HepG2细胞表达蛋白HCVC体外活化肝星状细胞的研究  

A study on the activation of hepatic stellate cells in vitro by HepG2 cells expressing HCV core protein

作　　者：孙英梅 李文杰 陶剑平[2] 

机构地区：[1]青岛市妇女儿童医院新筛实验室,山东青岛266034 [2]中山大学中山医学院,广东广州510080

出　　处：《中国病原生物学杂志》2013年第10期869-873,877,共6页Journal of Pathogen Biology

基　　金：广东省科技计划项目(No.2007B030702015)

摘　　要：目的观察HCV核心蛋白对肝星状细胞活化及合成细胞外基质的影响,为进一步研究HCV核心蛋白的致肝纤维化机制提供实验依据。方法采用双抗体夹心ELISA检测HepG2-HCV-C细胞株培养液中TGF-β1的表达。用HepG2-HCV-C和HepG2两株细胞培养5d的上清液培养人肝星状细胞系LX-2细胞,同时用DMEM培养LX-2细胞作对照,分别制备细胞爬片,采用免疫细胞化学染色(ICC)法检测LX-2细胞中人α-平滑肌肌动蛋白(α-SMA)、Ⅳ型胶原(ColⅣ)、结缔组织生长因子(CTGF)和纤维连接蛋白(FN)的表达;采用双抗体夹心ELISA检测LX-2细胞1~6d培养上清液中人ColⅣ、Ⅲ型前胶原肽(PⅢNP)、透明质酸(HA)和人层粘连蛋白(LN)的表达。所有数据采用SPSS11.0统计软件进行统计分析。结果双抗体夹心ELISA检测HepG2-HCV-C细胞1~6d培养液中的TGF-β1量为(110.00±11.45)pg/ml^(935.00±21.36)pg/ml,HepG2细胞为0^(124.16±11.81)pg/ml,差异有统计学意义(F=21984.81,P<0.01)。ICC检测用HepG2-HCV-C上清液培养的LX-2细胞α-SMA、ColⅣ、CTGF和FN均为弥漫阳性,细胞染成深褐色;用HepG2培养的LX-2细胞呈局灶性棕黄色,DMEM培养的LX-2细胞染色阴性。ELISA检测LX-2(HepG2-HCV-C)组ColⅣ、HA、LN和PⅢNP的值均显著高于LX-2(HepG2)组和LX-2(DMEM)组(均P<0.05或P<0.01)。结论 HCV C蛋白能上调TGF-βl的表达,表达C蛋白的细胞系能促进LX-2细胞多种细胞外基质(ECM)的合成。提示HCV C蛋白可能通过活化肝星状细胞,参与肝纤维化形成中多种ECM合成的调控。Objectives To observe the impact of HCV core protein on the activation of hepatic stellate cells （HSCs） and synthesis of extracellular matrix （ECM） in order to provide an experimental basis for further study of the mechanism of fi- brogenesis induced by HCV core protein. Methods The expression of TGF ~tl by cultured HepG2-HCV C and HepG2 cells was determined using double antibody sandwich ELISA. In addition, the supernatant of the two cell lines was used to cultivate LX-2 cells. LX 2 cells cultured with DMEM served as a control. Immunocytochemistry （ICC） was used to de- tect the expression of a SMA, CoIN, CTGF, and FN in LX-2 cells cultured from the two cells lines and control LX-2 cells cultured with DMEM. Double antibody sandwich ELISA was used to detect the expression of CollV, PllI NP, HA, and LN in supernatant. All data were analyzed using the statistical software SPSSll. 0. Results The concentration of TGF-I^I in the HepG2-HCV C culture was （110 ~ 11.45） pg/ml --（935 ~ 21.36） pg/ml and that in HepG2 culture was 0-- （ 124.16 ~ 11.81） pg/ml. The difference in concentration was statistically significant （F= 21984.81, P^0.01）. ICC results were strongly positive for LX-2 smears cultured with the supernatant from HepG2 HCV C cells, and cells were stained with anti- a SMA, anti-CollV, anti-CTGF, and anti-FN antibodies. However, ICC results were weakly positive for LX-2 cells cultured with the supernatant from HepG2 cells and those results were negative for LX-2 ceils cultured with DMEM. In addition, LX-2 （HepG2-HCV C） cells had a markedly higher level of CollV, HA, LN, and Pl]I NP com- pared to the I.X-2 （HepG2） cells and LX-2 （DMEM） cells （differences in levels were all P^0.05 or P^0.01）. Conclu- sion HCV C protein can up regulate the expression of TGF-I？I. A cell line expressing HCV C protein can promote a vari- ety of ECM synthesis in LX-2 cells. The current results suggested that C protein may participate in control of ECM syn- thesis in hepatic fibrosis by stimula

关 键 词：丙型肝炎病毒 核心蛋白 HEPG2细胞 LX-2细胞 活化 

分 类 号：R373.21[医药卫生—病原生物学]