary, we suggested that miR-361 acts as an activator in osteogenic differentiation of ESCs. / Die Embryonalentwicklung des Skelettsystems ist in Bezug auf programmierte Genaktivierung in Antwort auf physiologische Schlüsselreize strikten Kontrollen unterworfen. Studien zur Untersuchung solcher Kontrollelemente haben sich dabei vor allem auf die Identifikation von Mechanismen fokussiert, die einen bestimmten zellulären Phänotyp aktivieren. Zum Vorschein kamen microRNAs (miRNAs), die als negative Schlüsselregulatoren diverser biologischer und pathologischer Prozesse wirken, wie zum Beispiel der zeitlichen Regulation von Entwicklung, der Organogenese, Apoptose, zellulärer Proliferation und Differenzierung. Wie sie allerdings die Osteogenese, den Prozess der Knochenbildung, regulieren ist weitestgehend unbekannt.
MiRNAs sind kurze 22 Nukleotid lange endogene nicht-kodierende RNAs (ncRNAs), die an die 3\'' nicht translatierte Region (3\''UTR) einer Ziel mRNA binden und somit die Inhibition der Translation vermitteln, was letzten Endes zu einer Erniedrigung des Proteinlevels führt. Es bleibt allerdings zu etablieren, wie spezifische miRNAs zur Spezifikation in einen bestimmten Zell- oder Gewebephänotyps beitragen.
Einer der bisher identifizierten Akteure, der die Aktivierung von skelettalen Genen kontrolliert, ist der Wnt (wingless) Signalweg. Wnt Moleküle regulieren die Differenzierung vieler unterschiedlicher Zelltypen, aber lenken auch die Differenzierung von embryonalen Stammzellen (ESCs) in spezifische Richtungen, so z.B. in die Richtung von Knochenzellen, den Osteoblasten. Indem sie an die Zellmembran andocken, dirigieren Wnts eine Signalkaskade, die die Akkumulation von beta-catenin (CatnB) im Zellkern nach sich zieht, wodurch knochenspezifische Gene aktiviert werden. Obwohl die Wnt Signalkaskade weitestgehend beschrieben ist, ist der Beitrag globalerer Regulationsmechanismen, wie die der miRNAs, an der Osteogenese jedoch gleichfalls für das Verständnis von Gewebeentwicklung und -fehlfunktion von Bedeutung.
Das Ziel dieser Arbeit war es deshalb bestimmte miRNAs zu identifizieren, die differentiell in ESCs exprimiert werden, die zu Knochenzellen ausdifferenzieren. Desweiteren sollten diese miRNAs funktionell charakterisiert werden, um die molekularen Mechanismen, die der Osteogenese unterliegen, aufzudecken. Letztendlich war es ein weiteres wichtiges Ziel die Ziel mRNAs der knochenspezifischen miRNAs zu identifizieren und deren Bezug zum Wnt Signalweg zu charakterisieren.
miRNA Expression ist während der osteogenen Differenzierung herunter reguliert
Um solche miRNAs zu identifizieren, die potentiell in die Osteogenese eingreifen, wurden ESCs zu Osteoblasten differenziert und mit undifferenzierten ESCs mit Hilfe eines miRNA Microarrays verglichen. Das so durchgeführte miRNA Profiling zeigte, dass 25 miRNAs während der initialen Phase der osteogenen Differenzierung signifikant unterschiedlich exprimiert wurden. Die differentielle Expression von 4 getesteten miRNAs wurde in einem nächsten Schritt über quantitative real-time PCR (RT-qPCR) beispielhaft bestätigt. Generell zeigte sich, dass differenzierende ESCs viele miRNAs auf geringem Niveau exprimieren. Tatsächlich schien die Herunterregulation der miRNA Expression mit der Differenzierung der Zellen einherzugehen. Desweiteren zeigten miRNAs, die auf dem gleichen Chromosom kodiert sind, ähnliche Expressionsmuster. Zusammenfassend fanden sich etliche miRNAs, die in undifferenzierten Zellen im Vergleich zu differenzierenden Zellen unterschiedlich exprimiert werden, von denen schlussendlich 11 für weitere Analysen ausgewählt wurden (miR-22, miR-127, miR-130a, miR-183, miR-291b-5p, miR-293, miR-300, miR-361, miR-467b, miR-665 and miR-690).
