• Refine Query
  • Source
  • Publication year
  • to
  • Language
  • 11
  • 7
  • 4
  • 3
  • 3
  • 3
  • 3
  • 3
  • 3
  • 3
  • 3
  • 1
  • Tagged with
  • 32
  • 32
  • 24
  • 7
  • 7
  • 7
  • 7
  • 7
  • 6
  • 5
  • 5
  • 5
  • 4
  • 4
  • 4
  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
11

Investigating the effects of feeding soy protein and soy isoflavones on bone metabolism in female rats fed low dietary calcium

Farnworth, Sara January 2005 (has links)
No description available.
12

Investigating the effects of feeding soy protein and soy isoflavones on bone metabolism in female rats fed low dietary calcium

Farnworth, Sara January 2005 (has links)
The effects of feeding soy protein (SP) or SP plus isoflavones (IF) (150 and 400 mg IF/kg diet) on bone metabolism were assessed in female weanling and retired breeder (RB) rats fed low calcium (Ca) for five weeks. Young rats fed SP-based diets had significantly smaller reductions in bone mineral density (BMD) and bone mineral content (BMC) as a result of the low Ca diet compared to those fed casein-based diets. Added IFs had no further benefits. Soy protein also affected bone metabolism in both the young and RB rats as indicated by markers of bone resorption. Neither the SP nor the added IFs had any effects on BMD or BMC in the RB rats. Feeding SP to young rats resulted in beneficial changes in BMD, BMC, and biochemical markers of bone metabolism. This study indicates that SP positively affects bone metabolism and minimizes the negative effects associated with low Ca intakes in young rats.
13

The cardio-metabolic profile and bone mineral density in African and Indian postmenopausal women.

Moodley, Jayeshnee. January 2013 (has links)
AIMS. To determine the cardio-metabolic risk profile and incidence of low bone mineral density in African and Indian postmenopausal women attending the IALCH menopause clinic and to determine whether there is a correlation between cardio-metabolic parameters and low bone mineral density. METHODS. A retrospective, descriptive study involving all Indian and African postmenopausal women, above the age of 40, referred to the menopause outpatient clinic at IALCH from 01 July 2009 to 31 December 2010 was conducted. Data was collected from the medi-com database using a structured questionnaire. Cardio-metabolic data was analysed as continuous variables and summarized using means and standard deviations. Bone mineral density was treated as a quantitative variable and correlation analysis was used to assess relationships between the variables. This was done for each race group separately. The Students T-test was used to compare cardio-metabolic variables between the two ethnic groups. SPSS version 18.0 was used to analyse data. RESULTS. The records of 106 women were analysed (51 African and 55 Indian). In African and Indian women, the prevalence of hypertension was 54.9% vs 65.5%, the prevalence of diabetes was 31.4% vs 56.4%, the prevalence of dyslipidaemia was 17.6% vs 32.7% and the prevalence of ischaemic heart disease was 5.9% vs 14.9% respectively. The prevalence of low bone mineral density was higher in Indian women (40%) compared to African women (23.5%). The mean body mass index (BMI) of African women was significantly higher than Indian women, (33 vs 29). There were no significant differences between African and Indian postmenopausal women regarding their lipid profile, fasting glucose, fasting insulin and thyroid profile. The mean bone mineral density (BMD) in the hip and spine was lower in Indian women compared to African women, however the prevalence of osteopaenia and osteoporosis, as defined by T-scores, was not statistically significant. Statistically significant positive correlations were observed between an increasing BMI and BMD (p<0.001) and increases in weight and BMD (p<0.001). A statistically significant correlation were observed between serum LDL-cholesterol values and BMD (p=0.03), where serum LDL-cholesterol values were inversely proportional to BMD. There were no significant correlations between BMD and the remaining cardio-metabolic variables (ie blood pressure; waist-hip ratio; clinical stigma of dyslipidaemia; clinical stigma of insulin resistance; cholesterol; HDL; triglycerides; fasting glucose; fasting insulin and thyroid function). CONCLUSIONS. There is a high prevalence of cardiovascular risks and low BMD amongst the local menopausal population, irrespective of ethnicity. African and Indian postmenopausal women had a high prevalence of hypertension (60%), diabetes (44%), dyslipidaemia (25%) and obesity (54%). In African women, the incidence of low BMD was 35% in the hip, 53% in the neck of femur and 55% in the lumbar spine. In Indian women, the incidence of low BMD was 55% in the hip, 67% in the neck of femur and 69% in the lumbar spine. BMI and weight showed a positive correlation with bone mineral density. Regarding the cardio-metabolic variables, an increasing LDL value was negatively correlated with bone mineral density. It thus is apparent that a screening lipid profile during the peri-menopausal years, coupled with early and appropriate lifestyle management regarding body mass index/ weight may limit the burden of morbidity in later life. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2013.
14

Mechanisms of vitamin D receptor and retinoid X receptor mediated hormone resistance and cell differentiation in normal and cancer cells

Macoritto, Michael. January 2007 (has links)
Vitamin D is a precursor to a steroid hormone, 1,25 dihydroxyvitamin D (1,25(OH)2D). After its discovery and the characterization of its receptor, the vitamin D receptor (VDR), it was initially thought only to be involved in calcium homeostasis, but further research revealed an important role for vitamin D in the regulation of cell growth and differentiation of such cells as osteoblasts and bone marrow adipocytes. 1,25(OH)2D has also been shown to be a strong inhibitor and pro-differentiator of keratinocytes. The anti-proliferative and pro-differentiative properties of this hormone have led to studies where 1,25(OH)2D anticancer properties were assessed and initial findings that showed a requirement of other factors beyond VDR to induce 1,25(OH)2D signaling led to the identification of the retinoid X receptor, a common heterodimeric partner for several hormone receptors. The focus of thesis was to further elucidate the structure-function relationship of both the vitamin D receptor and the retinoid X receptor. Additionally, contributions to work directed towards further identifying the effects of vitamin D on osteoblast differentiation and survival. Interactions of 1,25(OH) 2D3 with its cognate receptor, identifying a key amino acid (Tryptophan 286) required for ligand contact and transcriptional activation, are described in Chapter 2. Mechanisms of vitamin D action on mesenchymal stem cell differentiation, promotion of osteoblast induction and maturation, and inhibition of adipocyte differentiation, are eluicidated in Chapter 3. Chapter 4 illustrates the effects of RAS/RAF/Mitogen-activated protein kinase mediated RXRalpha phosphorylation on the three-dimensional structure of the RXR/nuclear receptor partner heterodimers. Furthermore, this chapter reveals the inhibitory effect of the phosphorylation of a critical amino acid (serine 260) on the interaction of the AF-2 domain of the RXR with several coactivators, resulting in a decrease in the signaling potential of multiple steroid hormone receptors. The findings of this thesis further the knowledge of several areas of vitamin D biology, including both the canonical areas of bone formation, and the non-canonical area of vitamin D and cancer.
15

