Eine Vielzahl präklinischer Schlaganfallstudien zeigte die neuroprotektive und neuroregenerative Wirkung des hämatopoetischen Wachstumsfaktors G-CSF (Granulozyten-Kolonie stimulierender Faktor). Ein Wirkungsmechanismus des G-CSF ist die Mobilisation von protektiven Knochen-markzellen in die ischämische Läsion, wobei diese zeitverzögert nach G-CSF-Gabe stattfindet. Eine zusätzliche frühzeitige Transplantation mononukleärer Knochenmarkzellen (BM MNC) könnte diese therapeutische Lücke füllen. Ziel der vorliegenden Studie war es, die Wirksamkeit dieser Kombinations-therapie in einem Schlaganfallmodell der spontan hypertensiven Ratte (SHR) zu testen. Syngene BM MNC wurden aus dem Knochenmark von SHRs durch immunmagnetische Depletion der Granulozyten isoliert. Nach Verschluss der Arteria cerebri media wurde den Tieren über insgesamt 5 Tage G-CSF verabreicht und zusätzlich zu einem frühen (6h nach Schlaganfall) oder späteren (48h nach Schlaganfall) Zeitpunkt BM MNC intravenös appliziert. Unbehandelte Schlaganfalltiere sowie Tiere mit alleiniger G-CSF-Therapie dienten als Kontrolle. Das Infarktvolumen wurde weder durch die alleinige G-CSF-Gabe noch durch die zusätzliche Zelltherapie verändert. Dennoch wiesen Tiere mit G-CSF-Einzeltherapie eine anhaltende funktionelle Verbesserung des sensomotorischen Defizites auf. Während die zusätzliche frühzeitige Zelltransplantation (6h) keinen weiteren Therapieeffekt zeigte, führte die Zelltransplantation nach 48h zu einer Aufhebung des protektiven G-CSF Effektes. Die G-CSF-Therapie bewirkte erwartungsgemäß einen deutlichen Anstieg der zirkulierenden Leukozyten. Interessanterweise wurde der Granulozytengehalt im Blut und in der Milz durch die einmalige Zelltherapie nach 48h signifikant erhöht. Ein Großteil der transplantierten BM MNC (48h) konnte in der Milz nachgewiesen werden und führte dort vermutlich zu einer kompetitiven Hemmung des Granulozytenabbaus. Dies hatte sowohl den Anstieg der zirkulierenden Granulozyten als auch deren vermehrte Infiltration in das ischämische Hirngewebe zur Folge und könnte schließlich den negativen Einfluss auf die funktionelle Verbesserung erklären. Die beobachteten Interaktionsmechanismen werfen ein interessantes Licht auf die mögliche Wirkungsweise von Zelltherapien und unterstreichen die entscheidende Rolle des Immunsystems in der Pathophysiologie des Schlaganfalls.

Solid organ transplantation has evolved from being an experimental procedure to a life-saving treatment for patients with end-stage organ failure. The risk of losing a transplant due to acute rejection is very low with the use of modern immunosuppressive protocols and the short-term results are impressive. However, long-term outcomes are suboptimal and transplant recipients are at increased risks for severe complications such as cancers, opportunistic infections and cardiovascular events. The previous struggle to achieve short-term survival has turned into a search for new strategies to improve patient and transplant longevity. Regulatory T cells (TRegs), a subset of T cells, occur naturally in the immune system and have the capacity to down regulate immune responses. Under normal conditions they maintain self-tolerance and prevent excessive immune activation. Functional TReg defects lead to a massive autoimmune response and are not compatible with life. Preclinical data support that TRegs can be used as a cell therapy to prevent transplant rejection, with the potential to minimize the need for traditional immunosuppression and improve the long-term outcome. This thesis aims to enhance the translation of TReg cell therapy to clinical organ transplantation. In particular, strategies for isolation and expansion of TRegs from uremic patients awaiting kidney transplantation have been assessed. A non-invasive imaging technique to study T cell products after intravenous administration was developed, for use in future clinical trials. The performance of a novel cell purification technique was investigated to potentially improve the clinical production of TRegs. The thesis demonstrates that TRegs can be isolated and expanded from uremic patients to display potent suppressive properties in vitro. The mode of isolation and expansion affect the functional characteristics, where cells purified with cytometry based techniques and expanded with mature dendritic cells were the most advantageous. T cells can be labeled using the radioactive tracer [111In]oxine with preserved viability and subsequently followed in vivo with SPECT/CT for more than 1 week after intravenous administration. The use of microfluidic switch technology offers a novel way of purifying TRegs at high speed, purity and viability, under conditions compatible with clinical use.

