【作者】 王清平；

【导师】 罗湘杭；

【作者基本信息】 中南大学 ， 内科学， 2010， 博士

【摘要】 第一部分中国女性血清脂联素、瘦素水平与骨密度及骨转换生化指标的关系目的：作为脂肪组织分泌的主要循环肽,脂联素和瘦素对骨代谢有着重要的影响,但它们与女性骨密度(BMD)的关系仍不太明确。本部分研究就女性血清脂联素、瘦素水平与BMD及骨转换生化指标的关系进行探讨。方法：用ELISA测定265例绝经前、336例绝经后中国长沙地区健康成年女性血清脂联素、瘦素、血清骨特异性碱性磷酸酶(BAP)和血清Ⅰ型胶原交联氨基末端肽(NTX)等生化指标；用双能X射线吸收测定法(DXA)测定全身、腰椎L2-L4、总髋骨、总前臂的BMD以及脂重和瘦重；指标间的相关性采用Pearson’s相关分析和偏相关分析,并用多元线性回归模型分析哪些指标可作为BMD的独立预测因子。结果：多元线性回归分析表明,在绝经后的中国女性,绝经年限、瘦重、雌二醇(E2)和脂联素均为BMD的独立预测因子(P<0.05或P<0.01),而脂重和瘦素则均不是BMD的独立预测因子；但在绝经前的中国女性,脂联素与脂重和瘦素一样,也不是BMD的独立预测因子。Pearson’s相关分析表明,在绝经后中国女性,脂联素与血清BAP、血清NTX及NTX/BAP比值呈显著正相关(均为P<0.05),且这种相关性经校正年龄和脂重后依然显著(均为P<0.05)。结论：脂联素是绝经后中国女性BMD的独立预测因子,并与骨转换生化指标呈正相关；而在绝经前中国女性,这种关系并不存在。这提示脂联素对骨量产生负性作用,但脂联素的这一作用受绝经状态影响。第二部分E2对脂联素促人成骨细胞分化作用的影响目的：我们的研究表明,脂联素对BMD的影响与女性绝经状态有关。这提示血循环中的雌激素水平影响了脂联素对骨代谢的作用。本部分研究对雌激素(雌二醇,E2)在脂联素促人成骨细胞(HOB)分化中的影响进行探讨。方法：HOB用α-最小必需培养基(a-MEM)、含10μg/mL脂联素的a-MEM或含10μg/mL脂联素与10-10-10-8M E2的a-MEM分别培养48小时,观察E2不同剂量对脂联素诱导成骨细胞分化的影响；用含10μg/mL脂联素的a-MEM或含10μg/mL脂联素与10-8M E2的a-MEM分别培养12～48小时以观察E2不同作用时间对脂联素诱导成骨细胞分化的影响；另外,在含10μg/mL脂联素与10-8M E2的培养液中分别添加10ng/mLp38激酶激动剂或10-8M雌激素受体拮抗剂后,将细胞培养48小时,以观察E2对脂联素诱导成骨细胞分化的影响是否被p38激酶激动剂和雌激素受体拮抗剂阻断；分别用α-磷酸奈酚法检测成骨细胞ALP、用放射免疫测定法(RIA)测定骨钙素(OC)、用Western Blot检测p38和p-p38。结果：脂联素促进成骨细胞ALP活性、OC分泌和p38激酶磷酸化,E2则抑制成骨细胞ALP活性、OC分泌(均为P<0.01)和p38激酶磷酸化；E2与脂联素联合干预时,ALP活性、OC分泌均随E2浓度升高而降低(均为P<0.01),p38激酶磷酸化水平也随E2浓度升高而下降；而且,联合干预的时间越长,成骨细胞ALP活性与分OC泌量降低得越明显(P<0.05或P<0.01)。在添加p38激酶激动剂或雌激素受体拮抗剂的联合干预组,成骨细胞ALP活性和OC分泌量均较未添加p38激酶激动剂(P<0.05)或雌激素受体拮抗剂组(P<0.01)增加。结论：E2通过抑制MAPK信号通路p38激酶的磷酸化抑制了脂联素的促成骨细胞分化作用。第三部分E2对脂联素调控人成骨细胞OPG和RANKL表达的影响目的：探讨E2对脂联素调控HOB护骨素(OPG)与核因子κB受体活化因子配体(RANKL)表达的影响。方法：HOB用α-MEM、含10μg/mL脂联素或含10μg/mL脂联素与10-10～10-8M E2的a-MEM分别培养48小时,观察E2不同的剂量对脂联素调控成骨细胞OPG和RANKL表达的影响；用含10μg/mL脂联素与10-8M E2的a-MEM分别培养12～48小时以观察E2不同的作用时间对脂联素调控成骨细胞OPG和RANKL表达的影响；另外,在含1 0μg/mL脂联素与10-8M E2的培养液中分别添加10ng/mL p38激酶激动剂或10-8M雌激素受体拮抗剂后,将细胞培养48小时,以观察E2对脂联素调控成骨细胞OPG/RANKL的影响是否被p38激酶激动剂和雌激素受体拮抗剂阻抑；用实时定量PCR (RT-PCR)和酶联免疫分析(ELISA)检测成骨细胞OPG和RANKL mRNA及其蛋白表达。结果：脂联素抑制成骨细胞OPGmRNA表达和蛋白分泌而促进成骨细胞RANKL mRNA表达和蛋白分泌；相反,E2则促进成骨细胞OPGmRNA表达和蛋白分泌而抑制RANKL mRNA表达和蛋白分泌(均为P<0.01)；脂联素与E2共同干预时,成骨细胞OPGmRNA表达和蛋白分泌随E2浓度升高而升高,而RANKLmRNA表达和蛋白分泌则随E2浓度升高而降低(P<0.05或P＜0.01)；而且,联合干预的时间越长,E2对成骨细胞OPG mRNA表达和蛋白分泌的促进以及对RANKL mRNA表达和蛋白分泌的抑制作用越明显；与未添加p38激酶激动剂或雌激素受体拮抗剂的培养细胞相比,添加p38激酶激动剂或雌激素受体拮抗剂的培养细胞OPG mRNA表达和蛋白分泌均下降,而RANKL mRNA表达和蛋白分泌均升高(均为P<0.01)。结论：E2通过MAPK/p38途径阻滞脂联素对成骨细胞OPG/ RANKL表达的调控,从而使成骨细胞OPG表达升高、RANKL表达下降。这可能是绝经后女性BMD与脂联素呈负相关,而绝经前女性BMD与脂联素不相关的机制之一。更多还原

【Abstract】 Part one Relationships Between Serum Adiponectin, Leptin Levels and Bone Mineral Density, and Bone Biochemical Barkers in Chinese WomenObjectivesAdiponectin and leptin, as the main circulating peptides secreted by adipose tissue, are potential contributors to bone metabolism. However, their associations with bone mineral density (BMD) are unclear. The present study was to investigate whether these serum adipocytokines’ levels are