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目的通过敲低高尔基体α甘露糖苷酶2(GM2)基因的表达,探讨其对BGC-823人胃癌细胞黏附能力的影响。方法设计并构建3个针对GM2基因的短发卡RNA(shRNA)质粒表达载体,同时合成阴性对照载体,并将其转染至BGC-823细胞,实时定量PCR和Western blot法检测转染后BGC-823细胞GM2 mRNA及蛋白表达水平,筛选出敲低效果最佳的质粒;再分别运用同质细胞黏附试验、细胞-基质黏附实验、内皮细胞黏附实验观察GM2基因敲低后对BGC-823细胞黏附能力的影响;同时采用Western blot法检测GM2基因敲低后上皮钙黏素(E-cadherin)、CD44v6、细胞间黏附分子1(ICAM-1)及P选择素(P-selectin)等黏附分子的蛋白水平。结果与空白对照组及转染GM2-shRNA-NC组相比较,GM2-shRNA-2质粒可有效敲低GM2基因的表达;敲低GM2表达水平后,BGC-823细胞同质细胞之间黏附数目明显增加,而与基质和血管内皮细胞之间的黏附数目明显减少。敲低GM2基因表达后E-cadherin蛋白表达明显增加,P-selectin蛋白表达明显降低,而CD44v6和ICAM-1表达水平未见明显变化。结论敲低GM2基因表达水平后,胃癌BGC-823细胞间的黏附能力增强,而与细胞外基质和血管内皮细胞之间的黏附能力减弱,可能与敲低GM2后E-cadherin表达上调,而P-selectin的表达下调有关。
Objective To investigate the effect of GM2 gene on the adhesion of human gastric cancer cells BGC-823 by knocking down the expression of GM2 gene. Methods Three short hairpin RNA (shRNA) plasmid vectors targeting GM2 gene were designed and constructed. Negative control vectors were synthesized and transfected into BGC-823 cells. Real-time PCR and Western blot were used to detect the expression of BGC- 823 cells. The results of cell adhesion assay, cell-matrix adhesion assay and endothelial cell adhesion assay were used to observe the effect of GM2 gene knockdown on BGC-823 cell adhesion The expression of E-cadherin, CD44v6, ICAM-1 and P-selectin in GM2 gene were detected by Western blot. Protein level. Results Compared with the blank control group and GM2-shRNA-NC transfected group, the GM2-shRNA-2 plasmid knockdown the expression of GM2 gene effectively. After knockdown of GM2 expression, the adhesion of BGC-823 cells to homogenous cells Significantly increased, while the number of stromal and endothelial cells significantly reduced the number of adhesion. E-cadherin protein expression was significantly increased and P-selectin protein expression was significantly decreased after knockdown of GM2 gene expression, while no significant changes were observed in the expression of CD44v6 and ICAM-1. Conclusion The knockdown of GM2 gene expression in gastric cancer cell line BGC-823 enhanced the adhesion between the extracellular matrix and endothelial cell adhesion between the weakened, and knockdown GM2 E-cadherin expression may be up, and P -selectin expression related to the decline.