The influence of hormone suppression therapy and related factors on bone mineral density in cancer patients

Manning, Katherine L. January 2008 (has links)
Cancer-treatment-induced bone loss (CTBL) is a well-recognized co-morbidity that affects many cancer patients. Commonly used to treat breast and prostate cancer patients, hormone suppression therapy (HST) may accelerate bone loss, resulting in osteopenia or osteoporosis. Because of their broad clinical utility, lifestyle and dietary modifications, such as regular participation in bone-stressing exercise and calcium supplementation, are starting to play a much larger role in the prevention and treatment of CTBL. However, only limited information is available on the effects that these factors may have on bone mineral density (BMD). Purpose. The purpose of this investigation was to assess the degree of BMD change from the onset of HST to 6 months and to examine the impact that physical activity and calcium intake may have on BMD. Methods. Twelve subjects (8 females and 4 males) undergoing HST for breast or prostate cancer were enrolled in the study. BMD at the spine, dual femur, and total body was assessed by dual-energy x-ray absorptiometry at 0 and 6 months. In addition, subjects wore an accelerometer to assess physical activity level and completed a lifestyle questionnaire at baseline, 3, and 6 months after starting therapy. Aside from the 7 non-exercise subjects, 5 subjects chose to participate in The Cancer Exercise Program at Ball Memorial Hospital or complete bone-stressing exercises at home. Results. No significant changes in BMD were observed after 6 months of HST between the groups at any of the sites. When all subjects were examined together, a significant BMD decrease of 3.2% was observed at the lumbar spine. The accelerometer and lifestyle questionnaires revealed that the males were more active than the females and the exercisers were more active than the non-exercisers at both baseline and after 6 months of HST. Supplementation with calcium did not affect BMD changes at any site;although it is possible this is an effect of gender as all males were included in the same group. Lifestyle factors such as history of smoking and alcoholism were also examined, but were not correlated to changes in BMD. Conclusion. Treatment with HST results in decreases in BMD, particularly at the spine. Bone-stressing exercise helped maintain or improve total body BMD in 3 of the 5 subjects the exercise group. There appears to be no difference in BMD between those who supplemented with calcium and those who did not. / School of Physical Education, Sport, and Exercise Science
16

Papel dos receptores nucleares ativados por proliferadores de peroxissomos (PPAR) na periodontite induzida em ratos. / Role of peroxisome proliferator activated nuclear receptor (PPAR) in induced periodontitis in rats.

Rodrigo Martins Porto 03 July 2012 (has links)
Este estudo investigou o efeito da Roziglitazona (RTZ) sobre a perda óssea alveolar induzida pela periodontite (POAIP). Durante 3 semanas, ratos receberam sal puro de RTZ (i.p.) ou a formulaço comercial Avandia<font face=\"Symbol\">&#210; (v.o.); os grupos controles receberam os repectiovos veículos (DMSO ou CMC). Duas semanas após o inicio do tratamento, a periodontite (P) foi induzida. Após 7 dias da indução da P, as mandíbulas foram removidas para mediço da perda óssea alveolar. Amostras de osso alveolar foram analisadas por qPCR para RUNX2, Osterix, TRAF6, TRAF2, RANKL, óxido nítrico sintases (e, n e iNOS) e PPARs (<font face=\"Symbol\">a, <font face=\"Symbol\">b e <font face=\"Symbol\">g). A farmacocinética da RTZ para cada formulaço foi estudada por HPLC-MS/MS. Tanto o sal puro como a formulaço comercial de RTZ resultou no agravamento da POAIP. Apesar dos resultados similares nas concentrações plasmáticas de RTZ os mecanismos de sinalizaço parecem depender da formulaço administrada a qual pode ser devido a interferência do veículo. / This study investigate the effects of rosiglitazone (RTZ) on periodontitis-induced alveolar bone loss (PIABL). Rats received RTZ during 3 weeks, either as the pure maleate salt (i.p.) or the commercial formulation Avandia<font face=\"Symbol\">&#226; (p.o.); control animals received the respective vehicles (DMSO or CMC). Two weeks after the treatments begins, periodontitis (P) were induced. After 7 days after P induction, jaws were removed for ABL measurement. Alveolar bone samples were analyzed by qPCR for RUNX2, Osterix, TRAF6, TRAF2, RANKL, nitric oxide sintase (e, n and iNOS) and PPARs (<font face=\"Symbol\">a, <font face=\"Symbol\">b e <font face=\"Symbol\">g). RTZ pharmacokinetics from each formulation was also studied (HPLC-MS/MS). RTZ, either from the pure maleate salt or the commercial Avandia, resulted in aggravated PIABL. Despite resulting in similar plasma RTZ concentrations, signaling mechanisms seem to depend on the administered formulation which could be due to vehicle related effects interfence.
17

