AdIL-10对肝星状细胞α-SMA、TGF-β1及TNF-α表达的影响

The effect of AdIL-10 on the expression of α-SMA, TGF-β1 and TNF-α in HSC

目的应用携带大鼠白介素-10(IL-10)基因的重组缺陷型腺病毒(AdIL1-0)感染原代培养的大鼠肝星状细胞(HSC),通过体外实验观察外源性大鼠IL-10基因对HSC中αSMA、TGF-β1和TNFα表达的影响。方法以胶原酶原位灌注、密度梯度离心法分离大鼠原代HSC,并用AdIL10感染HSC(同时设AdGFP对照组及正常对照组),分别采用RTPCR法和WesternBlot法检测感染后HSC中IL10mRNA及蛋白水平的表达;WesternBlot法检测HSC中转化生长因子β1(TGF-β1)及肿瘤坏死因子α(TNFα)的表达;免疫组织化学法检测αSMA在HSC中的表达。运用图像处理系统对结果加以分析。结果感染AdIL10的HSC中IL10的mRNA及蛋白水平均较对照组上调(P<0.001);而其表达的TGFβ1、TNFα及αSMA与对照组相比明显降低(P均<0.001);正常对照组和AdGFP对照组中上述指标的表达均无明显差异(P均>0.05)。结论外源性大鼠IL10基因导入大鼠HSC后可能通过影响TGFβ1及TNFα的表达而抑制HSC的活化。

Objective To investigate the effect of recombinant nonreplicating adenovirus bearing the IL-10 gene （AdIL-10） on the expression of α-SMA, TGF-β1 and TNF-α in primary culture rat hepatic stellate cell （HSC） in vitro.Methods Isolation of rat HSC was achieved by using collagenase in situ perfusion and density gradient centrifugation. After the HSCs were infected with AdIL-10 （with AdGFP group and normal group setting up as control groups）, the mRNA and protein of IL-10 was analyzed by RT-PCR and Western Blot respectively. The expression of TGF-β1 and TNF-α protein was performed using Western-Blot and that of α-SMA was detected by immunohistochemical techniques. All the results were evaluated by image analysis. Results IL-10 mRNA expression and protein levels in HSCs infected with AdIL-10 were significantly higher than those of both AdGFP group and normal group （all P 〈 0.001 ）, while α-SMA, TGF-β1 and TNF-α protein levels were greatly lower in this group compared with those in both AdGFP group and normal group （all P 〈 0.001 ） ; there is no significant difference between the expression of IL-10 mRNA and IL-10, α-SMA, TGF-β1 and TNF-α protein levels in HSCs of the two control groups （all P 〉 0. 05）. Conclusion Exogenous rat interleukin-10 gene transfer into rat HSCs may impact the expression of TGF-β1 and TNF-α, which may lead to suppression of activation of HSCs.