摘要
观察锌指蛋白265(ZNF265)基因对睾丸Sertoli细胞溶脲脲原体(Ureaplasma urealyticum,UU)感染后IL-1α、IL-6及TGF-β1表达的影响,并探讨其可能的作用机制。构建沉默大鼠ZNF265基因表达的慢病毒载体,体外转染Sertoli细胞,对转染阳性的细胞进行UU感染,在感染后的第1、3、5天分别检测对照组和实验组中IL-1α、IL-6及TGF-β1表达。结果显示,成功构建干扰Sertoli细胞中ZNF265表达的慢病毒载体,在Sertoli细胞中转染效率高达62.8%,在mRNA水平和蛋白质水平干扰ZNF265表达的效率分别为65.73%和94%;干扰Sertoli细胞中ZNF265表达后,在UU感染的第1天和第3天,IL-1α、IL-6及TGF-β1表达升高;在UU感染第5天,ZNF265表达较对照组升高,TGF-β1表达升高,但IL-1α,IL-6表达下降。以上结果说明,构建的慢病毒载体成功地干扰ZNF265基因在大鼠Sertoli细胞中的表达,干扰其表达后,IL-1α和IL-6表达增加;ZNF265作为调控因子,可能通过作用于TGF-β1,在抑制炎症发展、维持睾丸局部免疫微环境的平衡中发挥重要作用。
To investigate the influence of ZNF265 on expressions of IL-1α, IL-6 and TGF-β1 during UU infection of rat Sertoli cells and its mechanism, we first applied lentiviral-mediated RNA interference （RNAi） to inhibit the expression of ZNF265 in Sertoli cells, which were then infected by UU and investigated for expression ofIL-1α, IL-6 , TGF-β1 at mRNA level and protein level on day 1, 3 and 5 post-infection. The infection rate of lentivirus in Sertoli cells was 62.8%, and the interference rate of ZNF265 expression at mRNA level and protein level was 65.73 and 94%, respectively. On day 1 and 3 post-infection, mRNA expression of IL-1α, IL-6 ,TGF-β1 was higher in the RNAi treated group than the untreated group. On day 5, expression of ZNF265 was higher than that of the control group, and so did TGF-β1, however, the levels of both IL-1α and IL-6 were deceased. These findings indicate that the lentiviral ZNF265 vector we constructed successfully inhibited expression of ZNF265 gene in Sertoli cells resulting in increased expression of IL-1α and IL-6. Therefore, ZNF265 may exerts its regulating role in inhibiting inflammatory and maintaining balance of immune microenvironment in the testis by upregulating expression of ZNF265. As ZNF265 a~ the fifth day going higher in shRNA-ZNF265 UU infection group than shRNA-NC UU infection group, which is in accordance to TGF-β1, the protein expression of IL-1α, IL-6 was down-regulated, and lower than that without RNAi. In conclusion, we successfully constructed lentiviral vector of RNAi to silence expression of ZNF265, and ZNF265 may combine with TGF-β1 to play a negative regulatory role by down regulating IL-1α, IL-6 in Sertoli cells during UU infection and control inflammation of Sertoli cell.
出处
《现代免疫学》
CAS
CSCD
北大核心
2013年第3期219-224,共6页
Current Immunology
基金
国家自然科学基金项目(81070479)
上海市教委重点项目(09ZZll7)