miR-127, miR-183, miR-291b-5p, miR-293, miR-361, miR-467b und miR-665 beeinflussen die Osteogenese
In einem nächsten Schritt wurden undifferenzierte und differenzierende ESCs für funktionelle Studien dieser 11 herrunterregulierten miRNAs herangezogen. Um die Funktion dieser miRNAs aufzudecken, wurden sogenannte Gain-of-function und Loss-of-function Studien durchgeführt. Die experimentelle Überexpression und der Knock-down dieser miRNAs führten zu Änderungen in der zellulären Morphologie, der Viabilität und der osteogenen Differenzierungskapazität wie durch einen Kalziumdepositionsassay, einen ALP Aktivitätsassay und die Expression knochenspezifischer Markergene gezeigt werden konnte. Im Besonderen erhöhte die Überexpression der miR-361 und der Knock-down der miR-665 den Mineralisierungsgrad der Zellen und die Expressionniveaus knochenspezifischer Gene. Daher ist zu schließen, dass beide miRNAs das Potential besitzen, die Osteogenese - besonders in den frühen Stadien der Keimbahnspezifikation - zu regulieren.
miRNAs als Modulatoren der Osteogenese
Um miRNA Zielkandidaten zu identifizieren, die die beobachteten Effekte auf die Zellviabilität und auf die osteogene Differenzierungen bedingen könnten, wurde ein kombinierter Ansatz aus Bioinformatischer Sequenz- und Prädiktionsanalyse, mRNA Expressionsanalyse und TurboGFP Reduktion nach miRNA Überexpression gewählt. Gepaart mit einer Literatursuche deutete diese Zielkandidatenanalyse darauf hin, dass die identifizierten miRNAs tatsächlich den Aktivierungsstatus des Wnt Signalwegs manipulieren könnten, da viele der prädiktierten Target mRNAs bekannt dafür sind, mit dem Wnt Signalweg zu interagieren.
Um zu bestätigen, dass miR-183, miR-293, miR-361, miR-665 und miR-690 die Osteogenese regulieren, wurde die mRNA/miRNA Interaktion indirekt mittels RT-qPCR studiert. Die Überexpression dieser miRNAs führte zu einer Erniedrigung des mRNA Expressionsspiegels von WIF-1 (Wnt inhibitory factor 1) durch miR-293, NFATc-3 (nuclear factor of activated T cells 3) und Prickle-1 durch miR-361, Dishevelled 1 (Dvl-1) durch miR-665, sowie forkhead box O3 (FoxO-3), Ras homolog gene family, member A (RhoA) und CatnB durch miR-690.
In einem nächsten Schritt konnte durch Nutzung eines speziellen Reportersystems (TurboGFP) eine direkte Interaktion zwischen miR-361 und Prickle-1 nachgewiesen werden. Wie bereits in anderen Studien gezeigt, ist Prickle-1 in der Lage, die Spiegel an Dvl-3 durch Ubiquitinierung des Proteins zu reduzieren, was zur Inhibierung des kanonischen Wnt Signalweges führt. Da Dvls als positive Regulatoren der Osteogenese bekannt sind, indem sie den CatnB Spiegel erhöhen und die lymphoid enhancer factor/T cell factor protein (LEF/TCF) abhängige Transkription stimulieren, könnte Prickle-1 als negativer Regulator fungieren, indem es Dvls von diesem Transkriptionskomplex entfernt.
Abschließend lässt sich zusammenfassen, dass miR-361 in dieser Arbeit als neuartiger Aktivator der osteogenen Differenzierung vorgeschlagen wird. Die molekulare Interaktion zwischen miR-361, Prickle-1 und Dvls bietet einen neuartigen Mechanismus der Wnt Signalaktivierung während der Osteogenese und kann für weitere Untersuchungen zur Identifizierung von Schlüsselkomponenten des Wnt Signalweges herangezogen werden.
