结缔组织生长因子反义寡核苷酸对肾小管上皮细胞纤溶酶原激活物抑制物1表达的影响

Effect of connective tissue growth factor antisense oligodeoxynucleotide on pl asminogen activator inhibitor-1 expression in renal tubular cells

目的探讨结缔组织生长因子(CTGF)反义寡核苷酸(ODN)对转化生长因子β1(TGF-β1)诱导的肾小管上皮细胞纤溶酶原激活物抑制物1(PAI-1)mRNA表达和蛋白产生的影响,以明确CTGF在肾脏细胞外基质(ECM)降解中的作用。方法体外培养人近曲小管上皮细胞(HKC),以脂质体介导方法将CTGF反义ODN转染细胞。以TGF-β1(5μg/L)刺激HKC不同时间后,用逆转录-聚合酶链式反应(RT-PCR)方法检测PAI-1mRNA表达;流式细胞仪法检测胞内PAI-1蛋白合成;Western印迹方法检测HKC分泌到上清中的PAI-1蛋白含量。结果TGF-β1可诱导HKC高表达CTGF和PAI-1。转染后6h,CTGF反义ODN可显著抑制TGF-β1诱导的HKCPAI-1mRNA表达。转染后24h,CTGF反义ODN可明显抑制胞内PAI-1蛋白合成,并减少了HKC分泌到胞外的PAI-1。结论CTGF在肾小管间质纤维化时ECM降解中起了关键调控作用,其反义ODN的导入可能是延缓肾小管间质纤维化的有效手段之一。

Objective To investigate the effect of connective tissue growth fa ctor(CTGF) antisense oligodeoxynucleotide(ODN) on plasminogen activator inhibito r-1(PAI-1) mRNA expression and protein synthesis induced by transforming growt h factor-a1 (TGF-a1) in renal tubular cells, and to explore the role of CTGF in the degradation of renal extracellular matrix(ECM). Methods A human proxim al tubular epithelial cell line(HKC) was cultured in vitro. Cationic lipid-medi ated CTGF antisense ODN was transfected into HKC. After HKC cells were stimulate d with TGF-a1(5 ìg/L), the mRNA levels of PAI-1 were detected by RT-PCR. In tracellular PAI-1 protein synthesis was assessed by flow cytometry. The secrete d PAI-1 protein in the media was determined by Western blot. Results TGF-a1 c ould increase tubular CTGF and PAI-1 mRNA expression. The CTGF and PAI-1 mRNA expression induced by TGF-a1 was significantly inhibited by CTGF antisense ODN . CTGF antisense ODN also inhibited the intracellular PAI-1 protein synthesis a nd lowered the secretion of PAI-1 protein in the media. Conclusion CTGF may pla y a crucial role in the degradation of excessive ECM during tubulointerstitial f ibrosis, and transfecting CTGF antisense ODN may be an effective way in preventi ng renal fibrosis.