【作者】 刘朝英；

【导师】 马克里；

【作者基本信息】 大连医科大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 目的:肝细胞生长因子(hepatocyte growth factor, HGF)是由间质细胞(如成纤维细胞、巨噬细胞)产生的多肽类细胞因子,又称间充质因子。HGF具有许多重要的生物学功能,如促进细胞的增殖、分裂;抑制细胞间隙连接的生成和细胞之间的粘附,导致细胞的离散;上调尿激酶型纤溶酶原激活物及受体(uPA/uPAR)的表达,促进细胞外基质降解;增加细胞骨架蛋白磷酸化,破坏细胞骨架,从而使细胞易于变形和迁移。因此,HGF又称促有丝分裂因子、离散因子、细胞形态发生素或促迁移因子。HGF在胚胎的发育、器官的形成、细胞的生长和迁移过程中发挥重要的调控作用,也与肿瘤的发生、进展、浸润、扩散和转移有着非常密切的关系。HGF的受体(hepatocyte growth factor receptor, HGFR)是原癌基因c-met编码的产物,又称c-Met。c-Met属于受体酪氨酸蛋白激酶家族成员,常由上皮细胞(如肝、肾、消化道细胞)表达。c-Met在肿瘤细胞表达增加,并与肿瘤细胞转移能力呈正相关。HGF由间质细胞分泌后,经旁分泌途径作用于周围靶细胞。HGF与受体c-Met结合后,导致受体二聚化和活化,进而激活细胞内不同的信号转导途径。已知HGF/c-Met活化的细胞内信号转导途经起码有三条,包括有丝分裂原激活的蛋白激酶信号转导通路(MAPK信号通路)、磷脂酶Cγ1/二脂酰甘油/蛋白激酶C信号转导通路(PLCγ1 /DAG/PKC信号通路)和三磷脂酰肌醇激酶/蛋白激酶B信号转导通路(PI3K/ PKB信号通路)。其中,PI3K/PKB和PLCγ/PKC信号转导途径是调控肿瘤细胞侵袭和转移的重要途径。KAI-1/CD82和MRP-1/CD9均属于4跨膜蛋白基因家族编码的产物。CD82是肿瘤转移抑制基因KAI-1编码的产物,目前被认为是广谱的肿瘤转移抑制因子。CD9又称细胞运动相关蛋白-1(motility-related protein-1)。二者均可下调癌细胞的运动性,从而抑制肿瘤细胞的浸润转移。对其作用机制研究表明,CD82和CD9通过与c-Met相互作用,抑制HGF诱导的c-Met酪氨酸蛋白激酶的活化,或抑制c-Met与整合素(integrins)的相互作用,下调c-Met对PI3K/PKB和PLCγ/PKC信号转导途径的活化,从而发挥其转移抑制作用。Hca-F细胞株和HCa-P细胞株为同一来源但具有不同淋巴结转移能力的两个细胞株。Hca-F为高淋巴道转移株,淋巴结转移率为80%,Hca-P为低淋巴道转移株,淋巴结转移率为0-20%。两细胞株淋巴道转移能力差别产生的分子机制尚不十分清楚。本课题主要比较两细胞株CD82/CD9和c-Met的表达以及PI3K/PKB和PLCγ1 /PKC信号转导通路的活性变化,以探讨肿瘤细胞淋巴道转移的分子机制。方法:小鼠腹水型肝癌Hca-F和Hca-P细胞株于小鼠腹腔培养。分别收集细胞后,分离提取细胞总蛋白,制备胞浆蛋白和颗粒结合蛋白。采用SDS-PAGE、Western Blotting技术及计算机扫描定量分析两细胞株CD82/CD9的表达和PI3K/PKB和PLCγ1 /PKC信号通路的活性。结果:1)CD82/CD9的表达:CD82在高淋巴道转移株Hca-F细胞和低淋巴道转移株Hca-P细胞中的表达没有显著性差别,Hca-P细胞CD9表达显著高于Hca-F细胞(Hca-P是Hca-F的1.31倍,P=0.4%,n=3),提示CD9可能是影响两细胞株淋巴道转移能力的主要因素。2)PI3K/PKB信号转导通路:采用Western Blotting技术对两细胞株PDK的表达和在细胞中的分布以及PKB磷酸化活化进行了比较分析。结果显示,两细胞株磷酸化PKB的表达无显著性差别;PDK在两细胞株的分布也无显著差异;说明PI3K/PKB信号转导通路不是影响两细胞株淋巴道转移能力主要途径。3)PLCγ1/PKC信号转导通路:采用Western Blotting技术对两细胞株PLCγ1及不同亚型PKCs的活性进行了比较分析。结果显示,淋巴道低转移株Hca-P细胞磷酸化的PLCγ1表达明显低于淋巴道高转移株Hca-F细胞(Hca-P是Hca-F的45.23%倍,P<0.05,n=3);Hca-P细胞中膜结合的PKCα、PKCβ1和PKCβ2含量明显低于Hca-F细胞(Hca-P细胞PKCα、PKCβ1和PKCβ2分别是Hca-F细胞的71.75%、76.80%和73.16%,P<0.05,n=3)。说明高转移株Hca-F细胞的PLCγ1/PKC信号转导通路的活性高于低转移株Hca-P细胞。PLCγ1/PKC信号转导通路可能是影响两细胞株淋巴道转移能力主要信号途径。结论:肿瘤转移抑制因子CD9在淋巴道低转移Hca-P细胞高表达;淋巴道低转移Hca-P细胞中的PLCγ1/PKC信号转导通路活性低。这可能是Hca-P细胞具有较低淋巴结转移能力的因素之一。推测可能是高表达的CD9与c-Met相互作用,抑制c-Met对Hca-P细胞PLCγ1/PKC信号通路的活化,从而降低Hca-P细胞的淋巴道转移能力。更多还原