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Aspectos celulares, teciduais e moleculares da osteogênese ectópica e ortotópica induzida pela matriz alogênica óssea e dentinária / Cellular, tissue and molecular aspects of the ectopic and orthotopic osteogenese induced by bone and dentine allogenic matrixCestari, Tania Mary 08 April 2009 (has links)
O objetivo do atual trabalho, foi correlacionar os eventos celulares e teciduais com a expressão das proteínas VEGF, BMP-7, RANKL e OPG durante a osteogênese ectópica e ortotópica, induzida pela matriz óssea (MO) e dentinária (MD) alogênica. Matrizes alogênicas desmineralizada em HCl a 0,6N, obtidas de fêmur e incisivo de ratos, fori implantada entre as fáscias musculares da coxa e em defeito trans-ósseo de 8mm de diâmetro nos ossos parietais. As análises radiográfica e histomorfométrica da neoformação óssea e, a imunohistoquímica e o western blotting para as proteínas VEGF, BMP, RANKL e OPG, mostraram que: a) o volume da região do enxerto nos sítios ortotópicos reduziu 19% em 42 dias; b) em ambos tipos de enxerto e locais de implantação, ocorreu formação de tecido cartilaginoso e ósseo; c) nos sítios intramusculares, a reabsorção da matriz alogênica e a remodelação do tecido cartilaginoso, ósseo e medular foi mais acelerado, em relação a implantação ortotópico; d) o aumento na densidade de volume dos vasos sanguíneos e no número de osteoblastos/osteócitos e osteoclastos ocorreu simultaneamente e estava associado à maior reabsorção da matriz alogênica e à formação do tecido medular (hematopoiético); e) as proteínas VEGF, BMP-7, RANKL, OPG foram expressas em condrócitos, osteoblastos ativos, osteócitos recém aprisionados na matriz e em células estromais próximas aos osteoblastos ou às áreas da matriz alogênica reabsorvida; e f) a expressão das proteínas VEGF, BMP-7, RANKL e OPG foi maior no grupo MO. O pico de expressão dessas proteínas ocorreu nos períodos de 14 aos 21 dias no grupo da MO e 21 e 28 dias no grupo da MD. Concluímos que, a capacidade osteoindutora da matriz alogênica desmineralizada está relacionado a origem da matriz e ao sítio de implantação e que, as proteínas VEGF, BMP-7, RANKL e OPG estão associadas a maior reabsorção da matriz implantada, promovendo uma rápida e contínua liberação dos morfógenos contidos em seu interior que, induzem temporal e espacialmente a formação óssea/medular. / The aim of the present work was to correlate the cellular and tissue events with the expression of VEGF, BMP-7, RANKL and OPG during ectopic and orthotopic osteogenesis, induced by bone and dentin allogeneic matrix. Allogenic matrices obtained from femur and incisor of rats and demineralized in 0.6 N HCl were implanted into a intramuscular pocket and a 8mm-diameter bone defect in the skull. The radiographic and histomorphometric analysis of new bone formation, and immunohistochemistry and western blotting for VEGF, BMP, RANKL and OPG proteins, showed that: a) the total volume of the graft region in orthotopic site decreased 19% at 42 days b) in both graft types and implantation sites occurred formation of cartilaginous and bone tissues, c) in intramuscular sites, the resorption of allogenic matrix and remodeling of the new formed cartilage and bone were faster, in relation to orthotopic implantation sites; d) the increase in the volume density of blood vessels and in the number of osteoblasts/osteocytes and osteoclasts occurred simultaneously and was associated with greater reabsorption of the allogenic matrix and hematopoietic bone marrow formation; e) VEGF, BMP-7, RANKL, OPG proteins were expressed in chondrocytes, active osteoblasts, newly osteocytes confined and stromal cells located near the osteoblasts or in the surface of the reabsorbed matrix; and f) the VEGF, BMP-7, RANKL and OPG expression was higher in MO grafts than in the MD. The peak of expression of these proteins each occurred at 14 and 21 days in MO and 21 and 28 days in MD. We concluded that, the osteoinductive capacity of allogeneic demineralized matrix is related to matrix origin and implantation site and that the VEGF, BMP-7, RANKL and OPG proteins are associated with greater reabsorption of the implanted matrix, promoting rapid and continuous matrix-release morphogens that induces spatially and temporally the bone and bone marrow formation.
