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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,其作用机制不详。NIH3T3成纤维细胞是一种多潜能干细胞,尚未见关于乙醇对NIH3T3成纤维细胞作用的有关报道。目的:观察乙醇对NIH3T3成纤维细胞分化为脂肪细胞的作用,及其对成脂转录因子过氧化物酶体增殖子活化受体-γ表达的影响。设计:单一样本观察。单位:郑州大学基础医学院生物化学教研室和郑州大学第一附属医院骨科。材料:NIH-3T3成纤维细胞由美国弗吉尼亚大学医院骨科实验室提供。方法:实验于2002-04/2003-06在郑州大学基础医学院生物化学教研室实验室完成。取NIH-3T3成纤维细胞随机分为6组,每天分别加入体积分数为0.998乙醇,剂量为0(对照组),0.03,0.06,0.09,0.15,0.21mol/L,培养至14d,苏丹Ⅳ染色,采用电子计算机图像分析软件ImageProPlus4.1,确定脂肪细胞百分比。采用反转录聚合酶链反应技术,检测细胞过氧化物酶体增殖子活化受体-γmRNA的表达。主要观察指标:①NIH3T3细胞分化为脂肪细胞结果。②乙醇组和对照组细胞基因表达结果。结果:以递增浓度(0.03,0.06,0.09,0.15,0.21mol/L)乙醇处理细胞14d。①各乙醇组中脂肪细胞百分比分别为11.9%,23.8%,28.2%,31.1%,42.6%,分别是对照组(9.6%)的1.2,2.5,2.9,3.2,4.4倍。0.03mol/L乙醇组与对照组的差别无统计学意义(P>0.05),其余4组与对照组及0.03mol/L乙醇组的差别有高度显著性(P<0.001)。②各乙醇组细胞中过氧化物酶体增殖子活化受体-γmRNA表达值与18S基因表达值的比值分别为0.218,0.411,0.486,0.473,0.453,分别是对照组(0.197)的1.1,2.1,2.5,2.4,2.3倍。0.03mol/L乙醇组与对照组的差别无统计学意义(P>0.05)。其余4组与对照组及0.03mol/L乙醇组的差别有高度显著性(P<0.001)。结论:乙醇能够直接诱导NIH3T3成纤维细胞大量分化为脂肪细胞,这可能是乙醇性骨坏死时骨髓内脂肪增多的原因之一。
Background: Animal experiments show that ethanol can cause fat accumulation in the femoral head, resulting in osteonecrosis, the mechanism of which is unknown. NIH3T3 fibroblasts are pluripotent stem cells, and no reports on the effect of ethanol on NIH3T3 fibroblasts have been reported yet. Objective: To observe the effect of ethanol on the differentiation of NIH3T3 fibroblasts into adipocytes and its effect on the expression of peroxisome proliferator-activated receptor-γ (CRP). Design: Single sample observation. Unit: Department of Biochemistry, School of Basic Medicine, Zhengzhou University, and Department of Orthopedics, First Affiliated Hospital of Zhengzhou University. Materials: NIH-3T3 fibroblasts were provided by the University of Virginia Hospital Orthopedic Laboratory. Methods: The experiment was performed at the Laboratory of Biochemistry, School of Basic Medicine, Zhengzhou University from April 2002 to June 2003. Take NIH-3T3 fibroblasts were randomly divided into 6 groups, were added to the volume fraction of 0.998 ethanol each day, the dose was 0 (control group), 0.03,0.06,0.09,0.15,0.21mol / L, cultured to 14d, Sudan Ⅳ staining , Using computer image analysis software ImageProPlus4.1, to determine the percentage of fat cells. Reverse transcriptase-polymerase chain reaction was used to detect the expression of peroxisome proliferator-activated receptor-γmRNA. MAIN OUTCOME MEASURES: ①NIH3T3 cells differentiated into adipocyte results. ② Ethanol group and control group cells gene expression results. Results: Cells were treated with increasing concentrations of ethanol (0.03,0.06,0.09,0.15,0.21mol / L) for 14 days. ① The percentages of adipocytes in each ethanol group were 11.9%, 23.8%, 28.2%, 31.1% and 42.6%, respectively, which were 1.2, 2.5, 2.9, 3.2 and 4.4 times of the control group (9.6%) respectively. 0.03mol / L ethanol group and the control group, the difference was not statistically significant (P> 0.05), the other four groups and the control group and 0.03mol / L ethanol group differences were highly significant (P <0.001). ② The ratio of peroxisome proliferator-activated receptor-γmRNA expression to 18S gene expression in each ethanol group was 0.218,0.411,0.486,0.473,0.453, respectively, which were respectively 1.1, 2.1 in the control group (0.197) 2.5, 2.4, 2.3 times. 0.03mol / L ethanol group and the control group, the difference was not statistically significant (P> 0.05). The differences between the other four groups and the control group and 0.03mol / L ethanol group were highly significant (P <0.001). Conclusion: Ethanol can directly induce the large number of NIH3T3 fibroblasts to differentiate into adipocytes, which may be one of the reasons for the increase of intramedullary fat in patients with alcoholic osteonecrosis.