Novel insights into the mechanistic gene regulation of STAT3 in bone cells

Corry, Kylie A. 25 June 2015 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / Many cells are involved in the orchestra that is bone homeostasis--particularly osteoclasts and osteoblasts, which mediate remodeling of bones. This creates a balance that must be kept in check, otherwise pathologies arise. The JAK-STAT signaling pathway is crucial to maintaining this balance. It has long been known that the transcription factor STAT3 has more profound effects on bone homeostasis than other members of the STAT family of proteins. Recently, a genetic condition called Job’s Syndrome has been specifically linked to point mutations in the Stat3 gene. These patients present with severe bone abnormalities, including prominent foreheads, broad nasal bridges, and abnormal eye spacing. For this reason, our lab has extensively studied conditional knockouts of Stat3 in all three types of bones cells in mice and observed severe deficiencies in numerous parameters of normal bone phenotypes. STAT3 seems to play a principal role in the signaling that takes place upon mechanical loading of bone tissues and calling cells into action where they are needed. Furthermore, STAT3 has been found to be up-regulated in the early-response gene cluster following mechanical loading. Our current approach to studying STAT3’s effects on bone includes both in vivo and in vitro comparisons of WT and KO STAT3 models. The conditional knock-out of STAT3 in 8-week old mice revealed significant phenotypic variations as compared to the WT controls, while no significant differences were observed in cKO newborn pups. We also looked at immortalized WT and STAT3 KO cell lines. The STAT3 KO cells had diminished proliferation rates and decreased differentiation capabilities. Furthermore, STAT3 KO cells showed significantly reduced mRNA levels of both Wnt3a and Wnt5a when exposed to fluid shear stress. By employing available ChIP-seq data, we were able to elucidate the genome-wide binding patterns of STAT3. From the peak distribution, we can begin to uncover novel downstream effectors of STAT3 signaling that are responsible for the observed phenotypes in our conditional knockout mouse model. A preliminary look at the ChIP-seq data reveals Wnt and Nrf2 signaling to be under the putative control of STAT3. In our further research, we endeavor to experimentally confirm the ChIP-seq data for STAT3 with RNA-seq experiments in the hopes of finding potential therapeutic targets for bone pathologies.
18

Mechanisms of vitamin D receptor and retinoid X receptor mediated hormone resistance and cell differentiation in normal and cancer cells

Macoritto, Michael. January 2007 (has links)
No description available.
19

Effect of calcium supplementation on bone mineral content and calcium absorption in Chinese children with habitually low calcium intake.