Growing evidence from animal and clinical studies suggests that cardiac cell therapy can restore perfusion and improve function in the ischemic/infarcted myocardium. However, cell therapy is hindered by insufficient cell numbers, inefficient cell homing and engraftment, and inadequate cellular interactions. Furthermore, the biological mechanisms and local effects of transplanted cells have not been well-elucidated. The research presented herein attempts to address some of these issues. In manuscript #1, a new subpopulation of circulating progenitor cells (CPCs), termed derived CD133+ cells, was generated from the CD133- fraction of human peripheral blood. The derived CD133+ progenitors appeared to have superior vasculogenic potential in vitro, which may prove to be beneficial in inducing vasculogenesis in ischemic tissues. Positron emission tomography (PET) with direct cell labeling and reporter gene techniques were employed to assess the fate of transplanted human CPCs in vivo at different subjects of investigation, and different stages of cell transplantation. In manuscript #2, PET imaging with 2-[18F]fluoro-2-deoxy-D-glucose (18F-FDG) direct cell labeling was used to demonstrate that collagen-based matrices improve the early homing and retention of delivered CPCs in a rat ischemic hindlimb model. This mechanism conferred by the matrix may have implications on cell therapy at the early stages after transplantation. In manuscript #3, a more efficient, stable and accurate labeling method, hexadecyl-4-[18F]fluorobenzoate (18F-HFB) direct cell labeling, was developed to quantify cell distribution of transplanted CPCs in a rat myocardial infarction model. PET imaging of 18F-HFB-CPCs revealed significant cell washout from the myocardium immediately after intramyocardial injection, with only a small proportion of transplanted CPCs remaining in the target area in the first 4 hours after delivery. In manuscript #4, human CPCs transduced with lentiviral vectors showed stable expression of PET reporter genes. This reporter gene based-cell labeling technique can be developed for noninvasive tracking cells within a bioengineered matrix by PET, while preserving cell phenotype, viability and function. These studies contribute important insights into the biology and physiology of transplanted stem cells and the ability of delivery matrices to improve transplanted cell engraftment, survival, and function. I believe with further refinement, cell expansion, tissue engineering and PET imaging could facilitate the clinical applications of cell therapies in years to come.

Cellular transplantation for photoreceptor replacement in retinal disease is limited by poor distribution, survival and integration of cells in vivo after standard delivery in saline vehicle. We were interested in addressing each of these barriers in order to improve transplant efficacy. To this end, we designed the first injectable biomaterial-based cell delivery vehicle to transplant adult stem cell progeny into the subretinal space of adult retina. A minimally-invasive and bio-resorbable blend of hyaluronan and methylcellulose (HAMC) was found to overcome cellular aggregation and non-contiguous distribution. The ability to direct stem cell differentiation toward a particular retinal lineage is another challenge facing clinical application. We showed that prospectively and clonally isolated multipotent mouse and human retinal stem cells (RSCs) could be directed toward a mature rod photoreceptor fate with the highest efficiency reported to date (>90%). Combinations of taurine and retinoic acid directed rod differentiation similar to rod development in vivo. RSC-derived rods exhibited morphology, protein and gene expression consistent with primary cultures of rods in vitro. When combined with the HAMC delivery vehicle, greater cell survival and improved integration of post-mitotic RSC-derived rods was observed in vivo compared to saline delivery. Improved donor rod survival was ascribed to the CD44 receptor – HAMC interaction in vitro and in vivo. Combined with HAMC delivery, disruption of the glial limiting membrane improved cell integration and resulted in the highest levels of integration of adult stem cell-derived rod photoreceptors relative to previous reports in the literature. Mature rod-lineage committed cells demonstrated higher integration potential compared to immature rods in this context. The integrated cells expressed Rhodopsin and elaborated outer segments. In the absence of the glial limiting membrane, rod integration depended on pro-survival signals from the environment. This work demonstrates that adult RSCs show promise for regenerative medicine strategies in the adult retina.

