国际生殖健康/计划生育杂志

国际生殖健康/计划生育 ›› 2016, Vol. 35 ›› Issue (2): 96-100.

• 论著 • 上一篇    下一篇

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

徐文丹，代晓南，朱倩，张蓓，高超，高莉，刘嘉茵，崔毓桂   

  1. 210029 南京医科大学第一附属医院生殖医学科
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2016-03-15 发布日期:2016-03-15
  • 通讯作者: 崔毓桂

Construction of LV-calb2 and LV-siRNA-calb2 and Expression in Leydig Cells of Teste

XU Wen-dan，DAI Xiao-nan，ZHU Qian，ZHANG Bei，GAO Chao，GAO Li，LIU Jia-yin，CUI Yu-gui   

  1. Clinical Center of Reproductive Medicine，First Affiliated Hospital of Nanjing Medical University，Nanjing 210029,China
  • Received:1900-01-01 Revised:1900-01-01 Published:2016-03-15 Online:2016-03-15
  • Contact: CUI Yu-gui

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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-siRNA-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。

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

Abstract: Objective：To explore the effect of CALB2 on steriodogenesis in testicular Leydig cell，we firstly established LV-calb2 and LV-siRNA-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-siRNA-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 R2C were measured by radioimmunaossay after MLTC-1 and R2C were infected with LV-calb2 and LV-siRNA-calb2 respectively. Results：PCR and gene sequencing confirmed that LV-calb2 and LV-siRNA-calb2 lentivirus vectors were established，with the capacity to infect efficiently MLTC-1 and R2C. Testosterone production was significantly increased in the MLTC-1 infected with LV-calb2 （P＜0.001）, while progesterone production was significantly decreased in R2C infected with LV-siRNA-calb2（P＜0.05）. Conclusions：This study successfully constructed LV-calb2 and LV-siRNA-calb2 lentivirus vectors which could infect Leydig cells with high-efficiency. The preliminary result indicated that CALB2 promoted steriodogenesis in Leydig cells.

Key words: Calcium-binding protein, vitamin D-dependent, Lentivirus, Testis, Leydig cells, Androgens