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目的:研究应用减毒沙门菌感染性转基因骨桥蛋白siRNA(OPNsiRNA)抑制Tca 8113细胞生长和转移的可行性和作用。方法:人工合成OPNsiRNA,同时,合成无义ControlsiRNA作为对照,进行以下平行试验;合成siRNA,分别构建表达载体pIRES2-EGFP,转染减毒沙门菌SL7207,获得的重组菌SL-OPNsiRNA和SL-ControlsiRNA分别体外感染Tca 8113细胞,观察细胞感染率及对细胞增殖、凋亡、迁移和侵袭力的影响,同时以Western印迹检测转基因OPNsiRNA对细胞内源性OPN、基质金属蛋白酶-2(MMP-2)和尿激酶型纤溶酶原激活因子(uPA)蛋白表达的影响。采用SPSS15.0软件包进行组间t检验。结果:重组减毒沙门菌感染Tca 8113的效率可达80.0%以上,两者之间无显著差异(P>0.05);OPNsiRNA可显著抑制Tca 8113细胞的生长和诱导凋亡;感染后24h,细胞OPNsiRNA和Control-siRNA的迁移指数分别为0.56±0.10和0.82±0.05,侵袭力指数分别为0.53±0.13和0.83±0.06,2组之间均存在显著差异(n=12,P<0.01);同时,OPNsiRNA感染可显著降低细胞内OPN、MMP-2和uPA蛋白的表达量。结论:携带OPN-siRNA的减毒沙门菌可有效通过细菌感染方式,转入舌癌细胞,发挥抗肿瘤生长和转移的作用。
AIM: To investigate the feasibility and role of OPNsiRNA in inhibiting the growth and metastasis of Tca 8113 cells by using attenuated Salmonella typhimurium infection. Methods: Synthesized OPNsiRNA and synthesized nonsense ControlsiRNA as a control. The following experiments were carried out in parallel. SiRNA was synthesized and the expression vector pIRES2-EGFP was constructed. The attenuated Salmonella typhimurium SL7207 was transfected into SL-OPN siRNA and SL-Control siRNA In vitro infection of Tca 8113 cells, the cell infection rate and the impact on cell proliferation, apoptosis, migration and invasiveness were observed. Western blotting was used to detect the expression of OPN, MMP-2, And urokinase-type plasminogen activator (uPA) protein expression. SPSS15.0 software package for t-test between groups. Results: The efficiency of recombinant attenuated Salmonella typhimurium infection of Tca 8113 was above 80.0%, with no significant difference (P> 0.05). OPNsiRNA significantly inhibited the growth and induced apoptosis of Tca 8113 cells. After 24h The migration index of OPNsiRNA and Control-siRNA were 0.56 ± 0.10 and 0.82 ± 0.05 respectively, and the invasiveness index was 0.53 ± 0.13 and 0.83 ± 0.06 respectively (P <0.01). Meanwhile, there was significant difference between the two groups (n = 12, P <0.01) OPNsiRNA infection can significantly reduce the expression of OPN, MMP-2 and uPA protein. CONCLUSION: The attenuated Salmonella carrying OPN-siRNA can effectively transfer into the tongue cancer cells through bacterial infection and exert anti-tumor growth and metastasis.