A insuficiência cardíaca é a doença crônica com maior impacto na sobrevida e qualidade de vida dos pacientes. Apesar do constante desenvolvimento de novas estratégias de tratamento, esta doença continua atingindo altos índices de mortalidade. O coração adulto tem capacidade de regeneração limitada e há grande evidência experimental de que os transplantes de células-tronco poderiam ser uma abordagem eficiente na recuperação do miocárdio lesado. Contudo, a maioria dos estudos são realizados em cardiomiopatias isquêmicas, existindo poucos estudos na cardiomiopatia dilatada (CMD). Em função disto, este trabalho foi realizado com o objetivo de avaliar a regeneração do miocárdio em coelhos com CMD induzida pela doxorrubicina, por meio do uso de células-tronco mesenquimais (MSC) obtidas de tecido adiposo, associadas ou não com plasma rico em plaquetas (PRP). Foram utilizadas 40 coelhas, Nova Zelândia, e um coelho macho doador das MSCs derivadas do tecido adiposo. As coelhas foram divididas em dois grupos: CMD induzida pela doxorrubicina e o grupo saudável. Cada grupo foi subdividido conforme o tratamento recebido: solução fisiológica, MCSs, PRP e MSCs associadas ao PRP. Os subgrupos receberam o tratamento por injeção diretamente no miocárdio no ventrículo esquerdo mediante toracoscopia vídeo assistida. Os coelhos foram avaliados por exames de ecocardiograma, eletrocardiograma, troponina I, no dia da chegada, após a indução da CMD e 15 dias após o recebimento das terapias. Nesta última avaliação, foi realizada a eutanásia e coletado o coração para análise histológica. Foi observado que após a indução, a troponina I se elevou, o segmento QRS visto no eletrocardiograma, aumentou e, no ecocardiograma, as frações de ejeção (FE) e encurtamento (FS) diminuíram e o diâmetro sistólico do ventrículo esquerdo (VEs) aumentou, em todos os animais avaliados. Após os tratamentos, o subgrupo MSCs obtiveram os melhores resultados em todas as análises citadas. Houve menor elevação da troponina I, o segmento QRS diminuiu, as FS e FE aumentaram e o VEs diminuiu. No exame histopatógico, analisado pela coloração de hematoxicilina-eosina, constatou-se que o subgrupo MSCs apresentou menos lesões, e nos subgrupos MSCs associadas com PRP, solução fisiológica e PRP as lesões aumentaram gradualmente, respectivamente. Os resultados sugerem que o uso das MSCs melhoraram a função cardíaca em coelhos com cardiomiopatia dilatada e que há necessidade de mais estudos no uso de PRP no miocárdio. / Heart failure is a chronic disease with major impact on survival and quality of patient’s life. Despite the constant development of new treatment strategies, this disease still affects high mortality rates. The adult heart has limited ability to regenerate and there is experimental evidence that large transplants of stem cells could be an effective approach in the recovery of injured myocardium. However, most studies are performed in ischemic cardiomyopathy, there are few studies in dilated cardiomyopathy. Because of this, this study aimed at evaluating the regeneration of the myocardium in rabbits with dilated cardiomyopathy induced by doxorubicin through the use of mesenchymal stem cell (MSC) derived from adipose tissue, associated or not with platelet-rich plasma (PRP). 40 New Zealand rabbits were utilized and a male rabbit donor MSCs derived from adipose tissue. The rabbits were divided into two groups: dilated cardiomyopathy doxorubicin-induced and the healthy group. Each group was divided according to treatment received: saline, MSCs, PRP and MSCs associated with PRP. The subgroups receiving treatment through an injection directly into the myocardium of the left ventricle through video-assisted thoracoscopy. The rabbits were evaluated by echocardiogram, electrocardiogram, troponin I, on the day of arrival, after induction of dilated cardiomyopathy and 15 days after receipt of therapies. This last evaluation, euthanasia was performed and the hearts collected for histological analysis. It was observed that after induction the troponin I increased, the QRS segment, seen on the electrocardiogram, increased, and, in echocardiography, the ejection and shortening fractions decreased, and left ventricular systolic diameter increased in all animals evaluated. After treatments, the subgroup MSCs have the best results in all tests cited. There was a lower elevation of troponin I, decreased QRS segment, the ejection and shortening fractions increased and left ventricular systolic diameter decreased. On examination histologic, analyzed by hematoxylin-eosin staining, the subgroup found that MSCs had fewer injuries, and in the subgroups MSCs associated with PRP, PRP and saline lesions gradually increased, respectively. The results suggest that the use of MSCs improved cardiac function in rabbits with dilated cardiomyopathy and that there is need for more studies on the use of PRP in the myocardium.

