Includes bibliographical references (leaves 165-225) / xx, 298 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, School of Molecular and Biological Sciences, Discipline of Physiology, 2004

引言: 青少年突發性脊柱側彎(Adolescent Idiopathic Scoliosis, AIS)是一種複雜的脊柱三維畸形，而目前病因未明。這病主要出現於11-14歲的青少年女性，罹患率約為四個百分比。未經治療的側彎可能逐漸加重並導致明顯的外觀畸形，嚴重者可導致心肺功能異常等其他併發症。目前對AIS的治療方法包括觀察，支具和手術內固定矯形。然而因為這些治療並非針對病因而仍然存在一定缺陷，如支具治療失敗和手術的併發症等。因此明確AIS的發病機制仍然是制定有效治療的關鍵。 / 根據過往研究報告，AIS患者有低體重、身形高大、臂展增加、低體重指數，月經初潮推遲與低骨量，這顯示AIS患者或有全身性生長異常。因為瘦素的重要生理功能如影響骨骼發育、開始青春期，能量消耗和身體成分，所以瘦素被假設為AIS的病因之一。之前的研究報告指出，AIS患者對比相同年齡和性別的人擁有較低水平的循環瘦素。然而，瘦素信號的強度不僅由瘦素水平所決定的，它也有可能受到可溶性瘦素受體(sOB-R)所影響。sOB-R是循環系統中瘦素的結合蛋白，可以調節血清瘦素水平，並影響對目標組織的生物活性游離瘦素之生物利用度。游離瘦素指數(FLI)的開發是為了幫助數據的詮釋，並提供游離瘦素水平的估計。 / 瘦素對骨代謝的影響是間接地由中央神經系統和直接地由周緣組織來施加的。瘦素被發現通過下丘腦神經系統對骨形成有分解效果。而瘦素的周緣作用被證實對骨形成有直接的合成作用，例如促進成骨細胞和軟骨細胞的增殖，刺激成骨細胞的分化和礦化，及抑制破骨細胞的生長和活性。根據先前對AIS患者的骨髓間充質幹細胞(MSCs)之研究，AIS患者對瘦素有較低的反應和在成脂和成骨幹細胞有較低的瘦素受體。這提供了在AIS患者的骨細胞和瘦素相關異常的初步證據。 / 當前研究的目的是: / 1）通過測量在AIS患者和正常對照組的血清總瘦素，sOB-R和FLI的水平來調查AIS患者是否有任何異常的游離瘦素生物利用度，及是否和異常的人體測量參數有關聯。 / 2）通過研究AIS患者和對照組之身體成分及與血清總瘦素，sOB-R和FLI的相互關係來調查游離瘦素生物利用度與AIS患者與對照組之關聯。 / 3）通過利用高分辨率周邊定量電腦斷層來研究與AIS患者與對照組的骨質量差異及與血清總瘦素，sOB-R和FLI的相互關系來調查游離瘦素生物利用度與AIS患者與對照組之關聯。 / 4）通過研究AIS患者與對照組對瘦素與成骨細胞增殖，分化和礦化的影響, 與成骨細胞上的瘦素受體來調查AIS患者是否對瘦素有異常反應和瘦素受體的異常表現。 / 方法: 1）游離瘦素的生物利用度和人體測量參數的研究包括了207名AIS女孩和155名年齡和性別匹配的正常對照組。因超重/肥胖者有比較高的血清瘦素水平，為避免來自於超重/肥胖者的影響，身體質量指數(BMI)大於23.0的實驗對象被排除。評估包括人體和性成熟的測量：如體重、身高、臂展、坐高，體重指數和坦納階段。血清總瘦素和sOB-R水平測定採用ELISA法，而游離瘦素指數(FLI)的計算為總血清瘦素/sOB-R的比值。 / 2）游離瘦素的生物利用度和身體成分的研究包括了148名AIS女孩和116名正常對照。身體成分的評估採用了生物電阻抗分析(BIA)。 / 3）游離瘦素的生物利用度和骨質量參數的研究包括了207名AIS女孩和155名正常對照。非優勢的遠端橈骨質量以高分辨率周邊定量電腦斷層(HR-pQCT)進行評估。體內骨骼強度以有限元分析(FEA)進行了評估。 / 4）對成骨細胞功能反應和瘦素受體表達的研究包括了12名接受矯正脊椎手術的AIS女孩和6名接受與側彎無關之手術的對照組。成骨細胞是從手術中拿取的骨活檢中分離，並進行培養和評估瘦素對細胞增殖，分化和礦化的影響(0，10，100，1000毫微克/毫升)，而分別採用MTT法，鹼性磷酸酶活性測定, 骨鈣素酶聯免疫吸附和von Kossa染色。瘦素受體（LEP-R）在基礎和成骨條件下的蛋白表達以Western blot檢測來進行AIS和對照組之間的比較。 / 結果: 1）在游離瘦素的生物利用度和人體測量參數的研究中，AIS女孩在調整了年齡和體重後有較高的sOB-R和較低的FLI，也和較低的體重和較低的BMI有關聯。AIS女孩還有在血清總瘦素，FLI和人體測量參數之間顯著較弱的相關性。 / 2）在游離瘦素的生物利用度和身體成分的研究中，AIS女孩有較低的骨骼肌質量，較低的身體脂肪和較低的身體脂肪百分比，以及在血清總瘦素，FLI和所有身體成分參數之間較弱的相關性。 / 3）在游離瘦素的生物利用度和骨質量的研究中，AIS女孩有較低的皮質體積骨密度，較低的骨小梁數量，較高的骨小梁分離度以及更高的結構模型指數（SMI）。在相關性分析中, AIS女孩在血清總瘦素和FLI與骨小梁參數的相關性有特殊的相關模式，這相關性沒有在正常對照組中出現。 / 4）在對成骨細胞的功能反應和瘦素受體的表達之研究中，對照組隨瘦素濃度升高而有增加的代謝信號，然而，AIS組沒有對瘦素出現增殖反應。在第六天和第十四天的分化實驗中，對照組的鹼性磷酸酶活性隨著瘦素濃度升高而表現出清晰而強烈的趨勢，而AIS組的鹼性磷酸酶活性沒有出現趨勢也沒有顯著差異。在骨鈣素酶聯免疫吸附實驗中, 對照組隨著瘦素濃度升高而表現出清晰而強烈的趨勢, 而AIS組沒有出現顯著差異。在礦化實驗中，對照組隨瘦素濃度升高而呈輕度上升的礦化趨勢, AIS組再次地沒有出現趨勢和差異。在基礎和成骨條件下, 瘦素受體的表達並沒有出現顯著差異。 / 討論：這是對AIS中瘦素的生物利用度與身體成分和骨質量之關係的第一個研究。本研究的結果表示，AIS女孩可能存在異常的游離瘦素生物利用度，並可能導致異常表型，例如較低的BMI，異常的身體成分和較差的骨質量。低骨量在AIS中的重要性和骨小梁參數與血清總瘦素和FLI的不正常的關係，使我們進一步研究瘦素對AIS的成骨細胞的影響。當與對照組相比,來自AIS女孩的成骨細胞對瘦素有非常低的反應。這異常反應可能是由於瘦素信號轉導途徑的功能障礙，其中可能包括異常的瘦素受體和下游信號分子。這是一個重要的發現，並可能有助於解釋與AIS相關的低骨量和較差的骨質量。這項研究提供了AIS發病機理的新見解和新的研究方向。未來的研究不妨包括瘦素信號轉導途徑中可能受到AIS影響的下游信號分子和動物模型來證實瘦素和瘦素生物利用度與AIS之間的因果關係。總而言之，這一系列的研究結果表示了瘦素和瘦素生物利用度在骨骼發育、體格，骨骼質量和AIS的發病機制中的重要性。 / Introduction: Adolescent idiopathic scoliosis (AIS) is a complex three-dimensional structural deformity of the spine and its etiology remains unknown. It mainly occurs in girls between 11 to 14 years old with a prevalence rate of 4%. This common spinal deformity can be associated with significant cosmetic, psychological, and clinical morbidities in severe cases. Current treatments for AIS are unsatisfactory, mainly because they are merely treating the phenotype of the spinal deformity but not the underlying etiology. Therefore, it is crucial to understand the etiopathogenesis of AIS so that effective therapeutic and preventive measures can be devised. / Previous studies have reported the association between AIS and low body weight, tall stature, increased arm span, low body mass index, delayed onset of menarche and low bone mass, which indicated abnormal systemic growth in patients with AIS. Leptin has been postulated as one of the etiologic factors of AIS because of its important physiological functions in neuro-osseous development affecting skeletal growth, the onset of puberty, energy expenditure and body composition. A previous study had reported lower circulating leptin in AIS girls than age- and sex-matched controls. However, the strength of leptin signal is not only determined by the leptin level, it could also be affected by soluble leptin receptor (sOB-R), its binding protein in circulation. sOB-R could modulate the serum leptin level, and affect the bioavailability of free leptin, the biologically active form, to its target tissues. Free leptin index (FLI) was developed to aid interpretation of data and provide an estimation of free leptin level. / The effects of leptin on bone metabolism are exerted indirectly through the central nervous system and directly on the peripheral tissues. Leptin was shown to have a catabolic effect on bone formation through the hypothalamus and the sympathetic nervous system (SNS). While the peripheral effects of leptin was shown to be anabolic to bone formation, promoting the proliferation of osteoblasts and chondrocytes, stimulating osteoblastic differentiation, mineralization, and inhibiting osteoclastogenesis and osteoclast activity. With regards to AIS, a previous study on bone marrow derived mesenchymal stem cells (MSCs) showed lower responses to leptin, and lower leptin receptor expressions in adipogenic and osteogenic MSCs. This study provided initial evidence that leptin related pathways might be abnormal in the bone cells of AIS patients. / This study aimed to: / 1) Investigate if there is any abnormality in free leptin bioavailability by measuring the serum total leptin, sOB-R levels, and FLI in AIS and control subjects, and if it is associated with abnormal anthropometric parameters in AIS. / 2) Investigate if free leptin bioavailability in AIS is associated with abnormal body composition by studying the difference in body composition and its correlations with serum total leptin, sOB-R, and FLI between AIS and control subjects. / 3) Investigate if free leptin bioavailability in AIS is associated with abnormal bone quality by studying the difference in bone quality as measured by high resolution pQCT and its correlations with serum total leptin, sOB-R, and FLI between AIS and control subjects. / 4) Investigate if there are abnormal functional responses to leptin and