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[目的]比较青少年特发性脊柱侧凸(AIS)患者与正常人骨髓间充质干细胞的成脂分化过程中基因表达的差异。[方法]收集本院脊柱侧弯中心2011年12月~2012年12月收治的AIS患者20例的骨髓血,同时采集10例年龄段性别接近的志愿者骨髓血作为对照组。采用密度梯度离心法提取MSC细胞,培养至P3流式细胞仪定性。诱导成脂分化14 d后提取全RNA进行Affymetrix 3’IVT表达谱芯片检测。对主要检查结果进行RT-PCR验证,并且对结果中特异性高表达的Thrsp蛋白的表达情况进行Western blot检验。[结果]受试者骨髓血均成功采集分离,密度梯度离心分离MSCs并培养至P3,流式细胞仪验证其性质,成脂分化培养14 d后苏木红染色定性为脂肪细胞,提取全RNA电泳条带清晰,表达谱芯片检测结果显示AIS患者MSC在成脂分化过程中有111条基因表达上调,189条基因表达下调。Western blot检验结果显示Thrsp蛋白在脂肪细胞中高度表达。[结论]AIS来源MSCs成脂分化相关差异表达的300条基因可能参与了AIS的发生和发展,Thrsp基因可能是导致MSCs分化差异性的关键因素。
[Objective] To compare the differences of gene expression in adipogenic differentiation between adolescent idiopathic scoliosis (AIS) patients and normal human bone marrow mesenchymal stem cells. [Methods] Bone marrow blood of 20 patients with AIS was collected from December 2011 to December 2012 in our hospital. At the same time, bone marrow blood of 10 age-matched volunteers was collected as control group. Density gradient centrifugation was used to extract MSC cells and cultured to P3 flow cytometry qualitatively. After induction of adipogenic differentiation for 14 days, total RNA was extracted for Affymetrix 3’IVT expression profiling. The main test results were verified by RT-PCR, and Western blot was performed on the expression of Thrsp protein with high specificity and specificity. [Results] The bone marrow blood of the subjects were successfully collected and separated. The MSCs were isolated by density gradient centrifugation and cultured to P3. The characteristics of the MSCs were verified by flow cytometry. After 14 days of adipogenic differentiation, hematoxylin and eosin With clear, the expression profile of the chip test results showed that AIS patients with adipogenic differentiation in 111 gene expression, 189 gene down regulation. Western blot results showed that Thrsp protein was highly expressed in adipocytes. [Conclusion] The 300 differentially expressed genes associated with adipogenic differentiation of AIS-derived MSCs may be involved in the occurrence and development of AIS. The Thrsp gene may be the key factor leading to the differentiation of MSCs.