摘要
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 Golgi α-mannosidase Ⅱ（ GM2） gene knockdown on adhesion abilities of BGC-823 human gastric carcinoma cells. Methods Three plasmid vectors expressing GM2 shRNAs and a negative control plasmid vector were designed,constructed and then transfected into BGC-823 cel s by LipofectamineTM2000. After transfection,the mRNA and protein levels of GM2 in BGC-823 cells were detected by real-time quantitative PCR（ qRT-PCR） and Western blotting to evaluate the transfection efficacy. The best plasmid for GM2 gene knockdown was selected and stably transfected into BGC-823 cells. Adhesion abilities of BGC-823 cells after GM2 gene silencing were observed by cell-cell,cell-matrix and cell-endothelial cell adhesion assays. At the same time,the expressions of E-cadherin,P-selectin,CD44v6 and intercellular adhesion molecule-1（ ICAM-1） proteins were detected by Western blotting after GM2 gene knockdown. Results The expression of GM2 was effectively knockdown in GM2-shRNA-2-transfected BGC-823 cells. Compared with the blank control group and the negative control group,the intercellular adhesion ability of the GM2-shRNA-2-transfected cells increased significantly,while their cell-matrix and cell-endothelium adhesion abilities markedly decreased. In GM2-shRNA-2 transfection group,E-cadherin expression was significantly elevated and the P-selectin expression was significantly reduced,while the expression levels of CD44v6 and ICAM-1 were not obviously changed. Conclusion After GM2 gene knockdown,the intercellular adhesion ability of gastric carcinoma BGC-823 cells is enhanced,while the adhesion abilities with the extracel ular matrix and endothelial cel s are weakened. The changes might be related to the up-regulated expression of E-cadherin and the down-regulation of P-selectin.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2017年第6期789-794,共6页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(30672431)
教育部高等学校博士学科点专项科研基金(20060631006)
重庆市自然科学基金(2010BB5361)