January 1995 (has links)
by Warren Tak-keung Lee. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 161-186). / Chapter CHAPTER 1: --- INTRODUCTION --- p.1 / Chapter 1.1 --- Background --- p.1 / Chapter 1.2 --- Objectives --- p.3 / Chapter CHAPTER 2: --- LITERATURE REVIEW --- p.4 / Chapter 2.1 --- Calcium intakes of Hong Kong Chinese: past and present --- p.4 / Chapter 2.1.1 --- Adults --- p.4 / Chapter 2.1.2 --- Children --- p.5 / Chapter 2.2 --- Calcium Metabolism --- p.6 / Chapter 2.2.1 --- Calcium and bone mass --- p.9 / Chapter 2.2.2 --- Ethnicity and bone mass --- p.9 / Chapter 2.2.3 --- Physical activity and bone mass --- p.10 / Chapter 2.2.4 --- Hormonal control of calcium metabolism --- p.11 / Chapter 2.2.5 --- Intestinal calcium absorption --- p.14 / Chapter (1) --- Calcium transport across the intestine / Chapter (2) --- Active Calcium Transport / Chapter (3) --- Passive calcium transport / Chapter (4) --- Age and calcium absorption / Chapter 2.2.6 --- Dietary components in relation to calcium bioavailability and absorption --- p.17 / Chapter (1) --- Habitual calcium intake / Chapter (2) --- Vitamin D nutritional status / Chapter (3) --- Protein / Chapter (4) --- Phosphorous and Ca:P ratio / Chapter (5) --- Sodium / Chapter (6) --- Lactose / Chapter (7) --- Glucose and Glucose Polymers / Chapter (8) --- Phytate / Chapter (9) --- Oxalate / Chapter (10) --- Plant estrogen (phyto-estrogen) / Chapter 2.2.7 --- Bioavailability from calcium salts --- p.23 / Chapter 2.3 --- Calcium intakes and requirements --- p.25 / Chapter 2.3.1 --- Calcium requirements in adulthood --- p.28 / Chapter 2.3.2 --- Calcium requirements in childhood --- p.29 / Chapter 2.3.3 --- Manifestation of calcium deficiency in children --- p.30 / Chapter 2.4 --- Assessment of Dietary Intakes --- p.32 / Chapter 2.4.1 --- Food weighing method --- p.32 / Chapter 2.4.2 --- Food Recording method --- p.34 / Chapter 2.4.3 --- 24-hour dietary recall --- p.35 / Chapter 2.4.4 --- Food frequency method --- p.36 / Chapter 2.4.5 --- Dietary history method --- p.38 / Chapter 2.4.6 --- Chemical analysis of duplicate meals --- p.39 / Chapter 2.4.7 --- Photographic method --- p.40 / Chapter 2.4.8 --- Selecting suitable methods for the present study --- p.40 / Chapter 2.5 --- Food composition database --- p.41 / Chapter 2.6 --- Evaluation of bone mass in vivo --- p.43 / Chapter 2.6.1 --- Single photon absorptiometry --- p.44 / Chapter 2.6.2 --- Dual photon absorptiometry --- p.46 / Chapter 2.6.3 --- Dual energy X-ray absorptiometry --- p.47 / Chapter 2.6.4 --- Quantitative computerized tomography --- p.47 / Chapter 2.6.5 --- The techniques selected to quantify bone mass in the present study --- p.48 / Chapter 2.7 --- Measurement of intestinal calcium absorption --- p.49 / Chapter 2.7.1 --- Metabolic balance study --- p.49 / Chapter 2.7.2 --- Isotopic techniques (radioisotope or stable isotope) --- p.50 / Chapter (1) --- Radio isotope vs stable isotope / Chapter (2) --- The single-label isotope technique / Chapter (3) --- The double-label isotope technique / Chapter (4) --- "Preparation of stable isotopes for human study, and determination of stable isotopes in body fluids" / Chapter (I) --- Dosage considerations / Chapter (II) --- Intrinsic or Extrinsic labelling / Chapter (III) --- Oral and intravenous administration of isotopes / Chapter 2.7.3 --- The technique selected to determine calcium absorption in the present study --- p.60 / Chapter 2.8 --- Mass spectrometry --- p.60 / Chapter 2.8.1 --- Thermal ionization mass spectrometry --- p.60 / Chapter 2.8.2 --- Fast atom bombardment mass spectrometry --- p.61 / Chapter 2.8.3 --- Inductively coupled plasma mass spectrometry --- p.61 / Chapter 2.8.4 --- Electron impact mass spectrometry and Gas chromatography mass spectrometry --- p.62 / Chapter 2.8.5 --- Neutron activation analysis --- p.62 / Chapter 2.8.6 --- The type of mass spectrometry used to determine stable isotopic ratios in the present study --- p.63 / Chapter 2.9 --- Assessment of physical activity in children --- p.63 / Chapter 2.9.1 --- Activity questionnaire or record --- p.64 / Chapter 2.9.2 --- Direct measurement of physical activity --- p.65 / Chapter (1) --- Accelerometer / Chapter (2) --- Pedometer / Chapter (3) --- Actometer / Chapter (4) --- Video-recording / Chapter (5) --- Heart-rate recording / Chapter 2.9.3 --- Selection of a suitable physical activity assessment method --- p.67 / Chapter CHAPTER 3 --- RANDOMIZED DOUBLE-BLIND CONTROLLED CALCIUM SUPPLEMENTATON TRIALS IN RELATION TO BONE AND HEIGHT ACQUISITION IN 7-YEAR OLD CHINESE CHILDREN FROM JIANGMEN (CHINA) AND HONG KONG --- p.71 / Chapter 3.1 --- Chapter summary --- p.71 / Chapter 3.2 --- Chapter Introduction --- p.72 / PART I --- p.73 / Chapter 3.3 --- Double-blind Controlled Randomized Calcium Supplementation and Bone and Height Acquisition in Chinese Children Accustomed to Mean Calcium Intake at About 300mg/d --- p.73 / Chapter 3.3.1 --- Introduction --- p.73 / Chapter 3.3.2 --- Objectives --- p.74 / Chapter 3.3.3 --- Subject and Methods --- p.74 / Chapter 3.3.4 --- Results --- p.81 / Chapter 3.3.5 --- Discussions --- p.83 / Chapter 3.3.6 --- Conclusion --- p.84 / PART II --- p.84 / Chapter 3.4 --- Randomized double-blind controlled calcium supplementation in relation to bone mineral accretion and height increment of Hong Kong Chinese children --- p.84 / Chapter 3.4.1 --- Introduction --- p.84 / Chapter 3.4.2 --- Objectives --- p.85 / Chapter 3.4.3 --- Subjects and Methods --- p.85 / Chapter 3.3.4 --- Results --- p.91 / Chapter 3.4.5 --- Discussions --- p.95 / Chapter 3.4.6 --- Conclusion --- p.97 / Chapter 3.5 --- Comparisons of the two calcium supplementation trials from Jiangmen and Hong Kong --- p.97 / Chapter 3.6 --- Chapter Discussions --- p.99 / Chapter 3.7 --- Chapter Conclusion --- p.103 / Chapter CHAPTER 4 --- TRUE FRACTIONAL CALCIUM ABSORPTION OF CHINESE CHILDREN AND THE EFFECTS OF DOUBLE-BLIND CONTROLLED CALCIUM SUPPLEMENTATION ON CALCIUM ABSORPTION IN CHILDREN MEASURED WITH STABLE ISOTOPES (42Ca and 44Ca) --- p.117 / Chapter 4.1 --- Chapter summary --- p.117 / Chapter 4.2 --- Chapter introduction --- p.118 / PART I --- p.119 / Chapter 4.3 --- True fractional calcium absorption in Chinese children measured with stable isotopes (42Ca and 44Ca) --- p.119 / Chapter 4.3.1 --- Introduction --- p.119 / Chapter 4.3.2 --- Objectives --- p.120 / Chapter 4.3.3 --- "Subjects, Materials and Methods" --- p.120 / Chapter 4.3.4 --- Results --- p.127 / Chapter 4.3.5 --- Discussions & Conclusion --- p.131 / Part II --- p.131 / Chapter 4.4 --- Effects of double-blind controlled calcium supplementation on calcium absorption in Chinese children measured with stable isotopes (42Ca and 44Ca) --- p.131 / Chapter 4.4.1 --- Introduction --- p.131 / Chapter 4.4.2 --- Objective --- p.132 / Chapter 4.4.3 --- "Subjects, Materials and Methods" --- p.132 / Chapter 4.4.4 --- Results --- p.135 / Chapter 4.4.5 --- Discussions --- p.137 / Chapter 4.4.6 --- Conclusion --- p.139 / Chapter 4.5 --- Chapter Conclusion and Discussions --- p.140 / Chapter CHAPTER 5 --- GENERAL DISCUSSIONS AND RECOMMENDATIONS FOR FURTHER STUDY --- p.156 / Chapter 5.1 --- Ethnic differences in bone acquisition and calcium absorption --- p.156 / Chapter 5.2 --- Calcium requirements for Chinese children --- p.157 / Chapter 5.3 --- Indications for further studies --- p.158 / REFERENCES --- p.161 / APPENDIXES / PUBLICATIONS
20

Development of siRNA delivery systems for approaching bone formation surfaces and for targeting osteoblasts.

