NDRG 1基因转染对HT-29人结肠癌细胞株增殖、侵袭和迁移能力的影响

Effects of NDRG1 on proliferation,invasion and migration of human colorectal cancer cell line HT-29

目的研究NDRG1基因转染对HT-29人结肠癌细胞株体外增殖、侵袭和迁移能力的影响。方法将重组真核表达质粒pEGFP-NDRG1-N3和空载体pEGFP-N3采用阳离子脂质体Lipofectamine转染HT-29人结肠癌细胞株,通过荧光倒置显微镜检测绿色荧光蛋白的表达。免疫组织化学染色检测转染前后HT-29细胞NDRG1的表达。并通过多种体外实验(流式细胞术法、24-transwell法、扫描电镜和透射电镜等)研究NDRG1对HT-29人结肠癌细胞株体外增殖、侵袭和迁移能力及表面和超微结构的影响。结果流式细胞术结果显示,与未转染组和转染pEGFP-N3空载体组比较,pEGFP-NDRG1-N3组细胞生长周期G0/G1期比例增高,S期明显减低,差异有统计学意义(P<0.05)。细胞侵袭和迁移实验的结果显示,转染pEGFP-NDRG1-N3组细胞与空载体和空白对照组比较,穿过微孔膜的细胞数均明显减少,差异有统计学意义(P<0.05)。扫描和透射电镜结果显示,转染pEGFP-NDRG1-N3组HT-29细胞生长增殖受抑制,细胞分化程度有增高趋势。结论 NDRG1基因可明显抑制HT-29人结肠癌细胞的体外增殖活性,降低其侵袭和迁移能力,促进其分化,提示其可作为一种候选的肿瘤转移抑制基因。

Objective To investigate the effects of NDRG1 on proliferation,invasion and migration of human colorectal cancer cell line HT-29 in vitro.Methods LipofectamineTM 2000 was used to transfect NDRG1 into human colorectal cancer HT-29 cells.The expression level of NDRG1 was measured by fluorescence microscopy and immunocytochemical staining.The cell cycle distribution was determined by fluorescence-activated cell sorting（FACS）.The ability of migration（without Matrigel） and invasion（with Matrigel） of HT-29 cells was measured by counting the number of cells transferring through the polycarbonate membrane with 8 μm pore in 24-transwell chambers.The outer structure of HT-29 cells was observed by scanning electron microscope（SEM）;The ultrastructure of HT-29 cell was observed by transmission electron microscopy（TEM）.Results Recombinant plasmids of pEGFP-NDRG1-N3 were successfully transfected into HT-29 cells.Compared with control group,an increase of the cell population at G0/G1 phase and a decrease at S phase was observed 48 hours after transfection（P0.05）.The number of cells transferring through the polycarbonate membrane in chambers with Matrigel or without Matrigel decreased significantly for transfected HT-29 cells（P0.05）.The proliferative activity was inhibited and differentiation was promoted in the NDRG1 transfected HT-29 cells.Conclusion NDRG1 gene inhibits cell proliferation,invasion,migration and promote cell differentiation of HT-29 cells in vitro,which suggests that it may serve as a tumor metastasis suppressor.