LV-calb2和LV-siRNA-calb2慢病毒载体的构建及其在睾丸间质细胞的表达  

Construction of LV-calb2 and LV-si RNA-calb2 and Expression in Leydig Cells of Teste

作　　者：徐文丹[1] 代晓南[1] 朱倩[1] 张蓓[1] 高超[1] 高莉[1] 刘嘉茵[1] 崔毓桂[1] 

机构地区：[1]南京医科大学第一附属医院生殖医学科,210029

出　　处：《国际生殖健康／计划生育杂志》2016年第2期96-100,I0001,共6页Journal of International Reproductive Health/Family Planning

基　　金：国家自然科学基金(81170559;81370754;31301182);江苏省卫生厅项目(ZX201110);江苏省妇幼保健重点学科(FXK201221)

摘　　要：目的:构建钙视网膜蛋白(CALB2)基因超表达及小干扰RNA(siRNA)慢病毒载体,观察CALB2对睾丸间质细胞雄激素合成的调节作用。方法:合成超表达及干涉的质粒,转入制备好的细菌感受态细胞,聚合酶链反应(PCR)鉴定阳性重组子后测序验证,确认构建成功的超表达及干涉慢病毒载体。用上述慢病毒载体分别感染MLTC-1及R2C间质细胞,观察感染效率,而后用放射免疫法检测睾酮和孕酮的合成。结果:PCR和基因测序证实LV-calb2和LV-si RNA-calb2慢病毒载体构建成功,并可高效感染MLTC-1及R2C细胞。超表达CALB2后,MLTC-1细胞睾酮生成显著增加(P<0.001);干涉CALB2表达后,R2C间质细胞孕酮生成显著减少(P<0.05)。结论:成功构建了calb2基因超表达及干涉慢病毒载体,并可体外转染睾丸间质细胞系。初步结果表明,CALB2可促进间质细胞类固醇激素生成。Objective：To explore the effect of CALB2 on steriodogenesis in testicular Leydig cell,we firstly established LV-calb2 and LV-si RNA-calb2 lentivirus vectors. Methods：Firstly,the over-expression and interference plasmids were synthysized,and the bacterial competent cells were prepared ahead. Secondly,polymerase chain reaction（PCR） and gene sequencing were administered to ensure the LV-calb2 and LV-si RNA-calb2 lentivirus vectors constructed successfully. At last,the infection efficiency was tested and then the level of testosterone in MLTC-1 and the level of progesterone in R2 C were measured by radioimmunaossay after MLTC-1 and R2 C were infected with LV-calb2 and LV-si RNA-calb2 respectively. Results：PCR and gene sequencing confirmed that LV-calb2 and LV-si RNA-calb2 lentivirus vectors were established,with the capacity to infect efficiently MLTC-1 and R2 C. Testosterone production was significantly increased in the MLTC-1infected with LV-calb2（P0.001）,while progesterone production was significantly decreased in R2 C infected with LV-si RNA-calb2（P0.05）. Conclusions：This study successfully constructed LV-calb2 and LV-si RNAcalb2 lentivirus vectors which could infect Leydig cells with high-efficiency. The preliminary result indicated that CALB2 promoted steriodogenesis in Leydig cells.

关 键 词：钙结合蛋白 维生素D依赖性 慢病毒属 睾丸 莱迪希细胞 雄激素类 

分 类 号：R339.2[医药卫生—人体生理学]