重组2型腺相关病毒-转化生长因子β3对实验性肝纤维化大鼠肝脏组织病理学和Ⅰ型胶原表达的影响

Effects of transforming growth factor beta 3 on the histopathology and expression of collagen Ⅰ in experimental hepatic fibrotic rats

目的通过重组2型腺相关病毒（rAAV2）和基因转导，探讨转化生长因子（TGF）β3对大鼠肝纤维化的影响。方法构建并验证rAAV2-TGFβ3。实验大鼠随机分为正常对照组、模型组、阴性对照组和TGFβ3干预组。40％的四氯化碳复制大鼠肝纤维化模型。在四氯化碳诱导前1周，将rAAV2-TGFβ3注入大鼠尾静脉，造模8周后处死大鼠，取肝脏组织做HE染色观察肝脏组织学改变，Masson染色观察胶原纤维分布，免疫组织化学染色观察Ⅰ型胶原的表达，对胶原纤维的阳性面积比和Ⅰ型胶原的平均吸光度值进行半定量分析。多组数据间比较采用单因素的方差分析，若各组总体方差齐同，采用SNK—q检验，若方差不齐，采用Dunnett’ST3检验。结果HE染色结果显示，rAAV2TGFp3干预后，肝组织纤维间隔增生减少；Masson染色结果显示，大鼠胶原纤维主要分布在血管和汇管区及狄氏间隙，TGFB3干预组大鼠肝内的胶原纤维阳性面积比为7．7％±1．5％，明显低于模型组的13．2％±2．2％和阴性对照组的12．3％±1．5％（q值分别为9．456和8．217，P值均〈0．01）。免疫组织化学染色结果显示，Ⅰ型胶原主要表达在血管和汇管区及狄氏间隙，TGFβ3干预组大鼠肝内的Ⅰ型胶原的表达为0．185±0．033，明显低于模型对照组的0．2524-0．042和阴性对照组的0．230±0．029（q值分别为6．228和4．346，P值均〈0．01）。结论TGFβ3可明显减轻实验大鼠的肝纤维化程度和组织学损伤，降低Ⅰ型胶原的表达。

Objective To investgate the effects of TGFβ3 on rat hepatic fibrosis. Methods The TGFβ3 cDNA was cloned into rAAV2 vector. Rats were randomly divided into four groups： normal control group, model group, negative control group and TGF β3 group. Hepatic fibrosis was induced by hypodermic injection of 40% CCh. Recombinant AAV2-TGF β3 viral particles were injected via the vena caudalis one week before CC14 treatment. Rats were executed 8 weeks after CCI4 treatment, global histological change was observed after HE staining, the distribution of the collagen fibers was observed after masson staining, histochemistry was done to observe the expression of collagen Ⅰ; The positive area rate of the collagen fibers and the average optical rate of collagen Ⅰ were quantified. Results HE staining indicated that collagen fibers were reduced in the TGF β 3 group. Masson staining shown that the collagen fibers were distributed around the blood vessel, in the portal area and disse space. Compared to the model group （13.2%± 2.2%） and negative control group （12.3% ± 1.5%）, the collagen fibers in liver tissues of TGF β3 group （7.7% ± 1.5%） were significantly decreased （q = 9.456, P 〈 0.01; q = 8.217, P 〈 0.01）. Histochemistry indicated that the collagen fibers of liver tissues of TGF β3 group （0.185 ± 0.033） were significantly higher than those in the model group （0.252 ± 0.042） and the negative control group （0.230 ± 0.029）, （q = 6.228, P〈 0.01; q = 4.346, P 〈 0.01）. Conclusion TGF β 3 alleviates the damage to hepatic cell and the level of fibrosis in CCl4 treated rats and inhibits the expression of collagen Ⅰ.