转化生长因子β1反义RNA对肾小球系膜细胞基质合成及分泌的影响

Effect of transforming growth factor beta 1 antisense RNA by adenovirus mediation on synthesis and secretion of extracellular matrix in cultured mesangial cells

目的 观察转化生长因子beta1（TGF-β1）反义RNA对系膜细胞基质合成的影响。方法 构建含TGF-β1反义RNA的重组腺病毒,将重组腺病毒转染系膜细胞,用Northern blot检测转染系膜细胞TGF-β1及ColⅣmRNA的含量,用免疫组化半定量分析转染的系膜细胞中TGF-β1、纤连蛋白（FN）及胶原（Col）Ⅳ蛋白水平,并与无转染的系膜细胞对照组比较其表达的变化。结果 构建了含TGF-β1反义RNA的重组腺病毒。重组反义TGF-β1腺病毒转染系膜细胞后,与对照组相比,在第24hTGF-β1及ColⅣ的mRNA无明显抑制,在48h TGF-β1及ColⅣmRNA抑制率分别为22.5％,18.2％;72h TGF-β1及ColⅣmRNA抑制率分别为29.5％,27.3％。免疫组织化学半定量结果显示:与对照组相比,在48h始转染系膜细胞TGF-β1、FN及ColⅣ蛋白含量开始下降,48hTGF-β1、FN及ColⅣ蛋白抑制率分别为16.5％,18.2％及14.6％;72hTGF-β1、FN及ColⅣ蛋白抑制率分别为23.5％,27.3％及26.8％。结论 重组反义TGF-β1可抑制系膜细胞合成细胞外基质,在肾小球肾炎及肾小球硬化的研究及治疗中有潜在的应用价值。

Objective To study the effect of transforming growth factor beta 1(TGF-B1) antisense RNA on synthesis and secretion of extracellular matrix in mesangial cell. Methods The recombinant anlisense TGF-Bl RNA adenovirus was generated. The cultured mesangial cells were transfected by recombinant adenovirus as therapy group, and the mesangial cells were not transfected as control group. The mRNA of TGF-B1 and a1(IV) collagen was analysed by Northern blot, and immunohistochemislry straining was employed for the protein of TGF-Bl, fibronectin and a1 (IV)collagen in both groups of mesangial cells. Results Recombinant antisense TGF-Bl adenovirus was constructed. The mRNA of TGF-B1 and a1(IV) collagen in mesangial cells was repressed by recombinant antisense TGF-Bl adenovirus at 48 hours after transfection in comparison with the control group. The repressed ratio of mRNA in TGF-Bl and a1(IV) collagen was 22. 5% , 18. 2% respectively in mesangial cells at 48 hours after transfection, and 29. 5% , 27. 3% respectively at 72 hours after transfection in comparison with the control group. The protein of TGF-B1, fibronectin and a1(IV) collagen was inhibited, and repressed ratio was 16.5%, 18. 2% and 14. 6% respectively at 48 hours after transfection, and was 23. 5% , 27. 3% and 26. 8% respectively at 72 hours after transfection in comparison with the control group. Conclusion Recombinant antisense TGF-B1 adenovirus inhibits synthesis and secretion of extracellular matrix in mesangial cells which may be effective in gene therapy for proliferative glomerulonephritis and sclerosing glomerulonephritis.