siRNA沉默结缔组织生长因子对鼠肝星状细胞生物学特性的影响  

Effects of silencing connective tissue growth factor by siRNA on biological characteristics of rat hepatic stellate cells

作　　者：李光明[1] 李定国[1] 谢青[2] 宗春华[1] 周慧娟[2] 姜山[2] 陆汉明[1] 

机构地区：[1]上海交通大学医学院新华医院消化内科,上海200092 [2]上海交通大学医学院瑞金医院感染科,上海200025

出　　处：《上海交通大学学报（医学版）》2008年第10期1258-1261,共4页Journal of Shanghai Jiao tong University:Medical Science

基　　金：上海交通大学医学博士点基金(BXJ0318)~~

摘　　要：目的研究化学合成的小干扰RNA(483 siRNA)对大鼠原代肝星状细胞(HSCs)结缔组织生长因子(CTGF)蛋白表达及细胞生物学特性的影响。方法以胶原酶原位灌注消化、密度梯度离心分离大鼠原代HSCs,以脂质体Oligofectamine包裹483siRNA转染培养72 h的原代HSCs作为转染组,并设空白对照组。收集孵育96 h的HSCs及培养上清液,采用Western blotting、免疫细胞化学染色、MTT和RT-PCR法分别检测细胞CTGF蛋白表达、α-SMA表达、细胞增殖和Ⅰ、Ⅲ型胶原mRNA表达;放射免疫法检测培养上清液中透明质酸及Ⅲ型前胶原含量。结果与对照组比较,转染组HSCs的CTGF蛋白表达下调(92±5)%,α-SMA染色阳性细胞减少(58±6)%,细胞增殖活性下调(18±5)%,Ⅰ、Ⅲ型胶原mRNA表达分别下调(53±6)%和(41±7)%;培养上清中透明质酸和Ⅲ型前胶原含量分别降低(48±5)%和(33±8)%。结论483 siRNA能显著下调原代HSCsCTGF蛋白表达,并由此抑制细胞活化、增殖及细胞外基质的合成和分泌。提示针对CTGF靶位的siRNA可能具有防治肝纤维化的潜力。Objective To investigate the effect of chemically synthetic small interfering RNA （483 siRNA） on the expression of connective tissue growth factor （CTGF） gene and the biological characteristics of rat hepatic stellate cells （HSCs）. Methods Primary HSCs were isolated from normal SD rats by portal vein collagenase in situ perfusion digestion plus density gradient centrifugation, siRNA targeting CTGF was transfected into primary HSCs which had been cultured for 72 h by Oligofectamine package （transfection group） , and untreated HSCs were served as control group. HSCs and culture supernatant were collected after incubation with siRNA for 96 h. The expression of CTGF and α-SMA genes in HSCs were detected by Western blotting and immunocytochemical staining, respectively. The cell proliferation was determined with MTT colorimetric assay. The expression of type Ⅰ and Ⅲ collagen mRNA was detected by RT-PCR. Contents of hyaluronic acid and type Ⅲ pro-collagen in supernatant were determined by radioimmunoassay. Results Compared with control group, the CTGF protein expression, the number of α-SMA positive cells, the activity of HSCs proliferation, the expression of type Ⅰ and Ⅲ collagen mRNA, and the contents of hyaluronic acid and type Ⅲ pro-collagen in supernatant were reduced in transfection group by （92±5）%, （58±6）%, （18±5）%, （53±6）%, （41±7）%, （48＋5）% and （33±8）%, respectively. Conclusion CTGF gene expression can be markedly down-regulated by 483 siRNA in primary HSCs, which results in the significant inhibition of HSC activation, proliferation and extracellular matrix production, siRNA targeting CTGF may be potential to prevent and treat liver fibrosis.

关 键 词：小干扰RNA 结缔组织生长因子 肝纤维化 肝星状细胞 

分 类 号：Q78[生物学—分子生物学] R575.2[医药卫生—消化系统]