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

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－shRNA－1、GV－P34H－shRNA－2和GV－P34H－shRNA－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 mRNA和蛋白的沉默效果。采用免疫荧光法观察P34 H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（P＜0．05）；其中以GV－P34H－shRNA－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（P＜0．05），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。%AIM:To investigate the effects of P34H gene silencing on the expression of P34H and activity of hyaluronidase (HYD) in mouse sperm.METHODS:The recombinant plasmid series of P34H targeted short hairpin RNA (shRNA) were constructed by GV248 plasmids vector.These recombinant plasmids were transformed into DH 5αcompetent cells, and the plasmids were taken from DNA sequencing analysis .The HEK293T cells were co-transfected with shRNA and lentiviral packaging plasmids .The 3 kinds of recombinant lentiviruses and negative control lentiviruses were used to in -ject into the mouse epididymis and the expression of P 34H at mRNA and protein levels was detected by real-time PCR and Western blot, respectively.The location of P34H protein on the mouse spermatozoa was determined by indirect immunofluo-rescent staining using P34H antibody.The positive rate and activity intensity of HYD was detected by modified sodium hya-luronate-gelatin membrane.RESULTS:DNA sequencing analysis confirmed that the 3 P34H-shRNA sequences were suc-cessfully inserted into the lentiviral vectors .P34H expression in epididymis tissue was significantly decreased at both mR-NA and protein levels compared with those of the non-transfected and normal control vectors (P<0.05).The GV-P34H-shRNA-1 played a significant role in reducing the percentage of P 34H positive rate and the activity of HYD in mouse sperm.The silencing effect did not significantly differ between the non-transfected and normal control vectors .CONCLU-SION:Silencing of P34H significantly inhibits the percentage of P 34H positive rate and the activity of hyaluronidase in mouse sperm.