摘要
目的研究G9PAMAM纳米载体介导的结缔组织生长因子(CTGF)短发夹RNA(shRNA)质粒(pCTGF-shRNA)对小鼠腹膜间皮细胞CTGF表达的影响。方法设计并构建质粒pCTGF1-shRNA、pCTGF2-shRNA和阴性对照pHK-shRNA;应用G9PAMAM将质粒分别转染4.25%葡萄糖刺激下的第3代小鼠腹膜间皮细胞;采用逆转录多聚酶链式反应(RT-PCR)检测细胞CTGFmRNA水平,采用WesternBlot检测CTGF蛋白含量。结果4.25%葡萄糖可刺激小鼠腹膜间皮细胞CTGF的mRNA和蛋白表达明显上调。pCTGF1-shRNA转染组和pCTGF2-shRNA转染组的CTGF表达较高糖组明显下调(P<0.05),尤其以pCTGF2-shRNA组显著。pHK-shRNA转染组与高糖组比较,差异无显著性(P>0.05)。结论G9PAMAM纳米载体可介导pCTGF-shRNA转染原代小鼠腹膜间皮细胞,并能抑制高糖诱导的CTGF表达上调,提示PAMAM介导的pCTGF-shRNA可有效地用于腹膜纤维化的基因治疗。
Objectives To investigate nano-carrier G9 PAMAM mediated plasmid containing CTGF-shRNA （pCTGF-shRNA） transfection on connective tissue growth factor （CTGF） expression in mouse peritoneal mesothelial cells （MPMCs）. Methods We first designed and constructed the plasmids pCTGFI-shRNA, pCTGF2-shRNA and the negative control pHK-shRNA. We transfected MPMCs （the 3rd generation and induced by 4.25% glucose） with the plasmids using G9 PAMAM. CTGF was determined by western blot, and its mRNA was detected by RT-PCR. Results CTGF and its mRNA in MPMCs were significantly up-regulated by the stimulation of 4.25% glucose. Introduction of pCTGFI-shRNA and pCTGF2- shRNA to MPMCs resulted in significant reduction of CTGF expression （P 〈 0.05）, and the reduction was more remarkable in MPMCs transfected with pCTGF2-shRNA. In contrast, transfection of pHK-shRNA showed no significant reduction in CTGF expression （P 〉 0.05）. Conclusions Nano-carrier G9 PAMAM efficiently introduces pCTGF-shRNA plasmids into primary MPMCs, by which the increased CTGF expression induced by high glucose can be inhibited. PAMAM mediated pCTGF-shRNA transfection may be used as an efficient method for gene therapy of peritoneal fibrosis.
出处
《中国血液净化》
2008年第4期203-206,共4页
Chinese Journal of Blood Purification