January 2012 (has links)
目前,骨形成低下的骨代謝異常在臨床中面臨巨大挑戰。治療這些疾病的途徑之一可通過小干擾核酸沉默骨形成抑制的基因。隨著核酸干擾技術的快速發展,採用核酸干擾策略進行治療的很多問題已被解決。然而,小干擾核酸的安全和有效遞送仍然是核酸干擾治療進行臨床轉化的瓶頸。其主要問題在於促進骨形成治療所需的小干擾核酸劑量較大,其系統給藥後可能對其他非骨組織產生副作用。所以,亟需針對具有促進成骨潛力的小干擾核酸開發安全有效的遞送系統。本研究的目的就是針對具有促進成骨潛力的小干擾核酸開發特定的遞送系統,以便應用於核酸干擾治療中的促進骨形成。策略之一是利用靶向骨形成表面的遞送系統攜載小干擾核酸到富集于骨形成表面的成骨系細胞。策略之二是直接把小干擾核酸遞送到成骨細胞,使其具有高度的細胞選擇性。在該研究中,我們採用具有成骨潛能的酪蛋白激酶2相互作用蛋白1小干擾核酸作為模型小干擾核酸以考察基因沉默效率。 / 靶向骨形成表面的(天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體-小干擾核酸遞送系統:首先對多肽序列(天門冬氨酸-絲氨酸-絲氨酸)₆靶向骨形成表面的特性進行鑒定。進一步將(天門冬氨酸-絲氨酸-絲氨酸)₆作為靶向分子與以DOTAP為主要成分的陽離子脂質體進行連接製備(天門冬氨酸-絲氨酸-絲氨酸)6-脂質體遞送系統。採用凍幹/再水化方法對小干擾核酸進行包裹並對其粒徑,ζ電位,包封率以及穩定性進行考察。最後分別在體外和體內模型對該遞送系統遞送效果以及其攜載小干擾核酸的基因沉默效率進行評價。 / 實驗結果證實(天門冬氨酸-絲氨酸-絲氨酸)₆是一種在體內可以有效靶向骨形成表面的多肽。(天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體的平均粒徑為140 nm左右,其包封率可高達80%。該遞送系統較穩定,可使攜載的小干擾核酸具有較高的基因沉默效率,而且沒有明顯的細胞毒性。體內試驗表明,該遞送系統在促進小干擾核酸在骨組織的分佈同時降低其被肝組織的攝取。該遞送系統所攜帶的酪蛋白激酶2相互作用蛋白1小干擾核酸可選擇性地沉默骨組織中的酪蛋白激酶2相互作用蛋白1基因,且對其他組織並沒有明顯影響。該結果表明(天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體可促進小干擾核酸靶向骨組織並在骨組織沉默攜載小干擾核酸相應的基因。免疫化學分析結果顯示(天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體可攜載小干擾核酸選擇性地到達骨形成表面的成骨系細胞,避免被前破骨細胞/破骨細胞吞噬。大鼠骨髓細胞採用Alp,Stro-1和Oscar抗體分選後的酪蛋白激酶2相互作用蛋白1 mRNA表達水平顯示該遞送系統可選擇性地沉默成骨系細胞。 / 靶向成骨細胞的L6適配子-脂質納米顆粒-小干擾核酸遞送系統:將針對大鼠成骨細胞(ROS 17/2.8細胞系)進行正向篩選,大鼠肝細胞(BRL-3A細胞系)和外周血細胞進行負向篩選的L6適配子與以DLin-KC2-DMA為主要成分的脂質納米顆粒採用膠束形式插入的方法進行連接製備L6適配子-脂質納米顆粒-小干擾核酸遞送系統。並對其粒徑,ζ電位,包封率和形態學進行考察。在體外評價實驗中,考察了該遞送系統的選擇性,細胞毒性,基因沉默效率以及細胞攝取機制。在體內實驗中,對小干擾核酸的組織分佈以及其攜載小干擾核酸在成骨細胞和肝細胞的分佈進行了評價。 / 實驗結果顯示L6適配子-脂質納米顆粒-小干擾核酸的平均粒徑為84.0±5.3 nm,其電勢為-23 ± 2 mV,包封率為80.8 ± 3.4%. 脂質納米顆粒表面的L6適配子可促進小干擾核酸在ROS 17/2.8細胞系(靶向細胞)中的攝取, 然而在BRL-3A 細胞系(非靶向細胞)中攝入很少。該遞送系統沒有明顯細胞毒性,在10 nM小干擾核酸的低濃度下,體外基因沉默效率可高達50 % 以上。由L6適配子引起的巨胞被證實是成骨細胞攝取L6適配子-脂質納米顆粒所攜載小干擾核酸的主要機制。體內實驗顯示該遞送系統可促進小干擾核酸在骨組織的分佈,降低其被肝組織的攝取。在肝组织冰凍切片中,肝血竇和肝細胞中沒有明顯的小干擾核酸分佈,進一步說明該遞送系統可降低對肝組織的影響。免疫化學分析結果顯示L6適配子-脂質納米顆粒-小干擾核酸可攜載小干擾核酸選擇性地到達成骨細胞,避免被前破骨細胞/破骨細胞吞噬。 / 重要意義:本研究中的兩種新型小干擾核酸系統可分別選擇性地遞送小干擾核酸靶向骨形成表面和成骨細胞。 (天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體-小干擾核酸遞送系統開拓了全新的途徑,實現選擇性地遞送小干擾核酸到骨形成表面從而降低對骨吸收的影響。 L6適配子-脂質納米顆粒-小干擾核酸遞送系統在成骨細胞表面特徵蛋白未知的情況下,首次採用適配子技術在細胞水準實現成骨細胞的選擇性遞送。該研究中的兩種遞送系統為核酸干擾治療的促進骨形成策略提供了強而有力的工具,為實現肌肉骨骼疾病相關領域的核酸干擾治療策略從基礎科學向臨床應用的轉化建立了堅實的基礎。 / Metabolic skeletal disorders that are associated with impaired bone formation are a major clinical challenge. One approach to treat these diseases was to silence bone formation-inhibitory genes by small interference RNAs (siRNAs). With the rapid development of RNA interference (RNAi) technology, more issues of RNAi-based therapy strategies have been addressed. However, the safe and effective delivery of siRNAs is still the bottleneck for its translation from bench to bedside. One major concern was that the large therapeutic doses of systemically administered siRNA to stimulate sufficient bone formation may carry a high risk for adverse effects on non-skeletal tissues. Therefore, development of specific siRNA delivery systems for safe and efficient transporting osteogenic siRNAs is highly desirable. The objective of the present study was to explore siRNA delivery systems for osteogenic siRNAs in RNAi-based bone anabolic therapy. One strategy was to develop siRNA delivery system targeting bone formation surfaces to facilitate delivery of siRNAs to osteogenic cells. Another approch was to develop siRNA delivery system targeting osteoblasts directly. Plekho1 siRNA targeting casein kinase-2 interacting protein-1 (Ckip-1) with osteogenic potential was employed as a representative siRNA in our current study. / (AspSerSer)6-liposome-siRNA for targeting bone formation surfaces: (AspSerSer)6 for targeting bone formation surfaces was firstly identified. Then, (AspSerSer)6 was conjugated with DOTAP-based liposome to produce (AspSerSer)6-liposome. (AspSerSer)6-liposome-siNRA was prepared by lyophilization/rehydration method and characterized in terms of particle size, zeta potential, encapsulation efficiency and the stability in serum. Finally, the delivery of siRNA and the corresponding gene silencing mediated by (AspSerSer)6-liposome-siRNA were evaluated in the in vitro and in vivo models. / The results indicated that the novel (AspSerSer)₆ was a promising peptide for targeting bone formation surfaces in vivo. (AspSerSer)₆-liposome with the average particle size of 140 nm encapsulating Plekho1 siRNA exhibited more than 80% encapsulation efficiency and good stability