趋化因子受体CXCR4及其配体CXCL12在肝癌侵袭转移中的作用及机制  

The mechanism and biological function of chemokine receptor CXCR4 and its ligand CXCL12 in hepatocellular carcinoma

作　　者：张引 余力信 张发鹏[1] 余晓雯 王文龙 罗志强[1] 

机构地区：[1]南昌大学第二附属医院肝胆外科,330000

出　　处：《天津医药》2018年第1期20-26,共7页Tianjin Medical Journal

基　　金：江西省自然科学基金资助项目(20132BAB205045)

摘　　要：目的探讨趋化因子受体CXCR4及其配体CXCL12(CXCL12/CXCR4)在原发性肝癌侵袭转移中的作用及机制。方法分别采用Western blot、免疫组化和Real-time PCR等方法检测60例肝癌及对应癌旁组织标本中CXCL12/CXCR4蛋白及m RNA表达水平。常规培养4种肝癌细胞(Huh7、MHCC97h、Hep G2、Hep3B)和正常肝细胞(7702),Real-time PCR检测上述细胞中CXCL12、CXCR4 m RNA的表达,筛选合适的实验细胞。将CXCR4干扰质粒(sh-CXCR4)和对应空载体(sh-control)分别转染至MHCC97h构建稳转细胞系。Transwell侵袭实验、细胞划痕实验、MTT实验分别检测2组细胞的侵袭、迁移和增殖能力。取稳定表达的sh-control和sh-CXCR4 MHCC97h细胞,接种至6只裸鼠皮下,观察瘤体生长情况。Western blot检测sh-control和sh-CXCR4 MHCC97h细胞及对应裸鼠移植瘤中血管内皮生长因子-C(VEGF-C)的表达,同时对转染CXCR过表达质粒的MHCC97h中VEGF-C的表达水平进行检测。结果 (1)Western blot、免疫组化和Real-time PCR的结果均证实肝癌组织中CXCL12/CXCR4蛋白及m RNA表达水平均高于癌旁组织。(2)Huh7、MHCC97h、Hep G2、Hep3B细胞中CXCL12/CXCR4 m RNA表达水平均高于7702细胞,选取MHCC97h为实验细胞。转染sh-CXCR4的MHCC97h细胞的侵袭、迁移和增殖能力均明显低于sh-control组,同时接种含sh-CXCR4的MHCC97h细胞的裸鼠移植瘤的生长速度也明显小于sh-control组。(3)体外和体内实验均证实sh-CXCR4组的VEGF-C表达水平均低于sh-control组,而过表达CXCR4后,VEGF-C的蛋白表达明显上调。结论 CXCL12/CXCR4在原发性癌组织和肝癌细胞中呈现高表达,CXCL12/CXCR4可能通过调控VEGF-C蛋白表达从而抑制肝癌细胞的增殖、侵袭和转移。Objective To investigate the mechanism and function of chemokine receptor CXCR4 and its ligandCXCL12(CXCL12/CXCR4) in primary hepatocellular carcinoma(PHC).MethodsWestern blot assay,immunohistochemistry and Real-time PCR were used to detect the protein and m RNA expressions of CXCL12/CXCR4 in 60 PHC and corresponding paracancerous tissue samples. Four kinds of hepatoma cells(Huh7, MHCC97 h, Hep G2 and Hep3 B)and normal hepatocytes(7702) were routinely cultured, and then real-time PCR was performed to detect the m RNAexpressions of CXCL12/CXCR4 in these cells to screen suitable experimental cells. CXCR4 interference plasmid(sh-CXCR4) and corresponding empty vector(sh-control) were transfected into MHCC97 h to construct stable transfected celllines. The ability of invasion, migration, and proliferation of the 2 groups of cells were detected by Tanswell invasionexperiment, cell scratch test and MTT test. The stably expressed sh-control and sh-CXCR4 MHCC97 h cells were taken intothe subcutaneous of six nude mice, and the growth of the tumor was observed. Western blot assay was used to detect theexpressions of vascular endothelial growth factor-C(VEGF-C) in sh-control and sh-CXCR4 MHCC97 h cell lines andcorresponding xenografts in nude mice, as the same in MHCC97 h, which was transfected with CXCR overexpressed plasmid.Results(1) The results of Western blot assay, immunohistochemistry and Real-time PCR showed that the expressions ofCXCL12/CXCR4 protein and m RNA were significantly higher in liver cancer tissues than those of paracancerous tissues.(2)The expression levels of CXCL12/CXCR4 m RNA were higher in Huh7, MHCC97 h, Hep G2 and Hep3 B cells than those of7702 cells. MHCC97 h was selected as the experimental cells. The ability of invasion, migration and proliferation ofMHCC97 h cells transfected with sh-CXCR4 were significantly lower than those of sh-control group. Meanwhile, the growthrate of nude mice transplanted with sh-CXCR4 MHCC97 h cells was also significantly lower than that of sh-control group.(3)Both in

关 键 词：肝肿瘤 趋化因子CXCL12 受体 CXCR4 肿瘤细胞 培养的 细胞增殖 动物实验 血管内皮生长因子受体 

分 类 号：R735.7[医药卫生—肿瘤]