论文部分内容阅读
背景:动物实验证明乙醇可引起股骨头内脂肪积聚,导致骨坏死。分子生物学实验证明乙醇能够诱导骨髓基质细胞成脂分化。若某种药物能够对抗乙醇诱导骨髓基质细胞的成脂分化作用,则可能防治酒精性骨坏死。目的:从细胞生物学角度观察葛根素对抗酒精诱导骨髓基质细胞成脂分化作用,探讨葛根素对酒精性骨坏死的防治作用。设计:随机对照实验。单位:郑州大学第一附属医院骨科和郑州大学基础医学院生物化学与分子生物学教研室。材料:实验于2000-04/2002-12在郑州大学基础医学院生物化学与分子生物学教研室实验室完成。选用清洁级健康昆明小鼠50只,6～8周龄,雌雄不拘。获取双侧股骨骨髓细胞,细胞接种密度1.5×106/cm2,置入6孔及24孔培养板内,随机分组,每孔作为1个样本,每组为24个样本。方法:分离培养小鼠骨髓基质细胞,随机分为3组:模型组(给予乙醇0.09mol/L处理细胞),实验组(乙醇0.09mol/L和葛根素终浓度为0.01g/L),对照组(无乙醇和葛根素)。苏丹Ⅲ染色,光镜下脂肪细胞计数;测定细胞内三酰甘油含量、碱性磷酸酶活性和细胞培养液中的骨钙素含量。主要观察指标:①3组小鼠骨髓基质细胞分化为脂肪细胞结果。②3组小鼠骨髓基质细胞内三酰甘油含量。③3组小鼠骨髓基质细胞内碱性磷酸酶活性值。④3组细胞培养液中骨钙素含量。结果:①处理细胞并培养21d后,实验组、对照组中脂肪细胞均比模型组显著为少。模型组中脂肪细胞数是实验组的8.9倍,是对照组的15.6倍[(319.17±19.92),(35.92±23.77)(20.42±12.15)个/cm2,P<0.001]。②处理细胞并培养21d后,实验组和对照组中三酰甘油含量均明显低于模型组[(4.15±1.92)和(3.42±1.60),(11.55±4.42)μg/孔,P<0.001]。③处理细胞并培养12d后,实验组和对照组细胞内碱性磷酸酶活性均明显高于模型组[(9.51±2.96),(11.18±3.11),(4.84±2.23)μkat/g,P<0.001]。④处理细胞并培养14d后,实验组和对照组培养液中的骨钙素含量分别是模型组的2.2倍和2.7倍,与模型组间的差异均非常显著[(11.11±4.57),(13.43±5.29),(4.95±2.31)μg/g,P<0.001]。结论:葛根素能够抑制酒精诱导骨髓基质细胞分化为脂肪细胞,维持其成骨分化,可能预防骨坏死。
BACKGROUND: Animal experiments have shown that ethanol can cause fat accumulation in the femoral head, leading to osteonecrosis. Molecular biology experiments show that ethanol can induce adipogenic differentiation of bone marrow stromal cells. If a drug can fight ethanol-induced adipogenic differentiation of bone marrow stromal cells, it may prevent alcoholic bone necrosis. Objective: To observe the effects of puerarin on the adipogenic differentiation of bone marrow stromal cells induced by alcohol from the perspective of cell biology, and to explore the preventive and therapeutic effects of puerarin on alcoholic osteonecrosis. Design: Randomized controlled trials. Department of Orthopedics, First Affiliated Hospital of Zhengzhou University and Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Zhengzhou University. MATERIALS: The experiment was performed at the Laboratory of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Zhengzhou University from April 2000 to January 2002. Fifty healthy clean Kunming mice were selected, 6-8 weeks old, male or female. Bilateral femoral bone marrow cells were obtained. The cell seeding density was 1.5×10 6 /cm 2 , and the cells were placed in 6-well and 24-well culture plates and randomly grouped. Each well was used as a sample, and each group had 24 samples. METHODS: Marrow stromal cells were isolated and cultured and randomly divided into 3 groups: model group (treated with 0.09 mol/L ethanol), and experimental group (ethanol 0.09 mol/L and puerarin 0.01 g/L). Group (without ethanol and puerarin). Sulfur III staining, light microscopy fat cell count; determination of intracellular triglyceride content, alkaline phosphatase activity and osteocalcin content in cell culture fluid. MAIN OUTCOME MEASURES: The results of differentiation of 13 bone marrow stromal cells into adipocytes. The content of triacylglycerol in bone marrow stromal cells of 23 groups of mice. Alkaline phosphatase activity in 33 bone marrow stromal cells. 43 groups of cell culture fluid osteocalcin content. RESULTS: After treatment of cells for 21 days, the adipocytes in the experimental group and the control group were significantly less than those in the model group. The number of adipocytes in the model group was 8.9 times that of the experimental group and 15.6 times that of the control group [(319.17±19.92), (35.92±23.77)(20.42±12.15)/cm2, P<0.001]. After treatment with cells for 21 days, the content of triacylglycerol in experimental group and control group was significantly lower than that in model group [(4.15±1.92) and (3.42±1.60), (11.55±4.42)μg/well, P<0.001] . 3 After treatment with cells for 12 days, the alkaline phosphatase activity in the experimental and control groups was significantly higher than that in the model group [(9.51±2.96), (11.18±3.11), (4.84±2.23) μkat/g, P< 0.001]. 4 After treatment of cells for 14 days, the osteocalcin content in the experimental and control groups was 2.2 and 2.7 times that of the model group, respectively, and the difference between the model group and the model group was very significant [(11.11±4.57), (13.43 ±5.29), (4.95±2.31) μg/g, P<0.001). CONCLUSION: Puerarin can inhibit the differentiation of bone marrow stromal cells induced by alcohol into adipocytes and maintain osteogenic differentiation, which may prevent osteonecrosis.