abnormal expression of leptin receptor in AIS by determining the effects of leptin on proliferation, differentiation, and mineralization of osteoblasts; and the expression levels of leptin receptor in osteoblasts isolated from intra-operative bone biopsies of AIS and control subjects. / Methods: 1) The study on free leptin bioavailability and anthropometric parameters included 207 AIS girls and 155 age- and gender-matched normal controls. Subjects with body mass index (BMI)>23.0 were excluded to avoid bias from the relatively high serum leptin level in overweight / obese subjects. Assessments included anthropometric and sexual maturity measurements: body weight, height, arm span, sitting height, BMI, and Tanner stages. Serum total leptin and sOB-R levels were measured with ELISA, and free leptin index (FLI) was calculated as the ratio of serum total leptin / sOB-R. / 2) The study on free leptin bioavailability and body composition included 148 AIS girls and 116 normal controls, while anthropometric and sexual maturity parameters included 207 AIS girls and 155 normal controls. Body composition was assessed with bioelectrical impedance analysis (BIA). / 3) The study on free leptin bioavailability and bone quality parameters included 207 AIS girls and 155 normal controls. Bone quality on the non-dominant distal radius was assessed with high resolution pQCT (HR-pQCT). In vivo bone strength was assessed with finite element analysis (FEA). / 4) The study on functional responses and leptin receptor expression in osteoblasts included 12 AIS girls undergoing corrective spinal surgery and 6 control subjects undergoing unrelated surgery. Primary osteoblasts were isolated from intra-operative bone biopsies, and were cultured and assessed for the effects of leptin (0, 10, 100, 1000 ng/mL) on cell proliferation, differentiation, and mineralization by MTT assay, alkaline phosphatase activity assay and osteocalcin ELISA, and von Kossa staining respectively. Protein expressions of leptin receptor (LEP-R) under basal and osteogenic conditions were compared between AIS and controls by Western blot. / Results: 1) In the study on free leptin bioavailability and anthropometric parameters, AIS girls had higher sOB-R and lower FLI after adjusting for age and body weight, and these were associated with lower body weight and lower BMI. Significant correlation between serum total leptin and sOB-R was found in controls, but no correlation was observed in AIS girls. AIS girls also showed markedly weaker correlations between serum total leptin, FLI, and anthropometric parameters. / 2) In the study on free leptin bioavailability and body composition, AIS girls had lower skeletal muscle mass, lower body fat, and percentage body fat, as well as weaker correlations between serum total leptin, FLI, and all body composition parameters. / 3) In the study on free leptin bioavailability and bone quality, AIS girls had lower cortical volumetric bone mineral density (vBMD), lower trabecular number, higher trabecular separation, and higher structural model index (SMI) value. In correlation analysis with serum total leptin and FLI, AIS girls