基因干扰技术抑制精原细胞GDNF mRNA表达的研究

Spermatogonial Stem Cells Expression of GDNF mRNA by RNA Interference Inhibits

目的观察精原细胞GDNF基因干扰后精原细胞GDNF m RNA的表达,了解GDNF在精原细胞中的作用机制。方法设计、构建多个针对GDNF的干扰性小RNA(small interfering RNA,si RNA),转染精原细胞,筛选出对GDNF基因干扰效率最高的GDNF si RNA,用酶标仪测定精原细胞的增殖,流式细胞仪检测精原细胞凋亡率。结果实验组和对照组精原细胞GDNF m RNA的表达强度分别为12.32±1.22%、54.25±1.34%,细胞凋亡率分别为(25.43±1.91)%、(5.61±0.16)%,两组比较,差异有统计学意义(P<0.01);表明构建的阳性表达载体有效抑制了GDNF m RNA表达并诱导细胞凋亡,抑制了精原细胞的增殖。结论 RNAi可有效抑制精原细胞GDNF m RNA和蛋白表达,GDNF在精原细胞增殖过程中具有重要作用。

Objective To investgate the expression of GDNF、RTKs、Fyn and FAK m RNA m RNA after RNA interferenced(RNAi) of GDNF,to know the mechanism of GDNF in SSCS. Methods si RNA expression vectors were designed and constructed to be directed at GDNF and transfected into SSCs,Flow cytometry、Western and RT-PCR were used to detect the expression of GDNF、RTKs、Fyn and FAK m RNA and the apoptosis of SSCs.Results The expression rates of GDNF were(12.32±1.22)% 、(54.25±1.34)%,and apoptosis rates of PC-3 cells were(25.43±1.91)% 、(5.61±0.16)% between experiment groups and contrasted group.There were significant different between two groups(P<0.01). Conclutions Expression of GDNF m RNA were effectively inhibited,then induced the spoptosis of PC-3 cells by RNAi. GDNF could improve the proliferation and differenciation of the spermatogonial stem cells,then it played an important role.