siRNA干扰转化生长因子β_1表达对SD大鼠系膜细胞纤维连接蛋白的影响  

Effects of siRNAs silencing transforming growth factor-β_1 on fibronectin in SD rat masangial cells

作　　者：毛华雄[1] 易著文[1] 吴小川[1] 党西强[1] 何小解[1] 曹艳[1] 莫双红[1] 

机构地区：[1]中南大学湘雅二医院小儿肾脏病研究室,长沙410011

出　　处：《中华儿科杂志》2007年第7期494-497,共4页Chinese Journal of Pediatrics

基　　金：教育部高等学校博士学科点专项科研基金(20040533043)

摘　　要：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 fibronectin synthesis in SD rat mesangial cells after transforming growth factor-β1（ TGF-β1） is silenced by the short interfering RNA （ siRNA ） expressed by reconstructed pGEFP-C1 vectors. Methods Depending upon the 538^th-556^th （A） and 895^th-913^th （B） nucleotides of rat TGF-β1 gene, a nucleotide （A or B） was constructed into a small hairpin nucleotide which was separately （A or B ） or together （ A plus B） inserted into a pGEFP-C1 vector with three reconstructed pGEFP-C1 vectors separately expressing the siRNAs for A or/and B. TGF-β1 and fibronectin were dynamically investigated for their interrelationship by ELISA in the supernatant and RT-PCR in their extracted total RNA. Results The siRNA hairpin-like molecules were constructed according to the 538^th- 556^th nucleotides of rat TGF-β1 gene were able to markedly silence the expression of TGF-β1 mRNA （ P 〈 0. 01） and protein （P 〈0. 01） at 48 h. Lipfectamin^TM 2000 transfection stimulated the peak secretion of fibronectin at 24 h in the control and the experimental group whose TGF-β1 was not silenced, but the silence of TGF-β1 in both experimental groups delayed the top values of fibronectin to 48 h （ P 〈 0. 01 ）. Conclusion The silence of TGF-β1 by siRNA decreased the fibronectin expression, but the latter was possibly not completely TGF-deoendent.

关 键 词：RNA 小分子干扰 转化生长因子Β 肾小球膜 大鼠 SPRAGUE-DAWLEY 纤维连接蛋白类 

分 类 号：R692[医药卫生—泌尿科学]