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Aspectos celulares, teciduais e moleculares da osteogênese ectópica e ortotópica induzida pela matriz alogênica óssea e dentinária / Cellular, tissue and molecular aspects of the ectopic and orthotopic osteogenese induced by bone and dentine allogenic matrixTania Mary Cestari 08 April 2009 (has links)
O objetivo do atual trabalho, foi correlacionar os eventos celulares e teciduais com a expressão das proteínas VEGF, BMP-7, RANKL e OPG durante a osteogênese ectópica e ortotópica, induzida pela matriz óssea (MO) e dentinária (MD) alogênica. Matrizes alogênicas desmineralizada em HCl a 0,6N, obtidas de fêmur e incisivo de ratos, fori implantada entre as fáscias musculares da coxa e em defeito trans-ósseo de 8mm de diâmetro nos ossos parietais. As análises radiográfica e histomorfométrica da neoformação óssea e, a imunohistoquímica e o western blotting para as proteínas VEGF, BMP, RANKL e OPG, mostraram que: a) o volume da região do enxerto nos sítios ortotópicos reduziu 19% em 42 dias; b) em ambos tipos de enxerto e locais de implantação, ocorreu formação de tecido cartilaginoso e ósseo; c) nos sítios intramusculares, a reabsorção da matriz alogênica e a remodelação do tecido cartilaginoso, ósseo e medular foi mais acelerado, em relação a implantação ortotópico; d) o aumento na densidade de volume dos vasos sanguíneos e no número de osteoblastos/osteócitos e osteoclastos ocorreu simultaneamente e estava associado à maior reabsorção da matriz alogênica e à formação do tecido medular (hematopoiético); e) as proteínas VEGF, BMP-7, RANKL, OPG foram expressas em condrócitos, osteoblastos ativos, osteócitos recém aprisionados na matriz e em células estromais próximas aos osteoblastos ou às áreas da matriz alogênica reabsorvida; e f) a expressão das proteínas VEGF, BMP-7, RANKL e OPG foi maior no grupo MO. O pico de expressão dessas proteínas ocorreu nos períodos de 14 aos 21 dias no grupo da MO e 21 e 28 dias no grupo da MD. Concluímos que, a capacidade osteoindutora da matriz alogênica desmineralizada está relacionado a origem da matriz e ao sítio de implantação e que, as proteínas VEGF, BMP-7, RANKL e OPG estão associadas a maior reabsorção da matriz implantada, promovendo uma rápida e contínua liberação dos morfógenos contidos em seu interior que, induzem temporal e espacialmente a formação óssea/medular. / The aim of the present work was to correlate the cellular and tissue events with the expression of VEGF, BMP-7, RANKL and OPG during ectopic and orthotopic osteogenesis, induced by bone and dentin allogeneic matrix. Allogenic matrices obtained from femur and incisor of rats and demineralized in 0.6 N HCl were implanted into a intramuscular pocket and a 8mm-diameter bone defect in the skull. The radiographic and histomorphometric analysis of new bone formation, and immunohistochemistry and western blotting for VEGF, BMP, RANKL and OPG proteins, showed that: a) the total volume of the graft region in orthotopic site decreased 19% at 42 days b) in both graft types and implantation sites occurred formation of cartilaginous and bone tissues, c) in intramuscular sites, the resorption of allogenic matrix and remodeling of the new formed cartilage and bone were faster, in relation to orthotopic implantation sites; d) the increase in the volume density of blood vessels and in the number of osteoblasts/osteocytes and osteoclasts occurred simultaneously and was associated with greater reabsorption of the allogenic matrix and hematopoietic bone marrow formation; e) VEGF, BMP-7, RANKL, OPG proteins were expressed in chondrocytes, active osteoblasts, newly osteocytes confined and stromal cells located near the osteoblasts or in the surface of the reabsorbed matrix; and f) the VEGF, BMP-7, RANKL and OPG expression was higher in MO grafts than in the MD. The peak of expression of these proteins each occurred at 14 and 21 days in MO and 21 and 28 days in MD. We concluded that, the osteoinductive capacity of allogeneic demineralized matrix is related to matrix origin and implantation site and that the VEGF, BMP-7, RANKL and OPG proteins are associated with greater reabsorption of the implanted matrix, promoting rapid and continuous matrix-release morphogens that induces spatially and temporally the bone and bone marrow formation.
