基础医学与临床 ›› 2015, Vol. 35 ›› Issue (9): 1209-1213.

• 研究论文 • 上一篇    下一篇

沉默Smo基因对大鼠原代软骨细胞增殖与凋亡的影响

朱志坚,于燕妮,陶欣,邓超男   

  1. 贵阳医学院
  • 收稿日期:2015-03-27 修回日期:2015-06-25 出版日期:2015-09-05 发布日期:2015-09-07
  • 通讯作者: 于燕妮 E-mail:gyyxybl2010@163.com
  • 基金资助:
    国家自然科学基金面上项目;教育部博士点基金(博导类)

The effects of silencing Smo gene on proliferation and apoptosis of rat primary chondrocyte

  • Received:2015-03-27 Revised:2015-06-25 Online:2015-09-05 Published:2015-09-07

摘要: 目的 观察沉默Smo基因后对大鼠原代软骨细胞增殖与凋亡的影响。方法 用机械-酶消化法获取原代大鼠软骨细胞,经免疫细胞化学Ⅱ型胶原鉴定后,将实验分为对照组、control siRNA组和Smo siRNA 1~3组,以慢病毒为载体将siRNA转入软骨细胞,72h后,MTT法检测细胞活力;反转录PCR (RT-PCR)及蛋白印迹(Western blot)检测Smo的表达量;流式细胞术检测细胞凋亡率。结果 各组序列经慢病毒载体成功转染到原代软骨细胞中,Smo siRNA1~3均不同程度的抑制Smo的表达,以Smo siRNA2组最为明显,其mRNA及蛋白的表达分别为0.19±0.03和0.39±0.07;同时,Smo siRNA2组细胞活力最低(77.38%±7.19%)而凋亡率最高(21.43%±2.97%)。结论 沉默Smo可抑制原代软骨细胞增殖,并诱导软骨细胞凋亡,Smo具有保护软骨细胞免遭凋亡的作用。

关键词: 原代软骨细胞, 慢病毒载体, siRNA, Hh信号通路, 凋亡, 增殖

Abstract: Objective To investigate the effects of silencing Smo gene on proliferation and apoptosis of rat primary chondrocyte in vitro. Method The primary chondrocyte was obtained by mechanical-enzyme digestion and identified by Immunohistochemical cells (ColⅡ). The experiment was divided into control group, control siRNA group and Smo siRNA 1~3 group. The siRNA were transfected into chondrocytes by lentivirus vector. After 72h, the cell viability was detected by MTT, Smo expressions were detected by RT-PCR and Western blot, and the apoptosis of chondrocyte was assessed by flow cytometry. Results All types of siRNA were transfected into primary chondrocyte by vectors, the Smo siRNA 1 ~ 3 could inhibit the expression of Smo mRNA and protein in chondrocytes, and Smo siRNA2 had the highest silencing rate (the expressions of Smo mRNA and protein were 0.19±0.03 and 0.39±0.07). The cell viability in Smo siRNA2 group was lowest (77.38%±7.19%), while the apoptosis rate of Smo siRNA2 was highest (21.43%±2.97%). Conclusion Silencing Smo gene in primary chondrocytes can inhibit proliferation and promote apoptosis, Smo may have a protecting role from apoptosis of the chondrocyte.

Key words: Primary chondrocytes, Lentiviral vector, siRNA, Hh signaling pathway , apoptosis, Proliferation