Methylglyoxal (MG), a highly reactive dicarbonyl accumulates after myocardial infarction (MI), causing adverse remodelling and cardiac dysfunction. We hypothesized that therapy using bone marrow cells (BMCs) overexpressing glyoxalase1 (Glo1), the main enzyme that metabolizes MG, injected into mouse MI model would translate into better survival of transplanted cells and improve their therapeutic effect. We found that Glo1 expression is significantly reduced at 7 days post-MI. Glo1 BMCs exposed to MG in vitro displayed greater angiogenic potential and reduced reactive oxygen species production compared to wild type (WT) BMCs. However, in the mouse MI model, Glo1 BMCs did not improve cardiac function or vascularity or reduce scar formation compared to WT BMCs and saline treatments. In conclusion, Glo1 overexpression in BMCs does not confer superior therapeutic efficacy for treating MI under the conditions tested.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Stem cells are undifferentiated cells capable of autorenovation and differentiation in several cell lines. These cells can be found in various tissues such as bone marrow, skin, nervous tissue, adipose tissue and dental pulp. Adipose tissue is an alternative source of mesenchymal stem cells because it consists in a less invasive collection and provides higher number of cells when compared to bone marrow. In humans, adipose tissue metabolism differs according to the anatomical location. In mice, differences in the cellular composition and differentiation capacity of mesenchymal stem cells derived from adipose tissue (ADSCs) were observed according to the anatomical regions. In order to assist researchers in choosing the site of collection and the best cell passage, this study aimed to compare the viability and quality of multipotent mesenchymal stromal (CMEM) derived from subcutaneous and omentum of a dog through indicator of cytotoxicity and oxidative stress in six different passages of culture. The samples of adipose tissue from the both sites were collected from a dog corpse. Cells were processed and cultured in a humidified atmosphere with 5% CO2 and subsequently stained with Trypan Blue and counted in a Neubauer chamber. To assess cytotoxicity the technique of fragmentation and fluorimetry with picogreen dye was used. Also, oxidative stress assays were performed by using fluorimetric assay 2-70-dichlorofluorescein diacetate technique (DCFH-DA) and measuring the levels of thiobarbituric acid reactive species (TBARS). The results showed greater levels of oxidative stress in the first and last passages of both groups, favoring cytotoxicity and cell death. / As células-tronco são células indiferenciadas com capacidade de autorrenovação e diferenciação em diversas linhagens celulares. Estas células podem ser encontradas nos mais variados tecidos, como medula óssea, pele, tecido nervoso, tecido adiposo e polpa dentária. O tecido adiposo é fonte alternativa de células-tronco mesenquimais, apresentando-se como método menos invasivo e permitindo coleta de maior quantidade celular em comparação a medula óssea. Nos humanos, o tecido adiposo apresenta diferenças de metabolismo, conforme a localização anatômica. Em camundongos, foram observadas diferenças em relação à composição celular e à capacidade de diferenciação das células-tronco mesenquimais derivadas do tecido adiposo (ADSCs), de acordo com as regiões anatômicas. Com o intuito de auxiliar pesquisadores na escolha do sítio de coleta e as melhores linhagens celulares, este trabalho teve como objetivo comparar a viabilidade e a qualidade de células mesenquimais estromais multipotentes (CMEM) derivadas do tecido adiposo subcutâneo e do omento maior de cão através de indicadores de citotoxicidade e estresse oxidativo em seis diferentes passagens de cultura. Para isso foram coletados tecido adiposo do omento e subcutâneo de um cadáver fresco de cão oriundo da rotina do Hospital Veterinário da Universidade Federal de Santa Maria. As células foram processadas e cultivadas em atmosfera umidificada a CO2 5%, sendo posteriormente coradas com Azul de Tripan e contadas em câmara de Neubauer. Para avaliar a citotoxicidade foi utilizada a técnica de fragmentação e fluorimetria com corante Picogreen. O nível de estresse oxidativo foi medido através do ensaio fluorimétrico do 2-70-dichlorofluorescein diacetate (DCFH-DA) e níveis de espécies reativas ao ácido tiobarbitúrico (TBARS). Os resultados mostraram que a primeira e última passagem em ambos os grupos são as passagens mais submetidas ao estresse oxidativo ficando mais sujeitas à citotoxicidade.