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Convivendo com a dor: a perspectiva da criança e do adolescente em cuidados paliativos / The perspective from children and adolescent´s in palliative careBorghi, Camila Amaral 19 December 2012 (has links)
A dor é um evento estressante para crianças e adolescentes e pode ter consequências negativas fisiológicas, psicológicas e comportamentais ainda mais quando é acompanhada por uma doença crônica, sem possibilidades de cura. Nesse sentido, o Cuidado Paliativo Pediátrico é uma filosofia de cuidado que deve ser instituída desde o diagnóstico da doença até que esta não responda mais às intervenções curativas. Assim, o foco do cuidado passa a ser a maximização da qualidade de vida que a criança e o adolescente e seus familiares necessitam, enquanto o sofrimento e a dor são minimizados. Considerando-se o caráter único da experiência de dor da criança e do adolescente, em cuidados paliativos, optou-se por desenvolver um estudo com abordagem qualitativa. Utilizamos como referencial teórico a Teoria de Desenvolvimento Cognitivo de Piaget e, como referencial metodológico, a História Oral. Tais referenciais são fundamentais para ancorar os resultados encontrados neste estudo e responder ao objetivo geral de conhecer a experiência da criança e do adolescente em cuidados paliativos no manejo diário da dor e aos objetivos específicos de conhecer como a criança e o adolescente em cuidados paliativos descrevem a intensidade, a qualidade e a localização da dor e de conhecer como a criança e o adolescente em cuidados paliativos manejam a dor em seu cotidiano. Permitem, igualmente, que crianças e adolescentes, de 6 a 17 anos 11 meses e 29 dias, portadores de uma doença crônica que causava dor e que estavam em cuidados paliativos e matriculados em um Ambulatório de Dor e Cuidados Paliativos de um Hospital Escola Pediátrico de caráter público de nível terciário tenham voz. Crianças em idade escolar descreveram sua dor a partir de componentes sensoriais e avaliativos. Os adolescentes, por outro lado, expressaram sua dor utilizando componentes sensoriais, avaliativos, afetivos e de miscelânea. Dos seis colaboradores deste estudo, cinco ainda frequentam a escola e relacionam-se com crianças e adolescentes da mesma faixa etária. Todos os colaboradores fazem uso de medicamentos e de alternativas não farmacológicas para o alívio da dor, como massagem, hidroterapia, acupuntura e crioterapia, constatando melhora em sua dor. Alguns colaboradores precisam lidar com sua aparência física prejudicada pela doença. Apesar da dificuldade de se entrevistar crianças e adolescentes, percebemos que eles têm muito a dizer e a nos ensinar, principalmente como eles lidam com a dor em seu cotidiano. Este trabalho é importante para que os profissionais de saúde compreendam que, com um adequado manejo da dor, crianças e adolescentes conseguem ter uma vida mais próxima da normalidade, reduzindo seu sofrimento. / Pain is a stressful event for children and adolescents and can have negative consequences - physiological, psychological and behavioral ones even more when it is accompanied by a chronic disease with no possibility of cure. In this context, the Pediatric Palliative Care is a philosophy of care that must be instituted from the diagnosis until the illness no longer responds to curative interventions. Therefore, the focus of care is to provide the highest quality of life possible to children and adolescents and their families while minimizing suffering and pain. Considering the uniqueness of the experience of pain in children and adolescents in palliative care, we chose to develop a qualitative study. We used the Theory of Cognitive Development Piaget as theoretical framework and the Oral History as the methodological one. Such references are essential to support the results found in this study and to address the overall objective of knowing the experience of the child and adolescent in palliative care for the daily management of pain as well as the specific goals of knowing how the children and adolescents in palliative care describe the intensity, quality and location of pain and of knowing how children and adolescents in palliative manage pain in their daily lives. Moreover, these frameworks allow that children and adolescents (from 6 to 17 years 11 months and 29 days), suffering from a chronic disease that caused pain and in palliative care and who were enrolled in an Outpatient Pain and Palliative Care of a public tertiary Pediatric Teaching Hospital character, have a voice. School children described their pain using sensory and evaluative components. Teenagers, on the other hand, expressed their pain using sensory, evaluative, affective and miscellaneous ones. Of the six collaborators to this study, five are still in school and relate to children and adolescents of the same age. All collaborators use drugs and non-pharmacological alternatives for pain relief such as massage, hydrotherapy, acupuncture and cryotherapy, reporting improvement in their pain. Some collaborators need to deal with their physical appearance which is affected by the disease. Despite the difficulty of interviewing children and teenagers, we have realized that they have a lot to say and to teach us, especially with regard to how they deal with pain in their daily lives. The present work is important for health professionals to understand that, with adequate pain management, children and adolescents can live a life as normal as possible, thus reducing their suffering.
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Lokalisation, Proliferation und Differenzierung von STRO-1-positiven Zellen aus dem Geweih von Damhirschen (Dama dama) / Localization, proliferation and differentiation of STRO-1-positiv cells of antlers of fallow deer (Dama dama)Seymour, Natascha 05 October 2010 (has links)
No description available.