associated with BMD and bone turnover markers.Materials and MethodsIn this study,336 postmenopausal and 265 premenopausal Chinese women were included. Serum concentrations of adiponectin, leptin, bone-specific alkaline phosphatase (BAP) and bone cross-linked N-telopeptides of type I collagen (NTX) were measured with ELISA. Serum levels of 25-hydroxyvitamin D, parathyroid hormone (PTH), insulin, estradiol (E2), total testerone were detected by radioimmunoassay (RIA). BMD values were measured using DXA at the total body, lumbar spine, total hip, and total forearm. Body compositions including lean tissue mass and body fat mass were also measured by DXA. The correlations between adiponectin, leptin and BMD, were made using Pearson’s correlation analysis and Partial correlation analysis. Multivariate linear stepwise analysis was performed to determine how much of the variance in BMD at various skeletal regions could be explained by age, BMI, lean mass, smoking habits, calcium intake, physical activity, adiponectin, etc.ResultsIn postmenopausal Chinese women, the multiple linear stepwise regression analysis showed that year since menopause, lean mass, E2 and adiponectin, but not fat mass and leptin, were independent predictors of BMD (P＜0.05 or P＜0.01). However, in premenopausal Chinese women, adiponectin was not the predictor of BMD. The significant positive correlations between adiponectin and BAP, NTX and NTX/BAP were found only in postmenopausal women even adjusted by both age and fat mass (all P<0.05).ConclusionsAdiponectin was an independent predictor of BMD, and positively correlated with bone turnover biochemical markers in postmenopausal women, but not in premenopausal women. It suggested that adiponectin might exert a negative effect on bone mass by promoting excessive bone resorption associated with bone loss. However, these effects may be mediated by menopausal status. Chapter two Effects of E2 on Adiponectin-Regulated Human Osteoblasts DifferentiationObjectivesOur study showed that adiponectin could exert a negative effect on bone mass in postmenopausal women, but not in premenopausal women. This suggested the action of adiponectin on BMD might be influenced by the levels of circulating estrogen in women. The present study was undertaken to investigate the effect of estrogen (Estradiol, E2) on adiponectin-regulated human osteoblast (HOB) differentiation.MethodsHOBs were treated with a-minimum essential medium (a-MEM) or a-MEM containing 10μg/mL adiponectin or 10μg/mL adiponectin together with 10-10～10-8M E2 for 48 hours. HOBs were also exposed toα-MEM containing 10μg/mL adiponectin alone or together with 10-8 M E2 for 12～48 hours. In addition, HOBs were treated with a-MEM containing 10μg/mL adiponectin and10-8M E2, adding p38 agonist, estrogen receptor antagonist or not respectively, for 48 hours. HOBs differentiation were