Made available in DSpace on 2014-06-11T19:31:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-07-17Bitstream added on 2014-06-13T20:41:32Z : No. of bitstreams: 1 sousa_mg_dr_jabo.pdf: 788549 bytes, checksum: 1d83d7d53925e7aa7edb678ee0f699f0 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / No trabalho em tela, avaliou-se o potencial terapêutico do transplante de células-tronco autológas em dois modelos experimentais de cardiomiopatia. Na primeira parte avaliou-se a função cardíaca de cães chagásicos crônicos submetidos ao transplante de células-tronco hematopoiéticas autólogas. Na segunda etapa, a terapia foi realizada em cães com cardiomiopatia dilatada induzida pela doxorrubicina. Nas duas situações, as células-tronco foram obtidas da medula óssea, que foi coletada e, subsequentemente, fracionada em gradiente de ficol. A seguir, as células mononucleares foram recuperadas, lavadas e diluídas para transplantação intracoronariana. A cateterização das coronárias foi realizada pela técnica de Seldinger e foram infundidas cerca de 60 milhões de células em cada animal. Ao longo de seis meses os cães foram avaliados mensalmente quanto aos parâmetros eletrocárdiográficos, ecocardiográficos e de pressão arterial. Nos cães chagásicos pouco parâmetros se modificaram ao longo do período de avaliação, embora tenham observadas variações nos parâmetros ecocardiográficos PVAO, PPE, TRIV e ITEI. Com relação aos cães com cardiomiopatia induzida pela doxorrubicina, o período pós-transplante foi caracterizado por variação importante nos parâmetros ecocardiográficos PE, IEPs, PPE, TRIV, ITEI, SIV, PLVE, FEJ, FEC, VMEF e IE, embora não ocorrido alterações nas variáveis eletrocardiográficas e de pressão arterial. Os resultados mostraram evolução favorável da função sistólica e diastólica do ventrículo esquerdo em ambos os modelos utilizados. / This study was conceived to evaluate the therapeutic potential of stem cells transplantation in two experimental models os cardiomyopathy. In the first of the study, the cardiac function of dogs with chronic Chagas disease sbumeitted hematopoietic stem cell therapy was assessed. In the second part, cell teraphy was carried out in dogs with doxorubicin-induced cardiomyopathy. In either situation, the animals included in the study underwent autologous bone marrow-derived stem cell transplantation into the coronary arteries. Bone marrow was aspirated and centrifuged in ficoll gradient. Mononuclear cells were collected, washed and diluted to permit intracoronary delivery. Coronary catheterization was accomplished with the Seldinger's technique, and approximately 60 million cells were transfunded in each animal. Dogs were assessed for electrocardiographic, echocardiographic and blood pressure parameters for six months at monthly intervals. Dogs with Chagas disease showed improvement in only a few parameters during the period of study, although a significant variation was seen in the echo parameters PVAO, PPE, TRIV and ITEI. Regarding the dogs with doxorubcin-induced cardiomyopathy, the post-transplantation riod was characterized by an important change in the echocardiographic parameters IVEs, SSPE, IEPs, PPE, TRIV, ITEI, SIV, PLVE, FEJ, FEC, VMEF and IE, although no change was documented in the electrocardiographic and blood pressure measurements. Results showed enhancement of left ventricular systolic and diastolic functions in both experimental models.

