沉默Smo基因对大鼠原代软骨细胞增殖与凋亡的影响

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

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（ColⅡ）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,72 h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。

Objective To investigate the effects of silencing Smo gene on proliferation and apoptosis of rat prima- ry chondrocyte in vitro. Methods The primary chondrocyte was obtained by mechanical-enzyme digestion and identified by Immunohistochemical cells （ Col Ⅱ ）. The animals were divided into control group, control siRNA group and Smo siRNA 1 - 3 group. The siRNA was transfected into chondrocytes by lentivirus vector. After 72 h, the cell viability was detected by MTT, Smo expression was 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 may 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% ）. Conclusions Silencing Smo gene in primary chondrocytes may inhibit proliferation and promote apoptosis, Smo may have a protecting role from apop- tosis of the chondrocyte.