靶向抗原递呈细胞激活Flk1重组减毒鼠伤寒沙门菌的构建及其抑制树突状细胞凋亡的初步研究  

Preliminary study on the establishment and role of dendritic cells activation-targeted recombinant attenuated Salmonella typhimurium strain

作　　者：冯珂珂[1] 戴科芳[1,2] 

机构地区：[1]天津市环湖医院神经外科,300060 [2]河北省邢台市第三医院神经外二科,054000

出　　处：《中国现代神经疾病杂志》2009年第6期557-561,共5页Chinese Journal of Contemporary Neurology and Neurosurgery

基　　金：国家自然科学基金资助项目(项目编号:30600633)

摘　　要：目的观察靶向抗原递呈细胞激活血管内皮生长因子受体-2(Flk1)的重组减毒鼠伤寒沙门菌对树突状细胞凋亡的抑制作用。方法构建含Flk1与靶向抗凋亡基因BAK siRNA的重组质粒载体pFlk1-U6BAK,将其转化至减毒鼠伤寒沙门菌株中,携带pFlk1-U6BAK和pFlk1的重组减毒鼠伤寒沙门菌分别感染经体外培养的小鼠树突状细胞;Western blotting法检测树突状细胞Flk和BAK表达水平,MTT法检测树突状细胞增殖活性。结果成功构建了含Flk1和靶向抗凋亡基因BAK siRNA的重组质粒载体pFlk1-U6BAK。经携带pFlk1-U6BAK的重组减毒鼠伤寒沙门菌感染后,树突状细胞在稳定表达Flk的同时,BAK表达水平显著降低;而经携带pFlk1-U6BAK的重组减毒鼠伤寒沙门菌感染后,树突状细胞增殖活性明显升高。结论携带pFlk1-U6BAK的重组减毒鼠伤寒沙门菌可以在稳定表达Flk的同时,通过下调凋亡基因BAK表达水平而抑制树突状细胞凋亡,有望成为较为有效的抗肿瘤血管生成疫苗。Objective To establish a recombinant attenuated Salmonella typhimurium strain targeting to dendritic cells （DCs） activation with expression of vascular endothelial growth factor receptor-2 （VEGFR-2, Flk1）, and to explore its role in inhibiting the apoptosis of DCs. Methods Established recombinant plasmid pFlk1- U6BAK containing Flk1 and targeting to anti-apoptosis gene BAK small interference RNA （siRNA）. Furthermore, pFlk1- U6BAK and Flk1 were respectively transformed to attenuated Salmonella typhimurium strain. The attenuated Salmonella typhimurium strain carried with pFlk1-U6BAK and pFlk1 were used to infect the in vitro cultured muriue DCs, respectively. Western blotting was used to detect the post-infected DCs Flk and BAK expressions, and methyl-thiazolyl-tetrazolium （MTT） method was used to examine the reproduction activity of DCs. Results Recombinant plasmid pFlkI-U6BAK was successfully established. Attenuated Salmonella typhimurium strain carried with pFlk1- U6BAK stabilized DCs Flk expression and obviously decreased BAK expression. After carried with pFlk1- U6BAK, the attenuated Salmonella typhimurium strain could significantly enhance the reproduction activity of DCs. Conclusion Attenuated Salmonella typhimurium carried with pFlk1-U6BAK can stabilize Flk expression and suppress DCs apoptosis by inhibiting apoptosis gene BAK, and it is hopeful to be an effective vaccine against angiogenesis in tumor.

关 键 词：受体 血管内皮生长因子 RNA 小分子干扰 沙门菌 鼠伤寒 疫苗 DNA 疫苗 减毒 树突细胞 细胞凋亡 

分 类 号：R392.1[医药卫生—免疫学]