【Abstract】 Objective: Hepatocyte growth factor is one kind of polypeptide cytokines generated from mesenchymal cells(such as mechanocyte and macrophage etc.). HGF has many important biological functions, such as promoting proliferation and division of cells; suppressing the formation of cell gap junctions and cell adhere lead to cell scattering ; up-regulating the expression of urokinase plasminogen activator and its receptor (uPA/uPAR), promoting the degradation of extracellular matrix; increasing the phosphorylation of cytoskeletal protein and interfering the rearrangment of cytoskeleton, which make cells apt to migration. So, HGF is also named as mitogenic factor、scattering factor and morphogen. HGF plays an important role in regulation of embryo development、organic formation、cells growth、proliferation and migration . It is also concerned with generation、development、diffusion and metastasis of tumor.The receptor of HGF is coded by proto-oncogene c-met and is also called c-Met. c-Met is one member of receptor tyrosine protien kinase family and often expresses in epithelial cell(such as liver、kidney、enteron). The expression of c-Met in tumor cells is higher and has positive correlation with metastasis potential of tumor cell. HGF acts on target cell by paracrine after secretion from mesenchymal cells. Once conjugated with HGF, the receptors are dimerizated and activated, then ignite the signaling pathway in cells. It is known that, at least, there are three signaling pathways activated by HGF/c-Met, including MAPK signaling pathway, PLCγ1/DAG/PKC signaling pathway and PI3K/PKB signaling pathway. Among these, PI3K/PKB signaling pathway and PLCγ/PKC signaling pathway are the key pathways for regulating the invation and metastasis of tumor cell. Both of KAI-1/CD82 and MRP-1/CD9 belong to the tetraspanin or trans- membrane-4 superfamily protens. CD82 is coded by tumor metastasis suppressor gene KAI-1. At present, It is konwed as a broad-spectrum suppressor of metastasis. CD9 is also called cell motility-related protein-1. Both of them can down-regulate the cell motility, and inhibit the invation and metastasis of tumor cell. A lot of experiment date has shown that CD82/CD9 can interact with c-Met, and thereby inhibits HGF-induced Met tyrosine kinase activity, as well as integrin to Met cross-talk, down-regulate PI3K/PKB and PLCγ/PKC signaling pathway. This is the mechanism by witch CD82 and CD9 down-regulate the cell motility, and inhibit the invation and metastasis of tumor cell.Both of Hca-F cell strain and Hca-P cell strain are established from the same parental cell line. Hca-F is the high lymphatic metastasis potential cell strain and its lymphatic metastasis rate is 80%; Hca-P is low lymphatic metastasis potential cell strain and its lymphatic metastasis rate is 0-20%. However the molecular mechanism involved in the different lymphatic metastasis potential between these two cell strains remains unclear. To investigate the molecular mechanism of tumor lymphatic metastasis, The expression of CD8, CD9 and c-Met on Hca-F and Hca-P cells was examined and the activity of PI3K/PKB and PLCγ/PKC signaling pathway in two cell lines was analyzed comparatively.Method: Hca-F cell and Hca-P cell were cultured in mouse abdominal cavities. Then the cells were harvested and the cytosol proteins and plasma membrane proteins were prepared. The expression of CD82/CD9 and activity of PI3K/PKB signaling pathway and PLCγ/PKC signaling pathway were analysed by using Western Blotting and computer scanning technique.Result: 1)expression of CD82/CD9:The contents of CD9 have no significant difference between Hca-F cell and Hca-P cell, while the content of CD9 in Hca-P cell increased by 1.31 fold comparing with Hca-F cell, It suggests that CD9 maybe the key factor effecting the lymphatic metastasis potential of Hca-F and Hca-P cells.2) PI3K/ PKB signaling pathway: The distribution of PDK and the phosphorylation of PKB in Hca-F and Hca-P cell were analysed by using Western Blotting. The results shown that there were no significant differences on the expression of the phosphorylated PKB between the two cell lines; It is the same with the expression and distribution of PDK; The results suggest that PI3K/PKB signaling pathway isn’t the main pathway effecting the tumor lymphatic metastasis potential. 3) PLCγ1/PKC signaling pathway:The acti- vety of PLCγ1and different hypotypes of PKCs among these two cell strains were analyzed by using Western Blotting. The results displayed that phosphorylated PLCγ1 in Hca-P cell significantly decressed by 45.23% comparing with Hca-F cell. The contents of PKCα、PKCβ1and PKCβ2 were obviously decressed by 71.75%,76.80% and 73.16% comparing with Hca-F cell; The results showed that the activity of PLCγ1/PKC signaling pathway in Hca-F cell is higher than that of Hca-P cell. Thus, PLCγ1/PKC signaling pathway maybe the major pathway effecting the the tumor lym- phatic metastasis potential.Conclusion:The expression of the tumor metastasis suppressor CD9 in Hca-P cell is higher comparing with Hca-F cell;Meantime, activity of PLCγ1/PKC signaling pathway in Hca-P cell has lower than that of Hca-F cell. These may be the important factors that effect the lymphatic metastasis potential of Hca-F and Hca-F cells.更多还原