论文部分内容阅读
结缔组织生长因子(CTGF)是一种新发现的促成纤维细胞分裂和胶原沉积的生长因子,在肝纤维化形成中的作用逐渐受到关注。RNA干扰技术是目前最有效的基因沉默技术,能特异性抑制靶基因的转录,进而下调相应蛋白水平及功能。本研究将能在体内自主合成短发夹状RNA(shRNA)的pEGFP-CTGF表达质粒转入大鼠肝星状细胞(HSC-T6),观察其对CTGF mRNA及蛋白表达的影响。一、材料与方法 1．主要试剂：pEGFP质粒购自武汉晶赛生物公司；脂质体转染试剂盒Metafectene购自德国Biothex公司；逆转录聚合酶链反应(RT-PCR)试剂盒购自日本TaKaRa公司；Trizol 试剂盒购自上海生工生物工程公司。小鼠单克隆β-肌动蛋白 (β-actin)第一抗体购自美国ABcam公司；羊抗CTGF多克隆抗体购自美国Santa Cruz公司；兔抗小鼠-辣根过氧化物酶
Connective tissue growth factor (CTGF) is a newly discovered growth factor that contributes to fibroblast division and collagen deposition, and its role in the formation of hepatic fibrosis has drawn increasing attention. RNA interference technology is currently the most effective gene silencing technology, can specifically inhibit the transcription of the target gene, and then down the corresponding protein levels and functions. In this study, the recombinant plasmid pEGFP-CTGF, which can synthesize short hairpin RNA (shRNA) in vivo, was transfected into rat hepatic stellate cells (HSC-T6) to observe its effect on CTGF mRNA and protein expression. First, materials and methods 1. The main reagents: pEGFP plasmid purchased from Wuhan Crystal 赛 bio-company; liposome transfection kit Metafectene purchased from Germany Biothex company; reverse transcription polymerase chain reaction (RT-PCR) kit purchased from Japan TaKaRa company; Trizol kit purchased Since Shanghai Biological Engineering Company. The mouse monoclonal β-actin first antibody was purchased from ABcam Company, USA; the goat anti-CTGF polyclonal antibody was purchased from Santa Cruz Biotechnology USA; the rabbit anti-mouse horseradish peroxidase