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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。
Objective To observe the effect of HCV core protein on activation of hepatic stellate cells and synthesis of extracellular matrix, and to provide experimental evidence for further study of hepatic fibrosis mechanism of HCV core protein. Methods Double antibody sandwich ELISA was used to detect the expression of TGF-β1 in HepG2-HCV-C cell line. The human hepatic stellate cell line LX-2 cells were cultured with HepG2-HCV-C and HepG2 cells for 5 days. LX-2 cells were cultured with DMEM as control to prepare cell slide respectively. Immunocytochemistry The expression of α-smooth muscle actin (α-SMA), collagen Ⅳ (Col Ⅳ), connective tissue growth factor (CTGF) and fibronectin (FN) in LX- Antibody sandwich ELISA was used to detect the expression of human col Ⅳ, procollagen Ⅲ (PⅢNP), hyaluronic acid (HA) and human laminin (LN) in culture supernatants of LX-2 cells. All data using SPSS11.0 statistical software for statistical analysis. Results The amount of TGF-β1 in HepG2-HCV-C cells was (110.00 ± 11.45) pg / ml ~ (935.00 ± 21.36) pg / ml and HepG2 cells was 0 ~ (124.16 ± 11.81) pg / ml, the difference was statistically significant (F = 21984.81, P <0.01). ICC showed that the α-SMA, ColⅣ, CTGF and FN of LX-2 cells cultured in HepG2-HCV-C supernatant were diffusely positive and the cells were stained dark brown; LX-2 cells cultured with HepG2 showed focal brown Yellow, DMEM cultured LX-2 cells stained negative. The values of ColⅣ, HA, LN and PⅢNP in LX-2 (HepG2-HCV-C) group were significantly higher than those in LX-2 (HepG2) and LX-2 ). Conclusion HCV C protein can up-regulate the expression of TGF-β1. The cell line expressing protein C can promote the synthesis of extracellular matrix (ECM) in LX-2 cells. It is suggested that HCV C protein may be involved in the regulation of various ECM synthesis in hepatic fibrosis by activating hepatic stellate cells.