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目的利用能够表达大鼠转化生长因子β_1(TGF-β_1)siRNA 的绿色荧光蛋白融合表达质粒载体(pGEFP-C_1)转染 SD 大鼠系膜细胞系、干扰 TGF-β_1的表达,观察系膜细胞纤维连接蛋白(FN)的相应变化,探讨防治肾脏纤维化的新途径。方法根据大鼠 TGF-β_1基因序列第538-556(A)和895-913(B)核苷酸序列,将 A、B 二段序列分别构建成小发夹结构,分别(A/B)或共同(A+B)插入一个 pGEFP-C_1载体中,得到3个重组载体,能够分别或同时表达针对 A 或(和)B 序列的 siRNA,分别转染系膜细胞后,通过 ELISA 和 RT-PCR 动态观察系膜细胞 TGF-β_1、FN 的 mRNA 和细胞培养上清中蛋白浓度的变化,探讨二者变化的相互关系。结果载体表达的 siRNA 与 TGF-β_1基因第538-556序列相同者,能够显著干扰48 h 时间段 TGF-β_1 mRNA(RT-PCR)(P<0.01)和蛋白分泌(细胞上清ELISA)(P<0.01),脂质体转染刺激系膜细胞 FN 分泌显著增加,表现为对照组和 TGF-β_1未被有效干扰组,FN 分泌于24 h 时段达到峰值,而 TGF-β_1被干扰的实验组,FN 峰值被推迟到48 h 才出现(P<0.01)。结论 siRNA 干扰系膜细胞 TGF-β_1的表达,能够减少 FN 的表达,但 FN 的表达可能并不完全依赖于 TGF-β_1。
OBJECTIVE: To investigate the effect of transfection of TGF-β1-siRNA on green fluorescent protein fusion plasmid pGEFP-C_1 in SD rat mesangial cells and to investigate the effect of mesangial cells Fibronectin (FN) corresponding changes in the prevention and treatment of renal fibrosis, a new way. Methods According to the nucleotide sequences of 538-556 (A) and 895-913 (B) of rat TGF-β 1 gene sequence, the A and B sequences were constructed into small hairpin structures respectively (A + B) were inserted into a pGEFP-C_1 vector to obtain three recombinant vectors capable of expressing siRNAs directed against A or (and) B sequences respectively or simultaneously. After transfection of mesangial cells respectively and by ELISA and RT-PCR Dynamic observation of mesangial cells TGF-β 1, FN mRNA and cell culture supernatant protein concentration changes, to explore the relationship between the changes. Results The siRNA of TGF-β 1 gene (538-556) and siRNA of TGF-β 1 gene could significantly interfere with the expression of TGF-β 1 mRNA (P <0.01) and the secretion of cell supernatant <0.01). The FN secretion of mesangial cells stimulated by liposome transfection was significantly increased, showing that the control group and TGF-β 1 were not effectively interfered, FN secretion peaked at 24 h, while the TGF-β 1 interfered group , FN peak was delayed until 48 h (P <0.01). CONCLUSION: siRNA interfered with the expression of TGF-β 1 in mesangial cells and decreased the expression of FN, but the expression of FN may not be completely dependent on TGF-β 1.