摘要
目的研究外源Smad7基因对原代肝星状细胞(HSC)活性和基因表达调控的影响。方法采用胶原酶原位灌注、密度梯度离心法分离SD大鼠原代HSC,并予转化生长因子β1(TGFβ1)刺激,同时分别以重组腺病毒AdSmad7和对照病毒AdGFP感染原代HSC,RTPCR法检测TGFβ1、Smad3mRNA、Smad7mRNA在HSC中的表达,免疫细胞化学法检测HSC中α平滑肌肌动蛋白(αSMA)和Smad7表达。结果RTPCR显示,与TGFβ1对照组比较,AdSmad7组Smad7mRNA表达显著上调(P<0.05),但TGFβ1和Smad3mRNA表达差异无统计学意义。免疫细胞化学染色显示,与其他各组比较,AdSmad7组HSC胞质中Smad7表达最高,αSMA表达则明显降低(P<0.01)。结论重组复制缺陷型腺病毒AdSmad7可在HSC中高效表达;外源Smad7基因可阻断TGFβ1对HSC的活化作用,但对Smad3和TGFβ1mRNA表达无明显影响。
Objective To investigate the effect of Smad7 gene on the activation of rat hepatic stellate cells （HSCs） in primary culture and on the regulation on gene expression of HSCs. Methods HSCs were isolated from male SD rats by collagenase perfusion of liver from portal vein and by 8.2% Nycodenz gradient eentrifugation, and thereby transfeeted with AdSmad7 and AdGFP （control） respectively. The mRNA expression of transforming growth factor （TGF） β1 , Smad3 and Smad7 were measured by RTPCR. Meanwhile, Smad7 and α-smooth muscle actin （α-SMA） expressions were detected by immunocytochemistry. Results The expression of Smad7 mRNA in AdSmad7 group increased remarkably compared with TGFβ1 control group, while the expression of Smad3 and TGFβ1 mRNA remains unchanged. The expression of Smad7 protein was significantly higher in AdSmad7 group than that in other three groups. Accordingly, the expression of α-SMA protein in the group of HSCs transfected with AdSmad7 was the lowest （P 〈0.0]）. Conclusions The replication-deficient recombinant adenovirus encoding Smad7 cDNA could be expressed with high efficiency in HSCs. Exogenous Smad7 gene delivered into HSCs by AdSmad7 could inhibit the activation of HSCs, but have no effect on the expression of Smad3 and TGFβ1 mRNA in HSCs.
出处
《中华消化杂志》
CAS
CSCD
北大核心
2005年第7期409-412,共4页
Chinese Journal of Digestion
基金
国家自然科学基金资助项目(30170412)
上海市科技发展基金资助项目(03ZR14050)
