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目的:通过构建基因真核表达载体,探讨人2型大麻素受体(human cannabinoid receptor 2,hCB2R)对人子宫颈癌HeLa细胞体外凋亡的作用及机制。方法:选用人脑组织的cDNA作为模板,进行hCB2R基因的RT-PCR扩增,构建重组质粒GV230-h CB2R及其对照空质粒GV230并转染HeLa细胞,Western blotting法及免疫荧光细胞化学染色联合激光扫描共聚焦显微镜技术检测h CB2R表达及细胞内定位;流式细胞术检测He La细胞凋亡,Western blotting法及实时荧光定量PCR检测HeLa细胞中hCB2R、Bcl-2、Bax、Bad的表达。结果:与空质粒转染组相比,GV230-hCB2R转染HeLa细胞后表达相对分子质量40 000的hCB2R蛋白,且细胞膜和细胞质中均有hCB2R的表达;GV230-hCB2R转染组的细胞凋亡率显著高于GV230空质粒对照组[(14.51±4.51)%vs(6.29±0.57)%,t=1.72,P<0.05];与空质粒对照组相比,h CB2R转染组细胞内Bax和Bad的表达水平明显上调(P<0.05),而Bcl-2的表达明显下调(P<0.05)。结论:hCB2R对子宫颈癌HeLa细胞的生长表现出明显的抑制作用,其作用机制可能与hCB2R直接参与了细胞凋亡相关蛋白的表达变化有关。
OBJECTIVE: To investigate the effect of human cannabinoid receptor 2 (hCB2R) on apoptosis of human cervical carcinoma HeLa cells in vitro and to elucidate its possible mechanism by constructing eukaryotic expression vector. Methods: cDNA of human brain tissue was used as a template for RT-PCR amplification of hCB2R gene. The recombinant plasmid GV230-h CB2R and its control plasmid GV230 were constructed and transfected into HeLa cells. Western blotting and immunofluorescent cytochemical staining Laser scanning confocal microscopy was used to detect the h CB2R expression and intracellular localization. The apoptosis of HeLa cells was detected by flow cytometry. The expressions of hCB2R, Bcl-2, Bax and Bad in HeLa cells were detected by Western blotting and real-time fluorescence quantitative PCR. Results: Compared with the empty vector transfected group, the expression of hCB2R protein with a relative molecular weight of 40 000 was expressed in HeLa cells transfected with GV230-hCB2R, and the expression of hCB2R in the cell membrane and cytoplasm was also observed. In the GV230-hCB2R transfected group, (14.51 ± 4.51)% vs (6.29 ± 0.57)%, t = 1.72, P <0.05]. Compared with the empty plasmid control group, the expression of Bax and Bcl-2 in the CB2R transfected group was significantly higher than that in the GV230 null plasmid control group The expression of Bad was significantly up-regulated (P <0.05), while the expression of Bcl-2 was significantly down-regulated (P <0.05). CONCLUSION: hCB2R can significantly inhibit the growth of HeLa cells. The possible mechanism is that hCB2R is directly involved in the expression of apoptosis-related proteins.