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Agradecemos a compreensão. on 2016-06-06T12:09:26Z (GMT) / Submitted by MARIA JOSE MALAGUTTI FERREIRA null (malagutti10@yahoo.com.br) on 2016-06-06T13:45:46Z No. of bitstreams: 1 dissertacao.pdf: 16505999 bytes, checksum: 2bad751dc7f5e6a56599b9731ecadd63 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-06-07T12:19:57Z (GMT) No. of bitstreams: 1 malaguttiferreira_mj_me_assis.pdf: 16424202 bytes, checksum: a8bc2a6fd56c317ab15caad2ca1f6025 (MD5) / Made available in DSpace on 2016-06-07T12:19:57Z (GMT). No. of bitstreams: 1 malaguttiferreira_mj_me_assis.pdf: 16424202 bytes, checksum: a8bc2a6fd56c317ab15caad2ca1f6025 (MD5) Previous issue date: 2016-03-17 / O tecido adiposo (TA) representa, atualmente, uma importante fonte de células-tronco mesenquimais (CTM), uma vez que pode ser obtido de forma relativamente fácil, por meio de procedimentos pouco invasivos, com baixa incidência de morbi-mortalidade e com grande disponibilidade de material para pesquisa aplicada. Para utilização das CTM derivadas do TA, em protocolos de terapia celular em pacientes humanos, emprega-se, em média, uma quantidade significativa de células, da ordem 107 a 108 células/kg do paciente. A partir de uma pequena fração de tecido adiposo autólogo ou heterólogo pode-se expandir o número de células, por meio de procedimentos de cultivo celular “in vitro”, a fim de se obter um número adequado de células que resulte em eficácia terapêutica. Os procedimentos de cultivo prolongado “in vitro”, podem, no entanto, provocar efeitos genotóxicos e mutagênicos. Neste sentido, é de extrema importância o monitoramento da análise da estabilidade genética durante as culturas de CT destinadas aos procedimentos de terapia celular em pacientes humanos. Em função destes aspectos, este trabalho teve por objetivo analisar, por meio do Ensaio Cometa (EC) e Teste de micronúcleo (MCN), a integridade genômica de células-tronco mesenquimais oriundas do tecido adiposo (CT-TA), mantidas em cultura até a décima primeira passagem, avaliando os possíveis efeitos genotóxicos e mutagênicos do cultivo celular prolongado. Os resultados mostraram que no teste EC, não houve diferença estatística entre os controles e células mantidas em cultivo por diferentes passagens. Os testes de MCN, no entanto, mostraram aumento de micronúcleos, a partir da terceira passagem de cultivo celular, com aumento significativo a partir da sétima passagem, em relação à primeira passagem. Os resultados mostram efeitos genotóxicos sobre células-tronco mantidas em cultura para expansão celular e indicam a necessidade de novas análises, a fim de se avaliar com maior segurança o emprego com células-tronco em terapia celular resultantes de culturas celulares expandidas “in vitro” durante diferentes passagens. / The adipose tissue (AT) currently represents a major source of mesenchymal stem cells (MSC), since it can be obtained relatively easily by means of minimally invasive procedures, with low incidence of morbidity and mortality and a large availability of material for applied research. For use of MSC derived from AT in cell therapy protocols in human patients, is employed, on average, a significant amount of cells of the order 107 -108 cells / kg patient. From a fraction of autologous adipose tissue or heterologous one can expand the number of cells through cell culture procedures "in vitro" in order to obtain an adequate number of cells resulting in therapeutic efficacy. Prolonged culture procedures "in vitro" may, however, cause genotoxic and mutagenic effects. Therefore, it is extremely important to monitor the analysis of the genetic stability during CT crops to cell therapy procedures in human patients. Based on these aspects, this study aimed to analyze, through the comet assay (AC) and micronucleus test (MCN), the genomic integrity of mesenchymal stem cells derived from adipose tissue (CT-AT), maintained in culture until the eleventh passage, evaluating the possible genotoxic and mutagenic effects of prolonged cell culture. The results showed that the AC test, there was no statistical difference between the control and cells maintained in culture for different passages. The MCN tests, however, showed an increase in micronuclei after the third passage of cell culture, significant increase from the seventh passage to the first passage. The results show genotoxic effects on stem cells maintained in culture for cell expansion and indicate the need for further analysis in order to assess with greater certainty employment stem cells in cell therapy resulting from expanded cell cultures "in vitro" during different passages.