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Convivendo com a dor: a perspectiva da criança e do adolescente em cuidados paliativos / The perspective from children and adolescent´s in palliative careCamila Amaral Borghi 19 December 2012 (has links)
A dor é um evento estressante para crianças e adolescentes e pode ter consequências negativas fisiológicas, psicológicas e comportamentais ainda mais quando é acompanhada por uma doença crônica, sem possibilidades de cura. Nesse sentido, o Cuidado Paliativo Pediátrico é uma filosofia de cuidado que deve ser instituída desde o diagnóstico da doença até que esta não responda mais às intervenções curativas. Assim, o foco do cuidado passa a ser a maximização da qualidade de vida que a criança e o adolescente e seus familiares necessitam, enquanto o sofrimento e a dor são minimizados. Considerando-se o caráter único da experiência de dor da criança e do adolescente, em cuidados paliativos, optou-se por desenvolver um estudo com abordagem qualitativa. Utilizamos como referencial teórico a Teoria de Desenvolvimento Cognitivo de Piaget e, como referencial metodológico, a História Oral. Tais referenciais são fundamentais para ancorar os resultados encontrados neste estudo e responder ao objetivo geral de conhecer a experiência da criança e do adolescente em cuidados paliativos no manejo diário da dor e aos objetivos específicos de conhecer como a criança e o adolescente em cuidados paliativos descrevem a intensidade, a qualidade e a localização da dor e de conhecer como a criança e o adolescente em cuidados paliativos manejam a dor em seu cotidiano. Permitem, igualmente, que crianças e adolescentes, de 6 a 17 anos 11 meses e 29 dias, portadores de uma doença crônica que causava dor e que estavam em cuidados paliativos e matriculados em um Ambulatório de Dor e Cuidados Paliativos de um Hospital Escola Pediátrico de caráter público de nível terciário tenham voz. Crianças em idade escolar descreveram sua dor a partir de componentes sensoriais e avaliativos. Os adolescentes, por outro lado, expressaram sua dor utilizando componentes sensoriais, avaliativos, afetivos e de miscelânea. Dos seis colaboradores deste estudo, cinco ainda frequentam a escola e relacionam-se com crianças e adolescentes da mesma faixa etária. Todos os colaboradores fazem uso de medicamentos e de alternativas não farmacológicas para o alívio da dor, como massagem, hidroterapia, acupuntura e crioterapia, constatando melhora em sua dor. Alguns colaboradores precisam lidar com sua aparência física prejudicada pela doença. Apesar da dificuldade de se entrevistar crianças e adolescentes, percebemos que eles têm muito a dizer e a nos ensinar, principalmente como eles lidam com a dor em seu cotidiano. Este trabalho é importante para que os profissionais de saúde compreendam que, com um adequado manejo da dor, crianças e adolescentes conseguem ter uma vida mais próxima da normalidade, reduzindo seu sofrimento. / Pain is a stressful event for children and adolescents and can have negative consequences - physiological, psychological and behavioral ones even more when it is accompanied by a chronic disease with no possibility of cure. In this context, the Pediatric Palliative Care is a philosophy of care that must be instituted from the diagnosis until the illness no longer responds to curative interventions. Therefore, the focus of care is to provide the highest quality of life possible to children and adolescents and their families while minimizing suffering and pain. Considering the uniqueness of the experience of pain in children and adolescents in palliative care, we chose to develop a qualitative study. We used the Theory of Cognitive Development Piaget as theoretical framework and the Oral History as the methodological one. Such references are essential to support the results found in this study and to address the overall objective of knowing the experience of the child and adolescent in palliative care for the daily management of pain as well as the specific goals of knowing how the children and adolescents in palliative care describe the intensity, quality and location of pain and of knowing how children and adolescents in palliative manage pain in their daily lives. Moreover, these frameworks allow that children and adolescents (from 6 to 17 years 11 months and 29 days), suffering from a chronic disease that caused pain and in palliative care and who were enrolled in an Outpatient Pain and Palliative Care of a public tertiary Pediatric Teaching Hospital character, have a voice. School children described their pain using sensory and evaluative components. Teenagers, on the other hand, expressed their pain using sensory, evaluative, affective and miscellaneous ones. Of the six collaborators to this study, five are still in school and relate to children and adolescents of the same age. All collaborators use drugs and non-pharmacological alternatives for pain relief such as massage, hydrotherapy, acupuncture and cryotherapy, reporting improvement in their pain. Some collaborators need to deal with their physical appearance which is affected by the disease. Despite the difficulty of interviewing children and teenagers, we have realized that they have a lot to say and to teach us, especially with regard to how they deal with pain in their daily lives. The present work is important for health professionals to understand that, with adequate pain management, children and adolescents can live a life as normal as possible, thus reducing their suffering.
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