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目的探讨MEK/ERK在结肠癌细胞NDRG1基因表达调控和体外侵袭、迁移中的作用;分析ERK通路、NDRG1基因、肿瘤侵袭及迁移三者间的潜在联系。方法将MEK/ERK抑制剂与HT-29结肠癌细胞共培养,光学显微镜下观察细胞形态变化;透射电子显微镜观察细胞超微结构变化;24孔-小室法检测癌细胞体外侵袭、迁移能力的改变;免疫细胞化学染色、Western blot检测NDRG1基因表达情况。结果与抑制剂共培养后,HT-29结肠癌细胞及胞核形态、大小趋于一致,排列多呈腺样结构。细胞表面微绒毛、高尔基复合体、线粒体增多,粗面内质网丰富,胞质内微腺腔常见,同时可见凋亡细胞。与对照组相比,加抑制剂组在侵袭迁移实验中穿过微孔膜的细胞数减少,差异有统计学意义(P<0.05)。免疫细胞化学结果显示,HT-29组与DMSO组NDRG1表达较少,加抑制剂组表达显著增多;Western blot结果显示,加抑制剂各组均出现NDRG1蛋白表达条带,而HT-29组与DMSO组则未见有NDRG1蛋白的表达。结论阻断ERK通路可诱导HT-29结肠癌细胞发生分化且促进细胞凋亡,同时可抑制其体外侵袭及迁移能力并明显上调其NDRG1蛋白的表达。
Objective To investigate the role of MEK / ERK in the regulation of expression of NDRG1 in colon cancer cells and invasion and migration in vitro, and to analyze the potential relationship between ERK pathway, NDRG1 gene and tumor invasion and metastasis. Methods MEK / ERK inhibitor was co-cultured with HT-29 colon cancer cells. The morphological changes of cells were observed under optical microscope. The ultrastructural changes of cells were observed by transmission electron microscopy. The invasion and migration of cancer cells were detected by 24- Immunocytochemical staining and Western blot were used to detect the expression of NDRG1 gene. Results After co-cultured with inhibitors, the morphology and size of HT-29 colon cancer cells and nuclei tended to be the same, arranged in a mostly adenoid structure. Microvilli cells, Golgi complex, increased mitochondria, rich rough endoplasmic reticulum, cytoplasmic micro-gland cavity common, while visible apoptotic cells. Compared with the control group, the number of cells passing through the microporous membrane in the inhibitor plus group decreased, with significant difference (P <0.05). The result of immunocytochemistry showed that NDRG1 was less expressed in HT-29 and DMSO groups, and the expression of NDRG1 was significantly increased in inhibitor group. Western blot results showed that NDRG1 protein bands were found in all groups, No DMSO group showed NDRG1 protein expression. Conclusion Blocking the ERK pathway can induce HT-29 colon cancer cells to differentiate and promote apoptosis, meanwhile, it can inhibit the invasion and migration of HT-29 colon cancer cells and up-regulate the expression of NDRG1 protein.