Submitted by Ana Carolina Picolo Pasian (ana_781@hotmail.com) on 2018-04-20T19:54:18Z No. of bitstreams: 1 PARA DESPÓSITO.pdf: 1930882 bytes, checksum: 397a91ec261857e9a2bea286ae3eeb52 (MD5) / Approved for entry into archive by ROSANGELA APARECIDA LOBO null (rosangelalobo@btu.unesp.br) on 2018-04-23T13:24:13Z (GMT) No. of bitstreams: 1 pasian_acp_me_bot.pdf: 1930882 bytes, checksum: 397a91ec261857e9a2bea286ae3eeb52 (MD5) / Made available in DSpace on 2018-04-23T13:24:13Z (GMT). No. of bitstreams: 1 pasian_acp_me_bot.pdf: 1930882 bytes, checksum: 397a91ec261857e9a2bea286ae3eeb52 (MD5) Previous issue date: 2018-02-27 / A medicina regenerativa é uma área em crescente expansão no Brasil e no mundo, a qual procura ampliar a capacidade natural de regeneração dos tecidos através da utilização de células, fatores de proliferação e biomateriais. Um dos ramos da medicina regenerativa é a terapia celular, vertente que utiliza células-tronco, visando a substituição de tecidos funcionalmente ou estruturalmente lesados, apresentando um caráter terapêutico. Na medicina LASERs e LEDs vem sendo estudados como ferramenta terapêutica, mostrando possuir capacidade bioestimulatória. Este campo é caracterizado por uma variedade de metodologias, que são utilizadas em uma gama considerável de aplicações. Na técnica de fotoestimulação, utiliza-se a luz para ativar moléculas e funções celulares, apresentando o potencial de afetar a proliferação e diferenciação e o metabolismo da célula, estimulando a fosforilação oxidativa e podendo reduzir a resposta inflamatória local. Entretanto para que essa resposta ocorra, inúmeros trabalhos afirmam sobre a importância da seleção de um comprimento de onda ideal, uma vez que a utilização de um comprimento inapropriado pode acarretar em resultados contrários aos esperados, como a bioinibição. Diante destes achados o presente trabalho propôs-se a avaliar a ação do LED 850nm, no regime pulsátil, nas doses de 3, 5 e 10J/cm² na cultura de célula-tronco mesenquimal (CTM) com Soro Fetal Bovino (SFB) e com Hormônios derivados de plaquetas (HDP), e na cultura de células de Linfoma linfoblástico tipo B, RAJI Cells na dose de 10J/cm². Em ambos experimentos de exposição a luz, o comprimento de onda 850 nm inibiu a proliferação celular. Na cultura de CTM o LED tornou o desdobramento celular mais lento e acarretou na diminuição da confluência celular, especialmente nas doses de 5 e 10J/cm². Na cultura de linfoma Linfoblástico tipo B, em apenas 1 semana de exposição o mesmo comportamento de bioinibição foi encontrado na dose de 10J/cm². O grupo não tratado apresentou 7,0 X 10 5 células, em média, por frasco enquanto que as células submetidas à irradiação sofreram diminuição do tempo de desdobramento sendo a concentração destas de 4,2X105 , em média, por frasco. / Regenerative medicine is a promising growing area worldwide, with the aim of restore and regenerate tissues and whole organs through the use of cells, proliferation factors and biomaterials. One branch of regenerative medicine is cell therapy, that uses stem cells, aiming at the substitution of functionally or structurally damaged tissues, presenting therapeutic fature. LASERs and LEDs are available as therapeutic tools, showing biostimulating ability. The photo-stimulation technique uses light to activate molecules and cellular functions, presenting potential to affect proliferation, cell differentiation and metabolism, stimulating oxidative phosphorylation and reducing the local inflammatory response. Data shows the importance of selecting an ideal wavelength, such as the use of an inappropriate choice, can lead to undisered results, such as bioinhibition. In the present work, we evaluated the action of LED 850nm, pulsatile, at the doses of 3, 5 and 10J/cm² in mesenchymal stem cells (CTM) with Bovine Fetal Serum (FBS) and with derived platelets – Hormones (HDP) and B-cell lymphoblastic cell culture, 10J /cm2 , RAJI cells. In all light exposure experiments, wavelength of 850 nm inhibited cell proliferation. CTM culture, LED had a low proliferation rate, resulting in a decrease in cellular confluence, especially at 5 and 10J/cm2 . Lymphoblastic lymphoma type B cells, in only one week of exposure presente the same behavior of bioinhibition at 10J/cm2 . The control group had 7.0 x 105 cells on per vial, while cells subjected to irradiation underwent the unfolding time at a concentration of 4.2 x 105 on average per vial.