pcDU_6质粒载体介导的TGFβ_1 shRNA抑制TGFβ_1在人腹膜间皮细胞中的表达

Inhibition of TGFβ_1 expression in human peritoneal mesothelial cells by pcDU_6 vector mediated TGFβ_1 shRNA

目的 :研究pcDU6质粒载体介导的转化生长因子β1短发夹RNA(shorthairpinRNA ,shRNA)对人腹膜间皮细胞 (HPMCs)转化生长因子 β1(TGFβ1)表达的影响。方法 :针对TGFβ1的以GGCC开始的 2 1碱基大小的片段 ,分别设计 2对寡核苷酸 ,形成双链后将其依次连入带有U6启动子的 pcDNA3.1( )载体 (命名为 pcDU6) ,2对DNA双链通过BamHI酶切位点连接后形成中间由 6碱基序列间隔的反向互补序列 ,构建成TGFβ1短发夹RNA的产生质粒。采用胰蛋白酶消化法从人腹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TGFβ1RNA的pcDNA3.1( )的载体质粒入 4 .2 5 %D 葡萄糖和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TGFβ1mRNA的表达。采用双抗夹心法酶联免疫吸附实验检测HPMC培养液中TGFβ1蛋白质水平。结果 :HPMC在 4 .2 5 %D 葡萄糖和LPS的刺激下可明显上调TGFβ1的表达 (P <0 .0 1)。pcDU6载体质粒介导的转化生长因子 β1shRNA干扰组较 pc DU6空载体组明显下调TGFβ1的表达 (P <0 .0 1)。pcDU6载体质粒介导的转化生长因子 β1shRNA干扰组间比较 ,差异无显著性 (P >0 .0 5 ) ;pcDNA3.1( )的载体质粒介导的反义RNA组与

Objective To investigate the influence of vector plasmid containing the shRNA of TGFβ 1 on the TGFβ 1 expression in human peritoneal mesotnelial cells. the TGFβ 1 expression in human peritoneal mesothelial cells(HPMCs) by a vector plasmid containing the shRNA of TGFβ 1. Methods The 2 selected fragments of coding sequence contained 21 nts,starting with ggcc.Two pairs of oligos were designed for these two fragments. After annealing double stranded DNA formed,they were ligated to plasmid pcDU 6(pcDNA3.1(-) with U 6 promoter) separately.The inverted motif contained 6 spacer and 4 Ts,which made it possible to form short hairpin RNA. Human peritoneal mesothelial cells were isolated from human omental specimens by trypsin disaggregation to establish a stable culture model. Plasmid pcDU 6 mediating the expression of TGFβ 1 and plasmid pcDNA3.1(-) mediating the expression of antisense TGFβ 1 RNA were transfected into the third passage HPMCs stimulated by 4.25% D-glucose and lipopolysaccharide(LPS,10 μg/ml) by lipofectamine 2000.The semi-quantification reverse transcriptive PCR (RT-PCR) was performed to detect the expression of TGFβ 1 mRNA. The TGFβ 1 level in the culture supernatant was measured with a sandwich enzyme-linked immunosorbent assay.Results The expression of TGFβ 1 was upregulated significantly in HPMCs stimulated by 4.25% D-glucose and LPS(P< 0.01).Both RT-PCR and ELISA showed that pcDU 6 vector plasmid mediated TGFβ 1 shRNAs inhibited the expression of TGFβ 1 in HPMCs significantly (P<0.01).pcDNA3.1(-) plasmid mediated antisense TGFβ 1 RNA did not remarkably inhibit the expression of TGFβ 1 in HPMCs compared with the pcDU 6 group(P>0.05).pcDU 6 vector plasmid mediated TGFβ 1 shRNAs may significantly down-regulated the expression of TGFβ 1 in HPMCs compared with the pcDNA3.1(-) plasmid mediated antisense TGFβ 1RNA(P< 0.01).There was no difference in the expression of TGFβ 1 between the 2 pcDU 6 vector plasmid mediated TGFβ 1 shRNAs (P> 0.05). Conclusion pcDU 6 vector plasmid mediated shRNAs may inhibit the expressin of TGFβ 1 in HPMCs stimulated by 4.25% D-glucose and LPS,suggesting the possible efficacy of pcDU 6 vector plasmid mediated shRNA in preventing peritoneal fibrosis in patients receiving peritoneal dialysis.