沉默附睾P34H基因对小鼠精子P34H表达和精子透明质酸酶活性的影响

发布时间：2019-05-19 13:41

【摘要】：目的:通过慢病毒介导的RNA干扰(RNAi)敲低P34H表达,分析其对小鼠精子P34H表达和精子透明质酸酶(HYD)活性的影响。方法:构建3种附睾精子P34H shRNA慢病毒表达载体GV-P34H-sh RNA-1、GVP34H-sh RNA-2和GV-P34H-sh RNA-3,提取阳性克隆质粒测序,转染HEK293T细胞,生产慢病毒颗粒。将3种重组慢病毒及阴性对照病毒分别注入小鼠附睾中,用real-time PCR和Western blot法分别检测其对P34H m RNA和蛋白的沉默效果。采用免疫荧光法观察P34H蛋白在小鼠精子上的定位,改良透明质酸钠-明胶底物膜法检测小鼠精子HYD活性(HYD阳性反应率和HYD活性强度)。结果:测序结果证实3种慢病毒载体均包装成功。感染小鼠附睾后,P34H的mRNA及蛋白表达量均较阴性感染组和正常对照组明显降低(P0.05);其中以GV-P34H-sh RNA-1作用最为显著,精子P34H阳性表达率和HYD活性均较阴性感染组和正常对照组明显降低(P0.05),而阴性感染组和正常对照组相比差异无统计学显著性。结论:附睾P34H基因沉默可抑制小鼠附睾中精子P34H阳性表达率和HYD活性。
[Abstract]:Aim: to analyze the effect of lentivirus mediated RNA interference on P34H expression and hyaluronidase (HYD) activity in mouse spermatozoa. Methods: three kinds of epididymal sperm P34H shRNA lentivirus expression vectors GV-P34H-sh RNA-1,GVP34H-sh RNA-2 and GV-P34H-sh RNA-3, were constructed and sequenced and transformed into HEK293T cells to produce lentivirus particles. Three recombinant lentiviruses and negative control viruses were injected into the epididymis of mice, and their silencing effects on P34H m RNA and protein were detected by real-time PCR and Western blot, respectively. The localization of P34H protein on mouse spermatozoa was observed by immunofluorescence method. The HYD activity (HYD positive rate and HYD activity intensity) of mouse spermatozoa were detected by modified sodium hyaluronate gelatin substrate membrane method. Results: sequencing results confirmed that all three lentivirus vectors were packaged successfully. The expression of mRNA and protein of P34H in epididymis of infected mice was significantly lower than that of negative infection group and normal control group (P 0.05). The positive expression rate of P34H and the activity of HYD in spermatozoa were significantly lower than those in the negative infection group and the normal control group (P 0.05), but there was no significant difference between the negative infection group and the normal control group. Conclusion: P34H gene silencing in epididymis can inhibit the positive expression rate of P34H and HYD activity in epididymis of mice.
【作者单位】：贵州医科大学组织胚胎学教研室;遵义医药高等专科学校组织胚胎学教研室;六盘水市人民医院病理科;
【基金】：贵州省科技计划项目(黔科合LH字[2015]7567号) 贵州省科技创新人才团队项目(黔科合人才团队[2014]4005号)
【分类号】：R339.2

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