Department of Nephrology, Navy General Hospital, Haidian, Beijing, PR China.
Ren Fail. 2011;33(3):334-40. doi: 10.3109/0886022X.2011.559679.
To investigate the effects of lentivirus-mediated shRNA targeting collagen type I on the mesangial cells of rats and the feasibility of lentivirus-mediated shRNA delivery through renal parenchyma injection.
Anti-collagen type I shRNA lentiviral vector was constructed, and rat mesangial cells were transfected with transfection enhancer (control group), blank lentiviral vectors (pSC-GFP group), and pSC-GFP/Col I lentiviral vectors (pSC-GFP/Col I group). Transfection efficiency and cell cycle were determined by flow cytometry. RT-PCR and Western blot were performed to detect the mRNA and protein expressions of Col I. Cell proliferation was evaluated by 3-(4,5)-dimethylthiahiazo-3, 5-di-phenytetrazolium-romide (MTT) assay and direct counting, and apoptosis was detected using AnnexinV/PE staining. The feasibility of renal parenchyma injection of lentiviral vectors was assessed.
The transfection efficiency was 75.42%. The expressions of collagen type I in pSC-GFP/Col I group was markedly decreased when compared with the other two groups. PSC-GFP/Col I group was higher than pSC-GFP group in the inhibition efficiency of mesangial cell after transfection. Results revealed that pSC-GFP/Col I transfection induced apoptosis to a certain extent. The proportion of cells in G2/M phase in pSC-GFP/Col I group and pSC-GFP group was higher than that in control group after of transfection. Moreover, cells arrested in S phase were markedly increased. Our results also revealed renal injection of lentivirus-mediated shRNA was feasible.
Lentivirus-mediated shRNA targeting collagen type I could stably and efficiently transfect rat mesangial cells and significantly suppressed collagen type I expressions with acceptable safety. Renal injection of Col I lentivirus-mediated shRNA was also feasible.
探讨靶向Ⅰ型胶原的短发夹 RNA（shRNA）慢病毒对大鼠系膜细胞的作用及经肾实质注射递送慢病毒载体的可行性。
构建靶向Ⅰ型胶原的 shRNA 慢病毒载体，转染增强剂（对照组）、空白慢病毒载体（pSC-GFP 组）和 pSC-GFP/Col I 慢病毒载体（pSC-GFP/Col I 组）转染大鼠系膜细胞，流式细胞术检测转染效率和细胞周期，RT-PCR 和 Western blot 检测 Col I 的 mRNA 和蛋白表达，3-(4,5)-二甲基噻唑-2,5-二苯基四氮唑溴盐（MTT）比色法和直接计数检测细胞增殖，AnnexinV/PE 染色检测细胞凋亡，评估肾实质注射慢病毒载体的可行性。
转染效率为 75.42%。与其他两组相比，pSC-GFP/Col I 组 Col I 的表达明显降低。转染后，pSC-GFP/Col I 组对系膜细胞的抑制效率高于 pSC-GFP 组。结果表明，pSC-GFP/Col I 转染在一定程度上诱导了细胞凋亡。转染后，pSC-GFP/Col I 组和 pSC-GFP 组细胞 G2/M 期比例高于对照组，S 期阻滞细胞明显增加。我们的结果还表明，经肾注射慢病毒介导的 shRNA 是可行的。
靶向Ⅰ型胶原的慢病毒介导 shRNA 可稳定有效地转染大鼠系膜细胞，显著抑制 Col I 的表达，且具有可接受的安全性。Col I 慢病毒介导的 shRNA 经肾注射也是可行的。
