PRP基因RNAi重组慢病毒制备及PRP基因与睾丸间质细胞增殖和睾酮产生的关系

Preparation of proliferin related protein gene RNAi recombinant lentivirus and its biological function

目的制备PRP RNAi重组慢病毒,并对PRP基因功能进行初步研究。方法采用miRNA方式抑制PRP基因表达,筛选最有效序列包装慢病毒,用于转染TM3睾丸间质细胞。转染实验分为TM3-negative对照组(阴性对照质粒组,转染pcDNA6.2-GW/EmGFP-miR-neg control质粒)以及A、B、C 3个干扰质粒组(分别转染pcDNA6.2-GW/EmGFP-miR-PRP-A,B,C质粒);RT-PCR与Western blot方法检测慢病毒对PRP基因表达影响,MTT方法检测细胞增殖情况,ELISA方法检测细胞睾酮产生情况。结果经测序鉴定,成功制备了PRP RNAi重组慢病毒;与阴性对照质粒组比较,转染pcDNA6.2-GW/EmGFP-miR-PRP-A,B组可有效下调TM3细胞PRP mRNA和蛋白表达(P<0.01,P<0.05)。MTT结果表明抑制PRP基因后,TM3细胞的增殖加快(P<0.01)。此外,相较于对照组,下调PRP基因抑制促黄体生成素(luteinizing hormone,LH)刺激产生睾酮的效应(P<0.05)。结论成功制备了PRP RNAi重组慢病毒,对其功能初步研究显示,PRP不仅与睾丸间质细胞增殖有关,而且参与睾酮产生。

Objective To prepare the proliferin related protein（PRP） RNAi recombinant lentivirus and study the biological function of PRP gene.Methods Expression of PRP gene was inhibited with mRNA.Effectively packaged lentivirus was selected to transfect TM3 interstitial cells.Transfection experiments were divided into TM3-negative control group（transfected with pcDNATM6.2-GW/EmGFP-miR-negative control plasmid） and 3 plasmid-interfering groups（groups A,B and C transfected with pcDNA6.2-GW/EmGFP-miR-PRP-plasmids A,B and C）.Effect of lentivirus on PRP gene expression was detected by RT-PCR and Western blotting,respectively.Proliferation of TM3 cells was assayed by MTT and production of testosterone in TM3 cells was detected by ELISA.Results PRP RNAi recombinant lentivirus was successfully prepared as confirmed by sequencing.The expression level of PRP mRNA and protein in TM3 cells was significantly lower in groups A and B than in control group（P〈0.05）.MTT assay showed that the proliferation of TM3 cells was faster when the expression of PRP gene was inhibited（P〈0.01）.In addition,down-regulation of PRP gene expression inhibited the LH-stimulated production of testosterone（P〈0.05）.Conclusion PRP RNAi recombinant lentivirus can be successfully prepared.PRP gene is not only related with the proliferation of interstitial cells but also participates in the production of testosterone.