pcDU_6质粒载体介导的TGFβ_1shRNA及pcDNA3.1介导的TGF-β_1反义RNA抑制人腹膜间皮细胞TGFβ_1的表达及细胞外基质的分泌  

Inhibition of TGF-β_1 expression and extracellular matrix secretion in human peritoneal mesothelial cells by pcDU_6 vector mediated TGF-β_1 shRNA and by pcDNA3.1(-) mediated antisense TGF-β_1RNA

作　　者：刘伏友[1] 刘虹[1] 彭佑铭[1] 袁芳[2] 刘映红[2] 段绍斌[2] 

机构地区：[1]中南大学湘雅二院肾内科,长沙410011 [2]中南大学湘雅二院肾脏病研究所,长沙410011

出　　处：《中国血液净化》2004年第10期549-555,共7页Chinese Journal of Blood Purification

摘　　要：目的　研究转化生长因子 β1(TGF - β1)短发夹RNA(shorthairpinRNA ,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 β1shRNA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 .2 5 %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 .2 5 %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 .0 1) ,TGF - β1反义RNA转染HPMC后 ,与对照组相比 ,转染 2 4小时后 ,FN、ColⅠ、PAI- 1mR NA分别下调 17.0 %、2 6 .0 %、9.6 % (P <0 .0 5 )。pcDU6载体质粒介导的转化生长因子Objective To investigate the effect of shRNA of TGF-β_1 carried by a vector plas mid and antisense TGF-β_1 RNA no the TGF-β_1 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 pcDU_6 [pcDNA3.1(-) with U6 promoter] separately to form short hairpin RNA. The recombinant human TGF-β_1 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-β_1 and plasmid pcDNA 3.1(-) mediating the expression of antisense TGF-β_1RNA 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-β_1mRNA and FN,collagenⅠ(colⅠ)and PAI-1 mRNA. The TGF-β_1 level in the culture supernatant was measured with a sandwich enzyme-linked immunosorbent assay. Results The expression of TGF-β_1 was upregulated significantly in HPMC stimulated by 4.25% D-glucoseand LPS(P<0.01). The mRNA of FN, colⅠ,PAI-1 in HPMC were repressed by TGF-β_1 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-β_1RNA did not remarkably inhibit the expression of TGF-β_1 in HPMC compared with pcDU_6 group(P>0.05). PcDU6 vector plasmid mediated TGF-β_1 shRNAs significantly down-regulated the expression of TGF-β_1 in HPMC compared with the PcDNA3.1(-) plasmid mediated antisense TGF-β_1RNA(P<0.01). Conclusion PcDU6 vector plasmid mediated shRNA inhibit the expressin of TGF-β_1 in HPMC stimulated by 4.25%D-glucose and LPS. These results suggested the possible efficacy of pcDU_6 vector plasmid mediated shRNA

关 键 词：RNA干扰 转化生长因子-β1 人腹膜间皮细胞 反义RNA 

分 类 号：R446.6[医药卫生—诊断学]