淋巴道转移潜能及ANXA7对肝癌细胞共培养淋巴管内皮细胞的调控作用  

Regulatory effects of lymphatic metastasis potential and ANXA7 on lymphatic endothelial cells co-cultured with hepatocarcinoma cells

作　　者：王静文 钱雪娇[2,3] 章明放 唐建武[4] WANG Jingwen;QIAN Xuejiao;ZHANG Mingfang;TANG Jianwu(Department of Pathology,Tianjin First Central Hospital,Tianjin 300192,China;不详)

机构地区：[1]天津市第一中心医院病理科,天津300192 [2]天津市第一中心医院呼吸科 [3]南开大学医学院 [4]大连医科大学基础医学院病理教研室

出　　处：《山东医药》2022年第28期1-6,共6页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(81071725)。

摘　　要：目的探讨淋巴道转移潜能及膜联蛋白A7(ANXA7)对肝癌细胞共培养淋巴管内皮细胞(LEC)的调控作用。方法将各肝癌细胞及LEC共培养,收集与F细胞(高淋巴道转移潜能)、P细胞(低淋巴道转移潜能)、F_(A7下调)细胞(ANXA7表达下调)、F_(无关序列)细胞、P_(A7上调)细胞(ANXA7表达上调)、P_(空载体)细胞共培养的LEC(L-F_(共)、L-P_(共)、L-F_(A7下调共)、L-F_(无关序列共)、L-P_(A7上调共)、L-P_(空载体共)细胞)以及正常LEC用于实验。分别用实时定量PCR、Western blotting检测共培养LEC内淋巴管内皮相关分子(VEGF-C、VEGF-D、VEGFR-3、NRP-2、PDPN、LYVE-1、SOX18)mRNA与蛋白表达。用细胞免疫荧光法检测共培养LEC内淋巴管内皮相关分子定位,用ELISA法检测细胞上清液VEGF-C、VEGF-D水平,用淋巴管成管实验测算淋巴管成管节点数和分支总长度。结果VEGF-C、VEGF-D、VEGFR-3、NRP-2、PDPN、LYVE-1、SOX18在mRNA和蛋白水平表达上,L-P_(共)、L-F_(共)细胞高于LEC(P均<0.05),L-F_(共)细胞高于L-P_(共)细胞(P均<0.05)。与L-F_(无关序列共)、L-P_(空载体共)细胞相比,L-F_(A7下调共)细胞上述分子表达均低(P均<0.05),L-P_(A7上调共)细胞上述分子表达均高(P均<0.05)。ANXA7调控前后,各细胞中VEGF-C、VEGFR-3、PDPN相对表达量与同类分子(VEGF-D、NRP-2及LYVE-1/SOX18)比较变化更显著(P均<0.05)。VEGF-C、VEGF-D主要在细胞质内表达;VEGFR-3/NRP-2主要在细胞膜表达,少量在细胞质表达;PDPN、LYVE-1主要在细胞质表达;SOX18主要在细胞核表达,少量于细胞质表达。ANXA7调控前,细胞上清液中VEGF-C、VEGF-D表达比较,L-F_(共)、L-P_(共)细胞高于LEC,L-F_(共)细胞高于L-P_(共)细胞(P均<0.05),且VEGF-C表达均高于VEGF-D表达(P均<0.05)。ANXA7调控前后,各细胞上清液中VEGF-C相对表达量与同类分子(VEGF-D)比较变化更显著(P均<0.05)。在节点数和分支总长度上,L-F_(共)、L-P_(共)细胞均多于LEC,L-F_(共)细胞多于L-P_(共)Objective To investigate the regulatory effects of lymphatic metastasis potential and Annexin A7(ANXA7)on lymphatic endothelial cells(LECs)co-cultured with hepatocellular carcinoma cells.Methods The liver cancer cells and LEC were co-cultured.The LECs(L-F,L-P,L-F_(A7DOWN),L-F_(SHUS),L-P_(A7UP),L-P_(NCEV))co-cultured with F cells(high lymphatic metastasis potential),P cells(low lymphatic metastasis potential),F_(A7DOWN) cells(F ANXA7 downregulated cells),F_(SHUS) cells(F-unrelated sequence cells),P_(A7UP) cells(P ANXA7 up-regulated cells),and P_(NCEV) cells(P empty vector cells),and normal LECs were collected and used in the experiment.Real-time quantitative PCR and Western blotting were used to detect the mRNA and protein expression levels of lymphatic endothelium-related molecules VEGF-C,VEGF-D,VEGFR-3,NRP-2,PDPN,LYVE-1 and SOX18 in co-cultured LECs.The lymphatic endothelium-related molecular localizations in co-cultured LECs were detected by cell immunofluorescence method,and the levels of VEGF-C and VEGF-D in cell supernatant were detected by ELISA.The lymphatic tube formation test was used to measure the number of lymphangiogenesis nodes and the total length of lymphangiogenesis branches.Results The expression levels of VEGF-C,VEGF-D,VEGFR-3,NRP-2,PDPN,LYVE-1,and SOX18 at mRNA and protein levels were higher in L-P and L-F than in LECs(all P<0.05),and those were higher in L-F than in L-P(all P<0.05).Compared with L-F_(SHUS) and L-P_(NCEV),the expression of the above molecules was lower in L-F_(A7 DOWN)(P<0.05),and was higher in L-P_(A7UP)(P<0.05).After ANXA7 regulation,the relative expression levels of VEGF-C,VEGFR-3,and PDPN in each co-cultured LEC group were significantly higher than those of similar molecules(VEGF-D,NRP-2,and LYVE-1/SOX18)(all P<0.05).VEGFC and VEGF-D were mainly expressed in the cytoplasm.VEGFR-3/NRP-2 was mainly expressed in cell membrane and a little in cytoplasm.PDPN and LYVE-1 were mainly expressed in cytoplasm.SOX18 was mainly expressed in the nucleus and a little in the cytoplasm.Bef

关 键 词：肝癌细胞 淋巴管内皮细胞 体外共培养 淋巴道转移潜能 膜联蛋白A7 淋巴管内皮相关分子 淋巴管成管 

分 类 号：R735.7[医药卫生—肿瘤]