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| 减毒沙门菌介导的TRAIL和VP3对胃癌细胞的生长抑制作用及其机制 |
| 曹红丹1,吕琳1,向廷秀2*,刘少宁1,王丽娟1,王丕龙1 |
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| (1.重庆医科大学附属第一医院消化内科,重庆 400016;2.重庆医科大学附属第一医院实验研究中心,重庆 400016) |
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| 摘要: |
| 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-7901，24 h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48 h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。 |
| 关键词: 减毒沙门菌 细胞凋亡 TRAIL基因 VP3基因 |
| DOI：10.3724/SP.J.1008.2008.01284 |
| 投稿时间：2008-03-20修订日期：2008-05-26 |
| 基金项目:国家自然科学基金（30500234）. |
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| Inhibitory effects of attenuated Salmonella typhimurium-mediated TRAIL and VP3 gene against gastric cancer cells |
| CAO Hong-dan1,L Lin1,XIANG Ting-xiu2*,LIU Shao-ning1,WANG Li-juan1,WANG Pi-long1 |
| (1.Department of Gastroenterology, First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China;2.Experiment Research Center, First Affiliated Hospital of Chongqing Medical University, Chongqing 400016) |
| Abstract: |
| Objective:To investigate the inhibitory effect of eukaryotic expression vector (attenuated salmonella typhimurium) carrying tumor necrosis factor-related apoptosis inducing ligand (TRAIL) and Chicken anemia virus VP3 gene on gastric cancer cells in vitro and in vivo.Methods: The cloning vectors pBud-TRAIL, pBud-VP3, and pBud-TRAIL-VP3 were transformed into attenuated Salmonella typhimurium by electric transformation technique. The S.typhimurium-based carriers were then transfected into gastric cancer cells. line SGC-7901 after stability assay. The expression of fusion green fluorescent protein was examined using fluorescent microscopy after 24 h. MTT assay was used to examine the inhibition of cell growth. Flow cytometry was used to detect cycle distribution and apoptosis rates of cells. The expression of caspase-3 and caspase-9 was assayed by immunohistochemistry method.Salmonella typhimurium carrying recombinant plasmid was administrated orally in sarcoma-bearing mice; 8 weeks later RT-PCR was used to detect the expression of cloning vectors in tumor tissue. Meanwhile, the sizes of tumors were also determined.Results: The recombinant plasmids were stably transformed into attenuated Salmonella typhimurium, and the plasmids was satisfactorily expressed in gastric cancer cells via attenuated Salmonella typhimurium. TRAIL and VP3 inhibited the proliferation of gastric cancer cells after 48 h. Flow cytometry analysis showed that the pBud-TRAIL-VP3 obviously enhanced apoptosis rates of gastric cancer cells. TRAIL and VP3 jointly increased the expression of caspase-3 and caspase-9. In vivo study showed that TRAIL and VP3 genes were expressed in tumor tissue and could inhibit the tumor growth（P<0.05）.Conclusion: Attenuated Salmonella typhimurium-mediated TRAIL and VP3 transfection of gastric cancer cells can inhibit cell growth in vitro and in vivo. The joint effect of TRAIL and VP3 is correlated with the increase of caspase-3 and caspase-9 expression. |
| Key words: attenuated Salmonella typhimurium apoptosis TRAIL gene VP3 gene |
