转化生长因子β1短发夹RNA对白蛋白刺激人肾近曲小管上皮细胞细胞因子表达的影响

Impact of pcDU6 vector-mediated TGF-β1 shRNA on cytokines expression induced by albumin in HK2 cells

目的 研究pcDU6载体质粒介导的转化生长因子β1（TGF-β1）短发夹RNA（shRNA）对人血清白蛋白（HSA）致人肾近曲小管上皮细胞（HK2细胞）增殖，TGF-β1、结缔组织生长因子（CTGF）和纤连蛋白（FN）表达的影响，并探讨有关机制。方法 构建TGF-β1shRNA的pcDU6载体质粒，体外培养HK2细胞株。采用脂质体转染将表达TGF-β1shRNA的pcDU6质粒载体（pcDU6-A1-A2和peDU6-B1-B2）分别导人实验组细胞。用HSA（5g／L）刺激HK2细胞12h或24h。四甲基偶氮唑盐（MTF）比色法测定细胞增殖水平。RT-PCR半定量分析HK2细胞中TGF-β1、CTGF和FNmRNA的表达水平；双抗夹心酶联免疫吸附法检测HK2细胞培养液中TGF-β1及FN蛋白质水平。结果 HK2细胞在HSA刺激下，其TGF-β1、CTGF及FNmRNA的表达明显上调，培养液中TGF-β1和FN的蛋白质含量亦明显升高（P〈0．05）。与pcDU6空载体组比较，pcDU6载体质粒介导的TGF-β1shRNA干扰组TGF-β1、CTGF及FNmRNA的表达明显下调（P〈0．05）。TGF-β1shRNA转染HK2细胞后12h或24h，细胞培养液中TGF-β1和FN蛋白质含量明显下降，HK2细胞增殖被部分抑制（P〈0．05）。在细胞增殖、TGF-β1、CTGF及FN基因表达方面，TGF-β1shRNA干扰组组间比较，以及pcDU6空载体转染组与HSA刺激组比较，差异均无统计学意义。结论 peDU6载体质粒介导的TGF-β1shRNA能够明显抑制HSA刺激下HK2细胞增殖、TGF-β1、CTGF和FN基因的表达。HSA刺激HK2细胞增殖及CTGF和FN基因的过表达可能通过TGF-β1介导。

Objective To study the effect of two TGF-β1 shRNA expression plasmids （pcDU6-AI-A2 and pcDU6-BI-B2） on proliferation, TGF-β1, CTGF and FN synthesis induced by human serum albumin （HSA） in HK2 ceils and to explore the mechanism concerned. Methods A vector plasmid containing the shRNA of TGF-β1 was generated. An HK2 cell line was used in the study. Two TGF-β1 shRNA expression plasmids were transfected into cultured HK2 cells by lipofectamine 2000. Cellular proliferation was assessed by tetrazolium salt colorimetry （MTT）. The semi-quantitative reverse transcriptive PCR was performed to detect the expression of TGF-β1, CTGF and FN mRNA. Levels of TGF-131 and FN protein were measured with a sandwich enzymelinked immunosorbent assay. Results After treating with 5 g/L HSA for 24 h in HK2 cells, cellular proliferating capacity significantly increased （P 〈 0.05）. The expression levels of TGF-β1, CTGF and FN mRNA were up-regulated in HK2 cells stimulated by 5 g/L HSA, and levels of TGF-β1 and FN protein in the culture supernatant increased （P 〈0.05）. The introduction of pcDU6-A1-A2 and pcDU6-B1-B2 resulted in significant reduction of cell proliferation activity. The expression levels of TGF-β1, CTGF and FN mRNA were down-regulated （P 〈 0.05）. Levels of TGF-β1 and FN protein in the culture supernatant decreased （P 〈0.05）. There were no significant differences of the expression levels of TGF-β1, CTGF and FN mRNA between two pcDU6 vector plasmid mediated TGF-β1 shRNA groups （P 〉0.05）. Conclusion pcDU6 vector plasmidmediated TGF-β1 shRNA inhibits the mRNA expression of TGF-β1, CTGF, FN and cellular proliferation stimulated by HSA in HK2 cells, which may be related to the mediation of TGF-β1.