Cx43基因干扰对鼠胎肝干细胞培养的优化效应

摘要

背景：胎肝干细胞具有分化成肝细胞、胆管细胞的潜能，参与肝脏的修复与重建，是肝细胞的重要来源，但是人体的胎肝干细胞含量极少，如何获取一定数量和较高纯度的胎肝干细胞是目前研究的热点。目的：构建能有效抑制大鼠胎肝干细胞Cx43基因表达的siRNA载体，探讨抑制 Cx43表达对体外培养的鼠胎肝干细胞增殖及细胞周期的影响。方法：体外培养鼠胎肝干细胞，设计及合成靶向 Cx43的 siRNA 序列(Cx43-siRNA)以及阴性对照序列(NC-siRNA)，采用电转法转染大鼠胎肝干细胞，即为实验组和对照组，未转染的胎肝干细胞为空白组。应用real-time PCR和Western blot法检测转染前后鼠胎肝干细胞Cx43基因和蛋白的表达；细胞生长曲线、CCK-8法观察细胞生长增殖情况；流式细胞术测定细胞周期分布的变化。结果与结论：转染Cx43-siRNA后，实验组与对照组、空白组相比Cx43基因和蛋白水平表达均明显降低，细胞的生长速度明显增快，G 0/G 1期细胞减少，S期细胞数增多，差异有显著性意义(P <0.05)，结果表明通过电转法转染靶向Cx43的siRNA能促进体外培养的鼠胎肝干细胞增殖，对其培养有优化作用。%BACKGROUND:Fetal liver stem cel s have the potential to differentiate into hepatocytes and bile duct cel s, and participate in the repair and reconstruction of the liver, which is an important source of hepatocytes. But there are a little amount of fetal liver stem cel s in human body, and how to obtain a certain number of high-purity fetal liver stem cel s is currently a hot research. OBJECTIVE:To construct a siRNA carrier that can effectively inhibit the expression of connexin 43 (Cx43) in rat fetal liver stem cel s, and to investigate the effect of Cx43 inhibition on the proliferation and cel cycle of fetal liver stem cel s cultured in vitro. METHODS:Fetal liver stem cel s were cultured by the suspension culture in vitro, siRNA sequences targeting Cx43 (Cx43-siRNA) and negative control sequence (NC-siRNA) were designed and synthesized. Then, rat fetal liver stem cel s were transferred electrophoretical y and divided into three groups:blank group, NC-siRNA group, Cx43-siRNA group. Real-time PCR and western blot were used to assess the knockdown efficiency. Cel ular proliferation was determined by cel growth curve and cel counting kit-8 assay. The cel cycle was analyzed by flow cytometry. RESULTS AND CONCLUSION:After transfection, the Cx43 gene and protein expression levels were declined dramatical y in the Cx43-siRNA, NC-siRNA and blank groups, and the cel s grew faster. The number of cel s at G0/G1 phase decrease, but the number of cel s in S phase increased. There were significant differences between the groups (P<0.05). Electrophoretic transfer of Cx43-siRNA can promote the proliferation of cultured fetal liver stem cel s and optimize the cel culture.