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目的:利用沙门菌作为真核质粒携带载体,在体内外观察肿瘤坏死因子相关凋亡诱导配体(TRAIL)和鸡贫血病毒VP3基因对胃癌细胞的生长抑制作用。方法:将重组质粒pBud-TRAIL、pBud-VP3、pBud-TRAIL-VP3电转化减毒沙门菌SL7207,经稳定性鉴定后直接转染胃癌细胞株SGC-7901,24h后利用荧光显微镜观察有无融合绿色荧光蛋白表达,MTT法检测表达载体对胃癌细胞的生长作用,流式细胞仪检测细胞凋亡率及周期变化,并用免疫组织化学方法检测该载体对胃癌细胞Caspase-3、Caspase-9表达的影响。荷瘤小鼠口服携带重组质粒的减毒沙门菌,8周后通过RT-PCR检测肿瘤组织内真核载体的表达状况,并测量荷瘤瘤体大小。结果:重组质粒能够在减毒沙门菌中稳定存在,经减毒沙门菌介导转染胃癌细胞后能够较好的表达,转染48h后可见到TRAIL和VP3对胃癌细胞生长有抑制作用,流式细胞仪观察到pBud-TRAIL-VP3能使胃癌细胞的凋亡率明显增高,TRAIL和VP3能够协同促进胃癌细胞Caspase-3、Caspase-9的表达。体内实验提示pBud-TRAIL-VP3沙门菌载体能够在肿瘤组织中表达并能抑制肿瘤生长(P<0.05)。结论:减毒沙门菌将外源基因VP3和TRAIL基因导入胃癌细胞后,体内外实验证实该载体对胃癌细胞的生长起明显抑制作用,TRAIL和VP3联合作用机制与促进Caspase-3、Caspase-9的表达有关。
OBJECTIVE: To investigate the inhibitory effect of Salmonella typhimurium on eukaryotic plasmid carrier in vitro and in vivo, and to observe the effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and chicken anemia virus VP3 gene on the growth of gastric cancer cells. Methods: The recombinant plasmid pBud-TRAIL, pBud-VP3 and pBud-TRAIL-VP3 were electrotransformed into attenuated Salmonella typhimurium SL7207, which was directly transfected into gastric cancer cell line SGC-7901 after being stably identified. After 24 hours, the fusion was observed by fluorescence microscopy The expression of green fluorescent protein (EGFP) was detected by MTT assay. The growth of gastric cancer cells was detected by MTT assay. The apoptosis rate and cell cycle were detected by flow cytometry. The expression of Caspase-3 and Caspase-9 in gastric cancer cells was detected by immunohistochemistry influences. Tumor-bearing mice were orally administered with attenuated Salmonella typhimurium. The expression of eukaryotic vector was detected by RT-PCR and the tumor size was measured 8 weeks later. Results: The recombinant plasmids could stably exist in attenuated Salmonella typhimurium and could be well expressed after attenuated Salmonella mediated transfection of gastric cancer cells. TRAIL and VP3 were found to inhibit the growth of gastric cancer cells 48h after transfection Cytotoxicity assay showed that pBud-TRAIL-VP3 could significantly increase the apoptosis rate of gastric cancer cells, and TRAIL and VP3 could synergistically promote the expression of Caspase-3 and Caspase-9 in gastric cancer cells. In vivo experiments suggest that the pBud-TRAIL-VP3 Salmonella vector can express in tumor tissue and inhibit tumor growth (P <0.05). CONCLUSION: The attenuated Salmonella can induce the growth of gastric cancer cells by inducing the expression of VP3 and TRAIL gene into gastric cancer cells in vitro and in vivo. The combined mechanism of TRAIL and VP3 and the promotion of the expression of Caspase-3, Caspase-9 Related to the expression.