以siRNA表达载体稳定抑制缝隙连接蛋白43表达的睾丸间质细胞和睾丸支持细胞系的建立

Establishment of Cx43-stably-downregulated leydig cell line and sertoli cell line by siRNA expression vector

目的构建Cx43-siRNA真核表达载体,获得连接蛋白43(connexin43,Cx43)被长期稳定抑制的睾丸间质细胞(TM3细胞)系和睾丸支持细胞(TM4细胞)系,为研究Cx43及其形成的细胞缝隙连接(gap junction,GJ)在睾丸组织中的作用提供有用模型。方法设计合成3对针对Cx43的短发夹样siRNA的DNA模板序列,定向连接到siRNA真核表达载体pSilencerTM2.1-U6neo上,通过测序鉴定后以脂质体法瞬时转染睾丸支持细胞,以Western blot方法检测Cx43蛋白表达水平,筛选出最有效的干扰序列,再将之分别转染睾丸间质细胞和睾丸支持细胞,G418筛选出能稳定表达siRNA的细胞系,以"Parachute"荧光传递示踪法检测细胞缝隙连接功能。结果 Western blot结果显示,第3对干扰序列对Cx43表达抑制效果最佳,以表达该序列的质粒稳定转染的TM3细胞和TM4细胞上Cx43蛋白表达水平均明显降低;荧光传递示踪法检测表明,两种细胞系的GJ功能均被明显抑制。结论以Cx43-siRNA真核表达载体稳定转染的方法能长期干扰TM3和TM4细胞上Cx43的表达,并抑制由其形成的GJ功能。

Aim To establish Cx43-stably-downregulated Leydig cell（TM3） line and Sertoli cell（TM4） line by siRNA expression vector.Methods Three pairs of hairpin siRNA template oligonucleotides targeting Cx43 were designed,chemically synthesized and inserted into pSilencer TM 2.1-U6 neo vector.After being confirmed by DNA sequencing,the recombinant plasmids were transfected into TM4 cells by liposomes.The interfering effect was determined by testing the Cx43 expression with Western blot,and the most effective vector was stably transfected into TM3 and TM4 cells by G418 selection,respectively.Gap junction function was assessed by＂parachute＂dye-coupling assay.Results The third siRNA sequence had the strongest interfering effect.Western blot confirmed that cells stably transfected with siRNA sequence 3 linked vector expressed less Cx43 than that of controls.Consistent with the results of Western blot,the gap junction function assessed by＂parachute＂dye-coupling assay was also depressed.Conclusions siRNA expression vector targeting Cx43 could be used to achieve long-term depression of Cx43 expression as well as gap junction function in both TM3 cells and TM4 cells,which will provide a useful tool for further investigation of the role of Cx43 and gap junction in testis tissue.