中国血液净化

›› 2004, Vol. 3 ›› Issue (10): 549-555.

• 基础研究 • 上一篇    下一篇

pcDU6 质粒载体介导的TGFβ1shRNA及pcDNA3.1介导的TGF-β1反义RNA抑制人腹膜间皮细胞TGFβ1的表达及细胞外基质的分泌

刘伏友 刘 虹 彭佑铭 袁 芳 刘映红 段绍斌   

  1. 410011 长沙， 中南大学湘雅二院肾内科(刘伏友，刘虹，彭佑铭)；肾脏病研究所(袁芳，刘映红，段绍斌) 
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2004-10-12 发布日期:2004-10-12

  • Received:1900-01-01 Revised:1900-01-01 Online:2004-10-12 Published:2004-10-12

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摘要： 目的 研究转化生长因子β1（TGF-β1）短发夹RNA（short hairpin RNA，shRNA）和TGF-β1反义RNA 对体外培养的人腹膜间皮细胞（HPMC）TGF-β1表达及细胞外基质分泌的影响。方法 利用带有U6启动子的pcDNA3.1(-)载体（命名为pcDU6）构建TGF-β1短发夹RNA的产生质粒，用pcDNA3.1(-)载体构建反义TGF-β1基因真核表达载体，采用胰蛋白酶消化法从人大网膜组织中分离间皮细胞，建立稳定的体外培养模型。用脂质体转染表达转化生长因子β1 shRNA的pcDU6载体质粒和表达反义TGF-β1RNA的pcDNA3.1(-)的载体质粒入高糖（4.25%D-葡萄糖）和细菌脂多糖（LPS）刺激下人腹膜间皮细胞。采用逆转录多聚酶链式反应（RT-PCR）半定量分析HPMC中TGF-β1mRNA的表达以及纤维连接蛋白（FN）、Ⅰ型纤溶酶原激活物抑制剂（PAI-1）和胶原Ⅰ（ColⅠ）的mRNA表达。采用双抗夹心法酶联免疫吸附实验检测HPMC培养液中TGF-β1蛋白质水平。结果 HPMC在高糖（4.25% D-葡萄糖）和细菌脂多糖（LPS）的刺激下可明显上调TGF-β1的表达（P<0.01），TGF-β1反义RNA转染HPMC后，与对照组相比，转染24小时后，FN、ColⅠ、PAI-1 mRNA分别下调17.0％、26.0％、9.6％（P<0.05）。pcDU6载体质粒介导的转化生长因子β1 shRNA干扰组较Gs+LPS组及pcDU6空载体组明显下调TGF-β1的表达（P<0.01），pcDNA3.1 (-)的载体质粒介导的反义RNA组与pcDU6空载体组比较差异无显著性（P>0.05），pcDU6载体质粒介导的转化生长因子β1 shRNA干扰组较pcDNA3.1(-)的载体质粒介导的反义RNA组明显下调TGF-β1的表达（P<0.01）。结论 pcDU6 载体质粒介导的转化生长因子β1 shRNA能够明显抑制在高糖（4.25% D-葡萄糖）和细菌脂多糖（LPS）的刺激下的HPMC转化生长因子β1的基因表达，可能为临床防治长期腹膜透析患者的腹膜纤维化提供一种较为有效的方法。

关键词: RNA 干扰, 转化生长因子-β1, 人腹膜间皮细胞, 反义RNA

Abstract:

Objective To investigate the effect of shRNA of TGF-β1 carried by a vector plas mid and antisense TGF-β1 RNA no the TGF-β1 expression in human peritioneal mesothelial cells (HPMC) and extracellular matrix secretion. Methods Two pairs of oligos were designed for the 2 selected fragments. After annealing double stranded DNA formed,they were ligated to plasmid pcDU6［pcDNA3.1(-) with U6 promoter］separately to form short hairpin RNA. The recombinant human TGF-β1 antisense eukaryotic expression vector was generated. Human peritoneal mesothelial cells were isolated from human omental specimens by trypsin digestion toestablish a stable culture model. Plasmid pcDU6 mediating the expression of TGF-β1 and plasmid pcDNA 3.1(-) mediating the expression of antisense TGF-β1RNA were transfected into the third passage HPMC stimulated by 4.25% D-glucose and lipopolysaccharide(LPS,10(g/ml) by lipofectamine 2000. The semi-quantification reverse transcriptive PCR (RT-PCR) was performed to detect the expression of TGF-β1mRNA and FN,collagenⅠ(colⅠ)and PAI-1 mRNA. The TGF-β1 level in the culture supernatant was measured with a sandwich enzyme-linked immunosorbent assay. Results The expression of TGF-β1 was upregulated significantly in HPMC stimulated by 4.25% D-glucose and LPS（P<0.01）. The mRNA of FN, colⅠ,PAI-1 in HPMC were repressed by TGF-β1 antisense RNA at 24 hours after transfection in comparison with the control group, they decreased 17%, 26%, 9.6% respectively.PcDNA3.1(-) plasmid mediated antisense TGF-β1RNA did not remarkably inhibit the expression of TGF-β1 in HPMC compared with pcDU6 group(P>0.05). PcDU6 vector plasmid mediated TGF-β1 shRNAs significantly down-regulated the expression of TGF-β1 in HPMC compared with the PcDNA3.1(-) plasmid mediated antisense TGF-β1RNA （P<0.01）. Conclusion PcDU6 vector plasmid mediated shRNA inhibit the expressin of TGF-β1 in HPMC stimulated by 4.25%D-glucose and LPS. These results suggested the possible efficacy of pcDU6 vector plasmid mediated shRNA for preventing peritoneal fibrosis in patients receiving peritoneal dialysis.

Key words: Transforming growth factor-beta-1(TGF-β1), Human peritoneal mesothelial cell(HPMC), Antisense RNA 

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