assessed by measuring ALP activity and osteocalcin (OC) secretion. ALP activity was determined by using p-nitrophenyl phosphate as substrate, OC secretion was measured by using a specific radioimmunoassay (RIA) kit, p38 and p-p38 were analysed by Western Blot.ResultsAdiponectin stimulated ALP activity, OC secretion and phosphorylation of p38 kinase, whereas E2 inhibited ALP activity, OC secretion (all P<0.01) and phosphorylation of p38 kinase significantly. In cells treated with adiponectin together with E2, E2 significantly decreased adiponectin-induced ALP activity, OC secretion (P<0.05 or P<0.01) and p38 activation in a dose-or time-dependent manner. However, the ALP activity and OC secretion depressed by E2 were increased significantly by addition of p38 agonist (P<0.05) or estrogen receptor antagonist (P<0.01).ConclusionThese data indicated that E2 supressed adiponectin-induced HOB differentiation via inhibiting the phosphorylation of MAPK p38 kinase. Chapter three The Effects of E2 on Adiponectin-Modulated the Expression of OPG and RANKL in Human OsteoblastsObjectivesAs it was showed, that E2 supressed adiponectin to promote HOB differentiation by inhibiting the phosphorylation of MAPK p38 kinase. This study was further undertaken to investigate the effects of E2 on adiponectin-regulated osteoprotegerin (OPG) and receptor activator of nuclear factor-KB ligand (RANKL) expression in HOB.Materials and MethodsHOBs were treated with a-MEM or a-MEM containing 10μg/mL adiponectin alone or together with 10-10～10-8 M E2 for 48 hours. Those were also exposed to a-MEM containing 10μg/mL adiponectin alone or together with 10-8M E2 for 12～48 hours. In addition, HOBs were treated with a-MEM containing 10μg/mL adiponectin and 10-8 M E2, adding p38 agonist, estrogen receptor antagonist or not respectively, for 48 hours. Real-time quantitative PCR (RT-PCR) was used to detect OPG and RANKL mRNA expression, and ELISA was used to detect their protein expression in cultured HOB respectively.ResultsAdiponectin inhibited OPG mRNA expression and protein secretion but stimulated RANKL mRNA expression and protein secretion (all P<0.01). Whereas, E2 stimulated OPG mRNA expression and protein secretion but inhibited RANKL mRNA expression and protein secretion (all P<0.01). In cells treated with adiponectin and E2 together, E2 enhanced adiponectin-regulated OPG mRNA and protein expression while attenuated RANKL mRNA and protein expression in a dose-and time-dependent manner (P<0.05 or P＜0.01),but these effects of E2 were reversed by addition of p38 agonist or estrogen receptor antagonist (all P＜0.01).ConclusionsE2 supressed the action of adiponectin on modulation of OPG and RANKL expression through MAPK/p38 pathway. This might be a mechanism that BMD was negatively correlated with adiponectin levels in postmenopausal women but not in premenopausal women.更多还原