against enzymatic degradation. It demonstrated high knockdown efficiency without obvious cytotoxicity. In in vivo study, the result of tissue distribution experiment indicated that (AspSerSer)6-liposome-siRNA enhanced the distribution of siRNA in bone, meanwhile reduced the uptake of siRNA in liver. The Plekho1 protein and mRNA expression in various tissues demonstrated that (AspSerSer)₆-liposome-siRNA could facilitate gene silencing in a bone-selective manner. The results of immunochemistry analyses indicated (AspSerSer)₆-liposome-siRNA facilitated delivering siRNA to osteogenic cells at bone formation surfaces and avoided siRNA to pre-osteoclast/osteoclast. Plekho1 mRNA expression in rat bone marrow cells sorted by fluorescence activated cell sorting (FACS) using Alp, Stro-1 and Oscar antibody, respectively, further suggested (AspSerSer)₆-liposome-siRNA could silence gene in a cell-selective manner in vivo. / L6-LNPs-siRNA for targeting osteoblasts: L6 aptamer for targeting osteoblasts (ROS 17/2.8 cell line) and using rat hepatocyte (BRL-3A cell line) and peripheral blood cells in negative selection was conjugated to DLin-KC2-DMA-based lipid nanoparticles (LNPs) to generate L6-LNPs-siRNA by post-insertion method in the form of micelles. L6-LNPs-siRNA was characterized with particle size, zeta potential, encapsulation efficiency and morphology. Its selectivity, cytotoxicity and knockdown efficiency were evaluated in vitro. The mechanism of L6-LNPs-mediated siRNA cellular uptake was further investigated. The tissue distribution of the injected siRNA and the localization of the siRNA with osteoblasts as well as hepatocytes were also evaluated in vivo. / The results showed L6-LNPs-siRNA have the average particle size of 84.0 ± 5.3 nm and zeta potential of -23 ± 2 mV. Its encapsulation efficiency was 80.8 ± 3.4%. The L6 aptamer on the surface of LNPs facilitated the cellular uptake of Plekho1 siRNA in ROS 17/2.8 cell line (target cells) but no uptake in BRL-3A cell line (non-target cells) in vitro. L6-LNPs-siRNA with low cytotoxicity exhibited above 50% knockdown efficiency at a low concentration of 10 nM in vitro. Macropinocytosis induced by L6 was demonstrated to be the predominant mechanism of L6-LNPs mediated siRNA uptake in osteoblasts. In in vivo study, it was shown that L6-LNPs-siRNA facilitated the distribution of siRNA in bone and decreased the hepatic uptake. No obvious siRNA fluorescent signals in sinus and hepatocyte was observed in liver cryosection further indicated the reducing influence on liver after administration of L6-LNPs-siRNA. Co-localization of fluorescence-labeled siRNA with Alp-positive cells was dominantly documented, whereas there were no instances of such overlapping staining with Oscar-positive cells after L6-LNPs-siRNA treatment, which suggested L6-LNPs-siRNA facilitated delivering siRNA in a cell-selective manner in vivo. / Significance: These two innovative siRNA delivery systems in the present study selectively targeted bone formation surfaces and osteoblasts, respectively. (AspSerSer)₆-liposome-siRNA opened up a new avenue to specifically deliver therapeutic siRNAs to bone formation surfaces without affecting bone resorption. L6-LNPs-siRNA achieved the osteoblast-specific delivery for siRNA at cellular level by aptamer technology for the first time, even without knowledge of characteristic protein on the surface of osteoblasts. The two delivery systems provided the powerful tools for RNAi-based bone anabolic strategy and established a solid foundation for translating RNAi-based therapies from basic science to clinic applications in the musculoskeletal field. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wu, Heng. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 130-142). / Abstract also in Chinese. / Acknowledgements --- p.i / Abstract --- p.iii / 論文摘要 --- p.vi / Table of contents --- p.ix / Publications --- p.xiv / List of tables --- p.xvi / List of figures --- p.xvii / List of abbreviations --- p.xxi / Chapter One Introduction --- p.1 / Chapter 1.1 --- Great challenges in skeletal disorders --- p.2 / Chapter 1.2 --- RNA interference (RNAi) as therapeutic strategy --- p.3 / Chapter 1.2.1 --- Mechanism of RNAi --- p.3 / Chapter 1.2.2 --- Potential triggers of RNAi-mediated gene silencing --- p.4 / Chapter 1.2.3 --- Current clinical trials using RNAi as therapeutic strategy --- p.7 / Chapter 1.2.4 --- Current