摘要
目的:研究结缔组织生长因子-5(CCN5)与肝星状细胞活化的关系及其作用机制。方法:以人肝星状细胞系LX-2为研究对象,以转化生长因子-β1(TGF-β1)刺激LX-2细胞,Western blot测定CCN5及CCN2表达变化;构建CCN5过表达载体,转染人肝星状细胞LX-2,使CCN5在LX-2中过表达;采用RT-PCR及Western blot测定α-平滑肌肌动蛋白(α-SMA)与Ⅰ型胶原表达变化情况;为进一步研究其作用机制,采用RT-PCR及Western blot对Smad2表达及Smad2磷酸化水平进行测定。结果:正常情况下,LX-2细胞中CCN2表达量远高于CCN5,TGF-β1刺激后CCN2明显增高,而CCN5无变化;与正常对照组和空载体组相比,转染组CCN5在LX-2中成功过表达后,α-SMA及Ⅰ型胶原表达量显著下降(P<0.01);Smad2磷酸化水平显著下降(P<0.01)。结论:CCN5具有抑制肝星状细胞活化的作用,与CCN家族另一重要分子CCN2的促纤维化作用截然相反,为肝纤维化的防治提供新的思路。
Objective： To investigate the relationship between connective tissue growth factor （CCNS） and hepatic stellate cell （HSC） activation as well as the mechanism of action. Methods： As the research object, LX-2 cells were stimulated with transforming growth factor-β1 （TGF-β1 ）, and the protein expression levels of CCN5 and CCN2 were determined by Western blot; Hepatocyte high expression system of CCN5 was constructed and transfected hepatic stellate cells （HSC） to make CCN5 overexpression; The expression levels of α-smooth muscle actin （α-SMA） and collagen I were determined by RT-PCR and Western blot. To further study its mechanism of action, Smad2 and phosphorylation level of Smad2 were determined by RT-PCR and Western blot. Results： Under normal circumstances, CCN2 expression levels were much higher than CCN5 in LX-2 cells, while CCN2 expression was significantly higher than CCN5 if LX-2 cells were stimulated by TGF-β1. However, there was no change for CCNS. Compared with the control group and the vector group, CCN5 was successfully overexpressed in the transfection group, and mRNA and protein levels of α-SMA and collagen Ⅰ were significantly decreased （P 〈 0.01）. Meanwhile, phosphorylafion level of Smad2 was also significantly decreased （ P 〈 0.01 ）. Conclusion： CCNS, which has the function that inhibits HSC activation, has the oppositerole compared with CCN2, therefore, a new idea was proposed for the prevention and treatment of liver fibrosis.
出处
《中国应用生理学杂志》
CAS
CSCD
2013年第5期411-415,共5页
Chinese Journal of Applied Physiology
