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目的:研究pcDU6载体质粒介导的转化生长因子β(TGF-β1)短发夹RNA(pcDU6-A1-A2和pcDU6-B1-B2)对人血清白蛋白(HSA)致人肾小管上皮细胞(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HSA刺激HK2细胞增殖及CTGF和FN基因过表达是否通过TGF-β1介导。方法:构建TGF-β1短发夹RNA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-β1 shRNA的pcDU6质粒载体(pcDU6-A1-A2和pcDU6-B1-B2)分别导入试验组细胞。使用HSA(5 g/L)刺激HK2细胞12 h或24 h。用四甲基偶氮唑盐比色法测定细胞增殖水平;逆转录多聚酶链反应半定量分析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:HSA对HK2细胞增殖在5 g/L作用24 h最明显;HK2细胞在HSA刺激下可明显上调TGF-β1,CTGF及FNmRNA的表达,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-β1 shRNA转染HK2细胞后12 h或24 h,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)。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(P>0.05)。结论:pcDU6载体质粒介导的TGF-β1shRNA能够明显抑制HSA刺激下HK2细胞增殖以及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 pcDU6 vector plasmid-mediated transfection of TGF-β1 short hairpin RNA (pcDU6-A1-A2 and pcDU6-B1-B2) on human renal tubular epithelial cells induced by human serum albumin HK2 cells), the expression of TGF-β1, connective tissue growth factor (CTGF) and fibronectin (FN), and to explore whether HSA can stimulate the proliferation of HK2 cells and whether the over-expression of CTGF and FN is mediated by TGF-β1. Methods: pcDU6 plasmid was constructed by transfecting TGF-β1 short hairpin RNA and HK2 cell line was cultured in vitro. The pcDU6 plasmid vector (pcDU6-A1-A2 and pcDU6-B1-B2) expressing TGF-β1 shRNA was transfected into the test group cells by lipofectamine. HK2 cells were stimulated with HSA (5 g / L) for 12 h or 24 h. The level of TGF-β1, CTGF and FN mRNA in HK2 cells was detected by reverse transcription-polymerase chain reaction (RT-PCR). The HK2 cell culture was detected by double-antibody sandwich enzyme-linked immunosorbent assay Fluid TGF-β1 and FN protein levels. Results: HSA had the most obvious effect on proliferation of HK2 cells at 5 g / L for 24 h. HK2 cells significantly upregulated the expression of TGF-β1, CTGF and FN mRNA under the stimulation of HSA. The protein content of TGF-β1 and FN in culture medium was also significantly increased Increased (P <0.05). Compared with the pcDU6 empty vector group, the expression of TGF-β1, CTGF and FN mRNA in pcDU6 plasmid-mediated TGF-β1 shRNA group was significantly decreased (P <0.05). TGF-β1 shRNA transfected HK2 cells 12 h or 24 h, the cell culture medium TGF-β1 and FN protein content was significantly decreased HK2 cell proliferation was partially inhibited (P <0.05). TGF-β1shRNA interference group comparison and pcDU6 empty vector transfection group and HSA stimulation group, the difference was not statistically significant (P> 0.05). CONCLUSION: The pcDU6 plasmid mediated TGF-β1shRNA can significantly inhibit the proliferation of HK2 cells and the expression of TGF-β1, CTGF and FN genes induced by HSA. HSA stimulated the proliferation of HK2 cells and over-expression of CTGF and FN genes through TGF-β1 mediate.