application of therapeutic siRNAs in skeletal disorders --- p.11 / Chapter 1.3 --- Challenges of siRNA in vivo delivery for targeting bone --- p.12 / Chapter 1.3.1 --- General challenges of siRNA delivery in vivo --- p.13 / Chapter 1.3.2 --- Challenges of siRNA delivery to bone --- p.15 / Chapter 1.3.2.1 --- Physiological property --- p.15 / Chapter 1.3.2.2 --- Targeting ligands for approaching bone --- p.16 / Chapter 1.4 --- Strategies of siRNAs in vivo delivery after systemic administration --- p.18 / Chapter 1.4.1 --- Naked siRNA and naked siRNA with chemical conjugation --- p.18 / Chapter 1.4.2 --- Nanoparticle delivery systems --- p.20 / Chapter 1.4.2.1 --- Liposome and lipid-like materials --- p.20 / Chapter 1.4.2.2 --- Polymers --- p.22 / Chapter 1.4.2.3 --- Targeted delivery system --- p.23 / Chapter 1.5 --- Strategies of osteogenic siRNAs delivery for stimulating bone formation --- p.24 / Chapter 1.6 --- Objective of present study --- p.25 / Chapter Chapter Two --- Preparation and characterization of (AspSerSer)₆-liposome-siRNA for targeting bone formation surfaces --- p.26 / Chapter 2.1 --- Introduction --- p.27 / Chapter 2.2 --- Materials and Methods --- p.28 / Chapter 2.2.1 --- Materials --- p.28 / Chapter 2.2.2 --- Identification of (AspSerSer)₆ --- p.29 / Chapter 2.2.3 --- Development of formulation --- p.30 / Chapter 2.2.3.1 --- Selection of the molar ratio of DOTAP --- p.30 / Chapter 2.2.3.2 --- Selection of the molar ratio of siRNA to lipids --- p.30 / Chapter 2.2.4 --- Preparation of (AspSerSer)6-liposome-siRNA --- p.30 / Chapter 2.2.5 --- Characterization of (AspSerSer)₆-liposome --- p.33 / Chapter 2.2.5.1 --- Particle Size and Zeta Potential --- p.33 / Chapter 2.2.5.2 --- Encapsulation Efficiency --- p.33 / Chapter 2.2.5.3 --- Stability in serum --- p.33 / Chapter 2.3 --- Results --- p.34 / Chapter 2.3.1 --- (AspSerSer)₆ as a targeting moiety --- p.34 / Chapter 2.3.2 --- Development of formulation --- p.37 / Chapter 2.3.3 --- Particle size, Zeta Potential and Encapsulation Efficiency --- p.38 / Chapter 2.3.4 --- Stability in serum --- p.38 / Chapter 2.4 --- Discussion --- p.40 / Chapter 2.5 --- Conclusion --- p.42 / Chapter Chapter Three --- Evaluation of (AspSerSer)₆-liposome-siRNA for cell-specific delivery and gene silencing in vitro and in vivo --- p.43 / Chapter 3.1 --- Introduction --- p.44 / Chapter 3.2 --- Materials and Methods --- p.45 / Chapter 3.2.1 --- Materials --- p.45 / Chapter 3.2.2 --- Biological evaluation in vitro --- p.46 / Chapter 3.2.2.1 --- Binding affinity with hydroxyapatite --- p.46 / Chapter 3.2.2.2 --- Cell culture --- p.46 / Chapter 3.2.2.3 --- Cellular uptake --- p.47 / Chapter 3.2.2.4 --- Knockdown efficiency in vitro --- p.47 / Chapter 3.2.2.5 --- Total RNA extraction, reverse transcription and quantitative real-time PCR --- p.48 / Chapter 3.2.3 --- Cytotoxicity --- p.49 / Chapter 3.2.4 --- Tissue distribution --- p.50 / Chapter 3.2.4.1 --- Experimental design --- p.50 / Chapter 3.2.4.2 --- Fluorescence image analysis --- p.50 / Chapter 3.2.4.3 --- Quantitative Analysis --- p.50 / Chapter 3.2.5 --- Localization of siRNA in liver --- p.51 / Chapter 3.2.5.1 --- Experimental design --- p.51 / Chapter 3.2.5.2 --- Histochemisty analysis --- p.51 / Chapter 3.2.6 --- Gene silencing in tissues --- p.52 / Chapter 3.2.6.1 --- Experimental design --- p.52 / Chapter 3.2.6.2 --- Determination of mRNA expression --- p.52 / Chapter 3.2.6.3 --- Western blot analysis --- p.52 / Chapter 3.2.7 --- Localization of siRNA with Osteoblasts/Osteoclasts --- p.53 / Chapter 3.2.7.1 --- Experimental design --- p.53 / Chapter 3.2.7.2 --- Immunohistochemistry analysis --- p.53 / Chapter 3.2.8 --- Gene silencing at cellular levels --- p.54 / Chapter 3.2.8.1 --- Experimental design --- p.54 / Chapter 3.2.8.2 --- Flow cytometry cell sorting --- p.54 / Chapter 3.2.9 --- Statistical analysis --- p.55 / Chapter 3.3 --- Results --- p.56 / Chapter 3.3.1 --- Binding affinity with hydroxyapatite --- p.56 / Chapter 3.3.2 --- Cellular uptake --- p.57 / Chapter 3.3.3 --- Knockdown efficiency in vitro --- p.57 / Chapter 3.3.4 --- Cytotoxicity --- p.59 / Chapter 3.3.5 --- Tissue distribution by imaging analysis --- p.60 / Chapter 3.3.6 --- Quantitative analysis of tissue distribution --- p.62 / Chapter 3.3.7 --- Localization of siRNA in liver --- p.63 / Chapter 3.3.8 --- Plekho1 mRNA and protein expressions --- p.64 / Chapter 3.3.9 --- Immunohistochemistry analysis --- p.65 / Chapter 3.3.10 --- Gene silencing at cellular level --- p.71 / Chapter 3.4 --- Discussion --- p.74 / Chapter 3.5 --- Conclusion --- p.77 / Chapter Chapter Four --- Preparation and characterization of aptamer-functionalized lipid nanoparticle for siRNA cell-specific delivery --- p.78 / Chapter 4.1 --- Introduction --- p.79 / Chapter 4.2 --- Materials and Methods --- p.80 / Chapter 4.2.1 --- Materials --- p.80 / Chapter 4.2.2 --- Synthesis of 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-di- oxolane (DLin-KC2-DMA) --- p.80 / Chapter 4.2.2.1 --- Synthesis of Linoleyl alcohol (1) --- p.81 / Chapter 4.2.2.2 --- Synthesis of Linoleyl bromide (2) --- p.81 / Chapter 4.2.2.3 --- Synthesis of Dilinoleylmethyl formate (3) --- p.82 / Chapter 4.2.2.4 --- Synthesis of Dilinoleyl Methanol (4) --- p.82 / Chapter 4.2.2.5 --- Synthesis of Dilinoleyl Ketone (5) --- p.83 / Chapter 4.2.2.6 --- Synthesis of 2, 2- Dilinoleyl- 4- (2-hydroxyethyl)-[1,3]-dioxolane (6) --- p.83 / Chapter 4.2.2.7 --- Synthesis of DLin-KC2-DMA --- p.83 / Chapter 4.2.3 --- Development of formulation --- p.84 / Chapter 4.2.3.1 --- Selection of the molar ratio of lipids --- p.84 / Chapter 4.2.3.2 --- Selection of the mass ratios of siRNA to lipids --- p.85 / Chapter 4.2.3.3 --- Selection of the molar ratios of L6-PEG2000-DSPE on L6-LNPs-siRNA --- p.85 / Chapter 4.2.4 --- Binding affinity with osteoblasts --- p.86 / Chapter 4.2.5 --- Preparation of L6-LNPs-siRNA --- p.86 / Chapter 4.2.5.1 --- Synthesis of L6-PEG2000-DSPE --- p.87 / Chapter 4.2.5.2 --- Preparation of LNPs-siRNA --- p.87 / Chapter 4.2.5.3 --- Post-insertion of aptamers on the surface of LNPs-siRNA --- p.88 / Chapter 4.2.6 --- Characterization of L6-LNPs-siRNA --- p.88 / Chapter 4.2.6.1 --- Particle size and Zeta Potential --- p.88 / Chapter 4.2.6.2 --- Encapsulation Efficiency (EE) --- p.88 / Chapter 4.2.6.3 --- Cryo-Transmission electron microscope --- p.89 / Chapter 4.3 --- Results --- p.90 / Chapter 4.3.1 --- Synthesis of DLin-KC2-DMA --- p.90 / Chapter 4.3.2 --- Formulation development --- p.93 / Chapter 4.3.3 --- Preparation of L6-LNPs --- p.95 / Chapter 4.3.4 --- Characterization of L6-LNPs-siRNA --- p.96 / Chapter 4.4 --- Discussion --- p.98 / Chapter 4.5 --- Conclusion --- p.101 / Chapter Chapter Five --- Evaluation of L6 aptamer functionalized lipid nanoparticles (L6-LNPs-siRNA) for osteoblast-specific delivery in vitro and in vivo --- p.102 / Chapter 5.1 --- Introduction --- p.103 / Chapter 5.2 --- Materials and Methods --- p.103 / Chapter 5.2.1 --- Materials --- p.103 / Chapter 5.2.2 --- Biological evaluation in vitro --- p.104 / Chapter 5.2.2.1 --- Cell culture --- p.104 / Chapter 5.2.2.2 --- Binding affinity with target/non-target cells --- p.105 / Chapter 5.2.2.3 --- Cellular uptake of siRNA in target/non-target cells --- p.105 / Chapter 5.2.2.4 --- Knockdown efficiency in vitro --- p.105 / Chapter 5.2.3 --- Cytotoxicity --- p.106 / Chapter 5.2.4 --- Mechanism of cellular uptake --- p.106 / Chapter 5.2.4.1 --- Spectral bio-imaging for endocytic pathways --- p.106 / Chapter 5.2.4.2 --- Chemical inhibition for endocytic pathways --- p.107 / Chapter 5.2.4.3 --- Determination of membrane ruffling --- p.107 / Chapter 5.2.5 --- Evaluation of specific delivery in vivo --- p.107 / Chapter 5.2.5.1 --- Experimental design --- p.107 / Chapter 5.2.5.2 --- Tissue distribution --- p.108 / Chapter 5.2.5.3 --- Localization of siRNA in liver --- p.108 / Chapter 5.2.5.4 --- Localization of siRNA with osteoblast/osteoclast --- p.108 / Chapter 5.2.6 --- Statistical analysis --- p.109 / Chapter 5.3 --- Results --- p.109 / Chapter 5.3.1 --- Binding selectivity of L6-LNPs-siRNA --- p.109 / Chapter 5.3.2 --- Selectivity of siRNA cellular uptake --- p.111 / Chapter 5.3.3 --- Knockdown efficiency in vitro --- p.112 / Chapter 5.3.4 --- Cytotoxicity --- p.113 / Chapter 5.3.5 --- Mechanism of cellular uptake --- p.113 / Chapter 5.3.6 --- Tissue distribution --- p.118 / Chapter 5.3.7 --- Localization of siRNA in liver --- p.119 / Chapter 5.3.8 --- Localization of siRNA with Osteoblasts/Osteoclasts --- p.120 / Chapter 5.4 --- Discussion --- p.123 / Chapter 5.5 --- Conclusion --- p.125 / Chapter Chapter Six --- Summary of the study and future research --- p.126 / Chapter 6.1 --- Summary of the study --- p.127 / Chapter 6.2 --- Future research --- p.128 / References --- p.130

Page generated in 0.0636 seconds