酒精性骨坏死发病机制的分子生物学实验研究

目的 :观察酒精诱导骨髓基质细胞 (Marrowstromalcells ,MSCs)的作用 ,揭示酒精性股骨头坏死新的发病机制。方法 :采用原代MSCs体外培养技术 ,取小鼠股骨骨髓细胞 ,分离获取MSCs。以酒精作为脂肪细胞分化诱导剂处理细胞 ,以苏丹Ⅲ染色 ,光镜下计数脂肪细胞。并测定细胞内甘油三酯、碱性磷酸酶活性和培养液中骨钙素含量。采用完整细胞斑点印迹分子杂交方法检测酒精 ( 0 .0 9mol/L)组和对照组细胞中 42 2(aP2 )mRNA和Ⅰ型胶原mRNA的表达。结果 :以递增浓度酒精 ( 0 .0 3、0 .0 9、0 .15mol/L)处理细胞 2 1d ,MSCs分化生成的脂肪细胞数量随酒精作用时间延长及浓度增大而增多 ,具有一定时间、剂量依赖性。与对照组相比 ,细胞内甘油三酯含量明显增高 ,碱性磷酸酶活性随酒精浓度增大而降低 ,培养液中骨钙素含量显著减少。实验组中 42 2 (aP2 )mRNA表达含量 72 0 7.8± 3 3 1.3 ,显著高于对照组 65 2 .2± 62 .6,P <0 .0 0 1。其Ⅰ型胶原mRNA表达含量 3 5 67.3± 3 0 0 .9,明显低于对照组 7487.0± 488.4,P <0 .0 0 1。结论 :酒精能够从基因调控水平诱导MSCs大量分化为脂肪细胞 ,成骨分化减少 ,这可能是长期酗酒后股骨头骨髓内脂肪组织增多 ,骨内压增加 ,血流灌注减少 ,导致缺血 ,同时骨修复能力不足 ,从而发生

An experimental study in molecular biology for alcohol-induced osteonecrosis

Objective: Observe the effect of alcohol on the differentiation of marrow stromal cells(MSCs)into adipocytes to elucidate the pathogenesis of the alcohol-induced osteonecrosis of the femoral head.Methods: The primary MSCs from the femora of the mice were procured and cultured. The samples were isolated after adherent growth culture in vitro, and were treated with ethanol that acted as adipocyte differentiation inducer. The cells in culture were stained with Sudan Ⅲ.The number of adipocytes was counted under light microscope. The level of intracellular triglyceride,alkaline phosphatase(ALP)activity in the cells, and osteocalcin in culture media were determined. The expression level of 422 (aP2) and type-Ⅰ collagen mRNA's in the cells treated with 0.09 mol/L ethanol and without with ethanol was investigated by means of intact cell RNA dot blot hybridization. Results: The cells in culture were treated with increasing concentration, (0.03?0.09?0.15 mol/L), of ethanol for 21 days. The number of adipocytes increased with longer durations of exposure to ethanol and with higher concentrations of ethanol, which showed the time-and dose-dependent relation. There were lowest adipocytes in the control group. The level of triglyceride in the cells treated with 0.15mol/L of ethanol markedly increased, which was 3.9-fold higher than that in the control cells. ALP activity in the cells decreased with higher concentration of ethanol. The level of osteocalcin in culture media of the cells treated with 0.15mol/L of ethanol. The 422(aP2)mRNA contents in the experimental group, 7207.8±331.3, were markedly higher than that in the control group, 652.2±62.6. By contrast, the type-I collagen mRNA contents in the experimental group, 3567.3±300.9, were significantly lower than that in the control group, 7487.0±488.4. Conclusion: Alcohol could induce the differentiation of MSCs into adipocytes and inhibit osteogenic differentiation by regulating gene expression. This might lead to an increase in volume of fat marrow in the femoral head following long-term alcohol abuse, with a concomitant increase in intraosseous pressure, which could decrease in vascular perfusion, eventually resulting in ischemia in the marrow, and without sufficient repair of the necrotic bone. The final result is osteonecrosis of the femoral head. It may be a new pathogenesis of the alcohol-induced necrosis of the femoral head.