论文标题:PKD1基因对体外培养肿瘤细胞侵袭和转移影响的分子机制研究
PKD1 Inhibits Cancer Cells Migration and Invasion via Wnt Signaling Pathway in Vitro
论文作者
论文导师 魏于全；周钦,论文学位 硕士,论文专业 细胞生物学
论文单位 四川大学,点击次数 176,论文页数 60页File Size4156K
2007-04-18论文网 http://www.lw23.com/lunwen_1980342/
PKD1 gene;; PC-1;; cell migration;; invasion;; Wnt signaling pathway
目的：常染色体多囊肾病(autosomal dominant polycystic kidney disease，ADPKD)是最为常见和多发的遗传性疾病，PKD1是主要的致病基因，占所有病人中的85％，其主要特征主要是在肾脏或肝脏的小管进行性扩张的囊肿。PKD1所编码的蛋白PC-1功能广泛，有研究证实PC-1参与了调节细胞的增殖、凋亡，钙离子通道的调节，参与了G-蛋白偶连的受体通路，PC-1的IG-g结构域参与细胞与细胞以及细胞与基质之间的粘附，被认为可能是细胞与细胞／基质的粘附受体。此外，有推测PC-1是做为肿瘤抑制因子蛋白家族中的一个新的成员。本实验在前人工作的基础上利用已经构建好的PKD1表达载体，通过人的肝癌、结肠癌、肺瘤细胞株为模型来研究PKD1基因的过表达对体外培养的肿瘤细胞生物学行为主要是涉及细胞迁移的影响及其分子机理研究。 方法：分别以人的肝癌、结肠癌、肺瘤细胞株为模型，建立人的PKD1 cDNA转基因稳定表达细胞株，并将其分为4组。实验组1，外源性人的PKD1转基因组；对照组2，空载pcDNA3.1转染组；对照组3，阴性对照组；对照组4，特异性信号通路抑制子处理组。分别观察过表达PDK1肿瘤细胞对细胞外基质的粘附情况，细胞间的粘附情况，以及肿瘤细胞的迁移情况和肿瘤对细胞外基质的的侵袭情况。Western blotting来探讨其可能涉及的大概分子机制。 结果：①PKD1处理组：由于PKD1基因的过表达使肿瘤细胞对Ⅰ型胶原粘附增强，细胞数目比未处理组及空载对照组高出0.5％(P＜0.05)。②细胞聚集实验表明，PKD1处理组细胞比其它对照组的细胞聚集能力增强，PKD1处理组聚集的细胞数量和细胞团块的大小都比未处理的要多。③细胞迁移实验显示，PKD1处理组细胞迁移速度显著变慢，在相同的时间内，阴性对照细胞划痕合拢，而PKD1处理组变化不大。④侵袭实验表明，PKD1处理组细胞侵袭力明显减弱，和其它对照组相比，侵入胶原的细胞数量减少大约3倍(P＜0.01)。⑤Westernblotting检测PKD1对体外培养肿瘤细胞粘附和侵袭力的影响作用可能部分通过Wnt信号通路。 结论我们通过体外实验首次报道，PC-1的过表达在体外实验影响了肿瘤细胞的迁移和侵袭，有可能是肿瘤抑制因子家族中一个新的成员，为推测提供了直接的证据。
BACKGROUND & OBJECTIVE: The PKD1 is the major disease-causing gene of autosomal dominant polycystic kidney disease (ADPKD) which is characterized by progressive formation of cysts in ductal organs principally in the kidneys and the liver.PC-1,which is encoded by PKD1, has a fairly wide tissue distribution, PC-1 is believed to participate in cell-cell/matrix interaction, regulation of cell proliferation, apoptosis, and cation transport, and G-protein-coupled signaling, PC-1 is thought to be a cell-cell/matrix adhesion receptor. Moreover, PC-1 is considered to be a novel member of the tumor suppressor family of proteins. This study was tying to provide direct evidence linking increased PC-1 expression to inhibited migration and invasion in cancer cells Invitro. METHODS: We established carcinoma cell model in human liver carcinoma cell line HepG2, human lung carcinoma cell line A549 and human colon carcinoma cell line SW480. Then we divided each cell line into four groups. The cells treated with PKD1 cDNA encoding PKD1 gene (therapy group), the cells treated with pcDNA3.1 group encoding null PKD1 gene (control group 2), negative control group (control group 3), Wnt inhibitor trdeated group (control group 4), respectively. The cancer cells adherence to the collagen, and the cancer cells aggregation, migration and the cancer cells invasion into the collegan was observed and photographed in three separate fields under×100 magnifications. And we also showed that polycystin-1 regulated these processes that may be at least partially through Wnt pathway by western blotting. RESULTS:①We found that cancer cells adhereto collagen 1-coated plated plates were enhanced by overexpression of PKD1 cDNA, the cells number was higher compared with other control about 0.5% (~#P<0.01) ;②We found that cancer cell aggregation was promoted by PKDl gene. Cells treated with PKDl underwent a greater aggregation as assessed by the number of aggregation platelets and by the size of the aggregation compared with the other two groups;③Cell migration assay showed that overexpression of PKD1 inhibits the migration of cancer cells Invitro. During a period of 16 hours, the negative control cells moved forward and closed the gap in the cell monolayer, however, the expression of PKD1 in the HepG2 cells resulted in a strong inhibition of migration compared with the other control cells;④The cell invasion assays revealed that the overexpression of PKD1 in cancer cells led to a decreased invasion ability to collagen. The cell number was decreased three times compared with the other control;⑤Our data suggest that the effects of PKD1 on cell adhesion and invasion may be at least partially mediated by Wnt pathway in cancer cells. CONCLUSION: Increased PC-1 expression to inhibited migration and invasion in cancer cells Invitro, suggesting the role of PKD1 as a novel member of the tumor suppressor family of genes.
|
