摘要
目的研究结缔组织生长因子（CTGF）和基质金属蛋白酶组织抑制因子-1（TIMP-1）的短发夹RNA（shRNA）表达质粒分别及共转染肝星状细胞（Hsc）对CTGF、TIMP-1、I型前胶原（PCI）mRNA表达及细胞外基质（ECM）分泌的影响。方法筛选并成功构建靶向大鼠CTGF和T1MP-1有效RNA干扰靶位的shRNA表达质粒，分别及共转染转化生长因子B1刺激活化的大鼠HSC-T6细胞，荧光定量PCR检测各组细胞中CTGF、TIMP-1、PCImRNA的表达；放射免疫法分析细胞上清液中Ⅲ型前胶原（PCⅢ）、透明质酸（HA）和层黏连蛋白（LN）的含量。组间比较采取方差分析；多组间两两比较采用SNK-q检验。结果CTGFshRNA转染和双质粒共转染HSC-T6的CTGFmRNA相对表达量分别为0．59±0．03、0．62±0．01，与空白对照组、CTGFshRNA转染组的1和1．00±0．07比较，CTGFmRNA表达量均明显下降，F=66．515，P值均〈0．05，差异有统计学意义；而TIMP-1shRNA组和双质粒共转染组的TIMP-1mRNA相对表达量分别为0．66±0．04、0．68±0．03，与空白对照组、CTGFshRNA转染组的1和1．05±0．03比较，TIMP-1mRNA表达量均明显下降，F=83．835，P值均〈0．05，差异有统计学意义;CTGF shRNA转染、TIMP-1shRNA转染和共转染组的PCImRNA相对表达量分别为0．55±0．02、0．57±0．02和0．41±0．01与空白对照组的1比较，PCI mRNA表达量均明显下降，F=709．905，P值均〈0．05，差异有统计学意义,双质粒联合转染对CTGF、TIMP-1、PCI的mRNA表达量抑制作用优于单质粒转染。CTGFshRNA转染、CTGFshRNA转染和双质粒共转染组的PCⅢ含量分别为（78．02±6．50）ng／ml、（79．03±5．47）ng／ml、（53．91±4．01）ng／ml，与空白对照组的（113．79±15．88）比较，PCⅢ含量均明显下降，P值均〈0．05，差异有统计学意义；CTGFshRNA转染、CTGFshRNA转染和双质粒共转染组的HA含量分别为（127．36±8．33）ng／ml、（116．55±3．37）ng／ml、（82．84±9．03）ng／ml，与空白对照组的（163．10±7．01）ng／ml比较，HA含量均明显下降，p值均〈0．05，差异有统计学意义CTGFshRNA转染、CTGFshRNA转染和双质粒共转染组的LN含量分别为（55．41±9．37）ng／ml、（49．77±6．70）ng／ml、（30．72±1．22）ng／ml，与空白对照组的（83．99±4．67）ng／ml比较，LN含量均明显下降，P值均〈0．05，差异有统计学意义。CTGF shRNA与TIMP-1shRNA双质粒联合转染对PCⅢ、HA、HA分泌的抑制作用优于单质粒转染。结论CTGFshRNA、TIMP-1 shRNA可明显抑制HSC的CTGF、TIMP-1、PCI基因表达及ECM的分泌，且shRNA联合干扰效果更显著，有望成为抗肝纤维化基因治疗的有效方法。
Objective To investigate the effect of short hairpin RNA （shRNA）-mediated silencing of CTGF and TIMP-1 in hepatic stellate cells （HSCs） on mRNA expression of TIMP-1, CTGF, and procollagen type-I （PC I）, as well as secretion of extracellular matrix （ECM） proteins. Methods Two recombinant expression plasmids harboring shRNAs against CTGF and TIMP-1 （psiRNA-GFP-CTGF and psiRNA-GFP-TIMP-1） were transfected alone or together into TGFβ1-activated HSC-T6 cells. The mRNA expression levels of CTGF, TIMP- 1, and PC I were detected by fluorescence quantitative PCR （FQ-PCR）. The concentrations of secreted PC type-III, hyaluronate （HA）, and laminin （LN） were measured by radioimmunoassay （RIA） of culture supematants. Results FQ-PCR analysis showed that CTGFshRNA and TIMP-lshRNA specifically inhibited the expression of CTGF, TIMP-1, and PC I mRNA in activated HSC-T6 cells. The concentrations of secreted PC III, HA, and LN were decreased significantly in HSC-T6 cells with shRNA-silenced CTGF or TIMP-1 （P〈0.01 orP〈0.05）. Moreover, HSC-T6 cells with shRNA-silenced CTGF and TIMP-1 showed a more robust decrease in synthesis of PC Ⅲ, HA and LN （all, P〈 0.01）, as well as in mRNA expression of PC I （P〈 0.05）. Conclusion CTGFshRNA and TIMP- 1 shRNA effectively inhibit expression of the respective target genes, as well as of PC I, and decrease secretion of ECM components from HSC-T6 cells. Silencing of both CTGF and TIMP-1 produces more robust effects than either in isolation. These data suggest that CTGF and TIMP-1 may be effective targets of shRNA-based gene therapy to treat liver fibrosis.
出处
《中华肝脏病杂志》
CAS
CSCD
北大核心
2012年第8期576-580,共5页
Chinese Journal of Hepatology
基金
四川省卫生厅科技项目（080197）
四川省重点学科项目（SZD0421）
