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目的探讨高尔基体囊泡转运蛋白P115基因沉默对胃癌BGC-823细胞凋亡的影响及其可能的相关机制。方法将含p115基因的重组质粒p115 shRNA-1318转染BGC-823细胞,并设shNC(阴性对照)转染组和空白对照组,免疫荧光显微镜检测转染效果;RT-PCR法检测细胞中p115和巨噬细胞游走抑制因子(Macrophage migration inhibitory factor,MIF)基因mRNA的转录水平;Western blot检测细胞中P115、MIF及核内肿瘤抑制蛋白质P53的表达水平;MTT法检测细胞的增殖活力;流式细胞术检测细胞凋亡及细胞周期的分布。结果转染后48 h,荧光显微镜下可见绿色荧光蛋白的表达,表明转染成功;与对照组相比,p115 shRNA-1318组细胞中的p115和MIF基因mRNA的转录水平及蛋白的表达水平均明显降低(P<0.05),细胞核内P53蛋白的表达水平明显增加(P<0.05),细胞的增殖活力明显下降(P<0.05),细胞凋亡数量明显增加(P<0.05),G1/G2期细胞数量明显增多,S期细胞数量明显减少(P<0.05)。结论沉默p115基因可促进胃癌细胞的凋亡,其调控机制可能是通过调控MIF因子来完成的。
Objective To investigate the effect of P115 gene silencing of Golgi vesical transporter (PGE1) on the apoptosis of gastric cancer cell line BGC-823 and its possible mechanism. Methods The p115 shRNA-1318 plasmid was transfected into BGC-823 cells and transfected with shNC (negative control) and blank control group. The transfection efficiency was detected by immunofluorescence microscopy. The expression of p115 And macrophage migration inhibitory factor (MIF) mRNA. Western blot was used to detect the expression of P115, MIF and nuclear tumor suppressor protein P53. MTT assay was used to detect the proliferation of cells Cytometry to detect apoptosis and cell cycle distribution. Results The expression of green fluorescent protein (GFP) was observed under fluorescence microscope 48 h after transfection, indicating that the transfection was successful. Compared with the control group, the transcriptional levels of p115 and MIF mRNA in p115 shRNA-1318 group and the protein expression levels (P <0.05). The expression of P53 protein in the nucleus significantly increased (P <0.05), the proliferation activity of the cells significantly decreased (P <0.05), the number of apoptotic cells increased significantly (P <0.05) The number of serous cells increased significantly, the number of S phase cells decreased significantly (P <0.05). Conclusion Silencing the p115 gene can promote the apoptosis of gastric cancer cells. The regulation mechanism may be through the regulation of MIF factor.