had distinctive correlation pattern with trabecular bone parameters that was not observed in the normal controls. / 4) In the study on functional responses and leptin receptor expression in osteoblasts, control group showed increasing MTT signals to leptin in a dose dependent manner, while AIS group showed no proliferative response to leptin. For differentiation, control group showed strong and significant trend in ALP activity to increasing leptin concentrations in both day 6 and 14, but this trend was not observed in the AIS group. Osteoblasts from the control group secreted osteocalcin in an increasing dose dependent manner to leptin, but AIS group showed weak responses to leptin. For mineralization, the control group showed a mild increasing trend to increasing leptin concentrations, and again no trend was observed in the AIS group. No significant differences in the expression of leptin receptor under basal and osteogenic conditions were found between AIS and control group. / Discussion: This is the first study on the relationship of leptin bioavailability with body composition and bone quality in AIS. The results in this study suggested that abnormal free leptin bioavailability might exist in AIS girls and could lead to abnormal phenotypes such as lower BMI, abnormal body composition, and deranged bone quality that often manifested in AIS simultaneously. The importance of low bone mass in AIS and the presence of correlations between trabecular bone parameters and serum total leptin and FLI which were not observed in controls, have led us to further investigate the effects of leptin on primary osteoblasts in AIS. The osteoblasts isolated from AIS girls had no or very low response to leptin when compared with controls, which was shown to be independent of the difference in leptin receptor expression levels. This lack of response might be due to dysfunction of leptin signaling pathway, which might include structural or binding abnormalities in the leptin receptor or downstream signal molecules. This is an important finding and might serve to explain the low bone mass and deranged bone quality that is associated with AIS. This study provided new insights and new research directions on the etiopathogenesis of AIS. Future research could include studies on the downstream signal molecules along the leptin pathways that might be affected in AIS, and animal models to confirm the causal relationship of leptin and leptin bioavailability to AIS. In summary, the findings in this series of studies demonstrated the importance of leptin and leptin bioavailability in skeletal growth, body build, bone quality, and the etiopathogenesis of AIS. / 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. / 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. / 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. / Tam, Man Shan Elisa. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 259-301). / Abstracts also in Chinese; appendixes includes Chinese.

Bibliography: leaves [124-153]

Bibliography: leaves [124-153] / x, 123 leaves : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Obstetrics and Gynaecology, 2001