CTGF shRNA对肝星状细胞细胞因子及细胞外基质表达的影响  

Effect of CTGF shRNA on expressions of TGF-β1,CTGF mRNA and extra-cellular matrix secretion in rat hepatic stellate cells

作　　者：主余华[1] 李肖红[2] 张春清[1] 任万华[1] 赵幼安[3] 马艳丽[1] 

机构地区：[1]山东大学山东省立医院消化内科,山东济南250021 [2]山东大学西校区校医院,山东济南250012 [3]山东大学齐鲁医院消化内科,山东济南250012

出　　处：《山东大学学报（医学版）》2007年第6期577-581,594,共6页Journal of Shandong University:Health Sciences

基　　金：山东省卫生厅青年基金(基金编号2005年第25号)

摘　　要：目的:研究结缔组织生长因子(CTGF)短发夹RNA(short hairpin RNA,shRNA)对肝星状细胞(HSC-T6)中TGF-β1、CTGF、Ⅰ型胶原、Ⅲ型胶原基因表达及细胞外基质分泌的影响。方法:将已构建并筛选有效含绿色荧光蛋白(EGFP)的CTGFshRNA质粒以转染试剂Metafectene转染HSC-T6,另设空质粒组、只加Metafectene组及空白对照组,24 h及48 h后,用RT-PCR法检测各组细胞中TGF-β1、CTGF、Ⅰ型胶原、Ⅲ型胶原mRNA的表达;放免法分析细胞上清液中Ⅲ型前胶原、Ⅳ型胶原、透明质酸和层粘连蛋白的含量;流式细胞仪分析转染效率。结果:CTGFshRNA质粒在肝星状细胞的转染效率24 h、48 h分别为(60±5)%(、42±3)%;与空白对照组相比,空质粒组、脂质体组对基因表达及细胞外基质的分泌均无影响,而CTGFshRNA能明显抑制CTGF、Ⅰ型胶原、Ⅲ型胶原mRNA的表达,降低上清液中Ⅲ型前胶原、Ⅳ型胶原、透明质酸和层粘连蛋白的含量(P<0.01或P<0.05),而对TGF-β1的基因表达则无影响。结论:CTGFshRNA可明显抑制肝星状细胞CTGF、Ⅰ型胶原、Ⅲ型胶原基因表达及细胞外基质的分泌。Objective： To investigate the effect of CTGF short hairpin RNA（shRNA） on the expression of TGF-β1, CTGF, type Ⅰ procollagen, type m procollagen mRNA and extracellular matrix secretion in HSC-T6. Methods： The CTGFshRNA plasmids containing EGFP successfully constructed and screened, were transfected to HSC-T6 with the transfection reagent Metafectene. There were another three HSC-T6 groups： one with plasmids and without CTGFshRNA, one with only Metafectene, and one untreated. The expression of TGF-β1, CTGF, type Ⅰ procollagen and type lit procollagen mRNA levels were determined by reverse transcription-polymerase chain reaction （RT-PCR） after 24 and 48 hours. The content of precollagen type Ⅲ, Ⅳ-type collagen, and hyaluronate （HA）, laminin （LN） in the supematants were determined by radioimmunoassay. Cell transfection efficiency was analyzed by flow cytometry. The tmnsfection efficiency of CTGFshRNA plasmids in HSC were （60 ± 5） %, （42 ± 3） % at 24 and 48 hours respectively.Compared with those of the blank control, there was no difference in the expression of genes and extracellular matrix secretion in HSC-T6 in the other groups, the group with plasmids and without CTGF shRNA and the group with only Metafectene. Expression of CTGF, type Ⅰ collagen and type m collagen mRNA were markedly down-regulated in CTGFshRNA transfected HSC-T6（P 〈0.01 or P 〈0.05）. But there was no difference in the expression of TGF-β1 mRNA. The contents of precollagen type Ⅲ, Ⅳ-type collagen, hyaluronate （HA） , and laminin （LN） in the supematants decreased significantly in CTGFshRNA transfected HSC-T6（ P 〈0.01 or P 〈0.05）. Conclusion： CTGFshRNA can regulate the gene expression of the growth factor and the extra-cellular matrix molecule and inhibits the extra-cellular matrix secretion in HSC-T6.

关 键 词：RNA干扰 短发夹RNA 转化生长因子Β 结缔组织生长因子 肝纤维化 

分 类 号：R503[医药卫生—内科学] R575.2[医药卫生—临床医学]