反义转化生长因子βⅠ型受体表达质粒对大鼠肝星状细胞功能的影响  

Effects of antisense transforming growth factor β receptor-Ⅰ expressing plasmid on rat activated hepatic stellate cells in vitro

作　　者：蒋炜[1] 王吉耀[1] 杨长青[1] 刘文滨[1] 王逸青[1] 

机构地区：[1]复旦大学附属中山医院消化科,上海200032

出　　处：《肝脏》2004年第3期145-147,共3页Chinese Hepatology

摘　　要：目的　观察反义转化生长因子 (TGF) βⅠ型受体 (TβRⅠ )表达质粒对大鼠肝星状细胞 (HSC)增殖及细胞外基质分泌的影响。方法　双酶灌注和梯度离心法分离大鼠HSC ,将构建的反义TβRⅠ真核细胞表达质粒与 pcDNA3空质粒经脂质体转染培养活化的HSC ,通过RT PCR、Western印迹检测外源导入质粒在HSC中的表达 ,采用MTT法、3 H TdR掺入法检测细胞增殖情况 ,ELISA法检测TGF β1含量变化 ,并应用Western印迹检测HSC中Ⅰ、Ⅲ型胶原表达。结果　反义TβRⅠ真核细胞表达质粒可抑制活化HSC中TβRⅠmRNA及蛋白表达。与 pcDNA3转染组相比 ,反义TβRⅠ质粒表达可抑制HSC增殖 (P <0 .0 1) ,降低TGF β1含量 (P <0 .0 5 ) ,抑制Ⅰ、Ⅲ型胶原分泌 (P <0 .0 5 )。结论　重组反义TβRⅠ表达质粒可在HSC中获得较好表达 ,并可显著抑制HSC增殖及细胞外基质分泌。Objective To investigate the effect of antisense transforming growth factor(TGF) β receptor Ⅰ(TβRⅠ) expressing plasmid on proliferation and extracellular matrix secretion of rat hepatic stellate cell (HSC) in vitro.Methods HSC were isolated from normal rat liver by pronase and collagenase digestion and purified by centrifugal elutriation. The constructed antisense TβRⅠ expressing plasmid and the pcDNA3 empty plasmid were transfected in activated HSC by Effectene reagent separately. Cells were selected after growing in DMEM containing 400μg/ml G418 for 2 3 weeks. Expression of TβRⅠ in HSC was determined by RT PCR and Western blot. Cell proliferation was assessed by 3H TdR incorporation as well as MTT colorimetric assay. The content of TGF β 1 was also measured by ELISA and quantified collagen types Ⅰ and Ⅲ in HSC by Western blot.Results The exogenous antisense TβRⅠ recombinant plasmid could block the mRNA and protein expression of TβRⅠ significantly in vitro. Its expression could inhibit the proliferation of HSC ( P <0.01 ) and reduced the contents of TGF β 1 ( P <0.05 ). Its expression could also markedly inhibit the secretion of collagens tyep Ⅰ and Ⅲ of HSC ( P <0.05 ). Conclusion The results demonstrate that the antisense TβRⅠ recombinant plasmid could express well in HSC. It could inhibit the proliferation of HSC and thus inhibit the secretion of collagens type Ⅰ and Ⅲ of HSC.

关 键 词：HSC 反义 大鼠 Ⅰ型受体 鼠肝 肝星状细胞 真核细胞表达质粒 增殖 分泌 Ⅲ型胶原 

分 类 号：R575.2[医药卫生—消化系统]