过表达Rab7基因对LPS和Poly I:C活化的巨噬细胞RAW264.7中iNOS/NO的影响  

The impact of Rab7 gene overexpression on the production of iNOS/NO in activated RAW264.7 cells by LPS and Poly I:C

作　　者：王宁飞[1] 蔡富娟[1] 刘帅[1] 宋亮[1] 谢业功[2] 王玉珍[1] 

机构地区：[1]内蒙古农业大学生命科学学院,呼和浩特010018 [2]天津医科大学,天津300070

出　　处：《中国免疫学杂志》2011年第6期497-501,共5页Chinese Journal of Immunology

基　　金：国家自然科学基金(30801001);内蒙古自然科学基金(2010MS0513)

摘　　要：目的:通过建立稳定表达Rab7及其突变体的巨噬细胞系,分析稳定表达细胞系的生物学特性,研究Rab7及其突变体基因过表达后对LPS和Poly I:C刺激的巨噬细胞表达iNOS/NO的影响。方法:将Rab7及其突变体Rab7(T22N)的真核表达载体通过脂质体法转染RAW264.7细胞,G418选择性培养基筛选,建立稳定表达Rab7及Rab7T22N的细胞系。通过RT-PCR和Western blot方法鉴定稳定表达细胞系。通过观察细胞形态及MTT法分析稳定表达细胞系的生长特性。以LPS和PolyI:C刺激稳定表达细胞系,不同时间后检测NO和iNOS的表达量变化。结果:与转染空质粒的细胞相比,转染Rab7和Rab7T22N的细胞中Rab7的mRNA和蛋白水平都显著增高。Rab7过表达后引起细胞形态变化并显著抑制了细胞增殖,Rab7T22N过表达后促进细胞增殖。Rab7过表达后,巨噬细胞在LPS和Poly I:C刺激后分泌的iNOS和NO显著降低,而Rab7T22N过表达后iNOS和NO的分泌又恢复。结论:成功建立了稳定表达Rab7及其突变体的细胞系。Rab7过表达后抑制了细胞增殖,抑制了Poly I:C和LPS刺激后巨噬细胞中NO和iNOS的表达。该研究为进一步阐明Rab7在TLRs信号通路中的作用奠定了基础。Objective： To establish and analyze the biological characteristics of trmtsformed macrophage cell lines stably expressing Rab7 and its dominant negative mutant Rab7T22N, and study the production of iNOS/NO in transformed macrophage cell lines after stimulation with LPS and Poly I ： C. Methods： The eukaryotic expression vector of Rab7 and Rab7T22N were transfected into RAW264.7 cells by liposome, and screened with C418. The G418-resistant colonies were obtained and amplified. The trmlsfonned cell lines were identified by RT-PCR, RealTime PCR and Western blot. The characteristics of transformed cell lines were analyzed by observing the cell morphology and MTI methods. The production of iNOS and NO were measured after transformed cell lines stimulation with I PS and Poly I：C. Results：Compared with the control group, the mRNA and protein expression of Rab7 were significantly improved in cell lines transfected with Rab7 and Rab7T22N. Overexpression of Rab7 changed the cell morphology and inhibited lhe proliferation of RAW264.7 cells, while overexpression of Rab7T22N promoted the proliferation of RAW264.7 cells. Overexpression of Rab7 significantly inhibited iNOS and NO production in LPS and Poly I： C stimulated macrophages. The production of iNOS and NO were restored in Rab7T22N cell line. Conclusion： Overexpression of Rab7 inhibits the proliferation of macrophages, and suppresses the production of iNOS and NO in macrophages stimulated with LPS and Poly I：C. The study may lay a foundation for our further study the role of Rab7 in TLRs signaling pathways.

关 键 词：多聚胞苷酸 脂多糖 一氧化氮合酶 一氧化氮 

分 类 号：R392.11[医药卫生—免疫学]