Rab7调节巨噬细胞Toll样受体信号转导通路的研究.docVIP

摘 要 Toll样受体家族可以识别几乎所有的病原微生物的一些结构组分和代谢产物，通过信号转导通路诱发细胞因子和趋化因子等启动机体的天然免疫，构成了机体抵抗病原微生物的第一道防线，在免疫学研究中一直是一个很受关注的家族。不同TLRs分子有不同的配体，如TLR3主要识别病毒dsRNA和Poly I:C；TLR4主要识别LPS等。TLR的活化是一把双刃剑过渡活化或者活化异常可能引起急慢性疾病TLR信号转导通路的负相调控成为天然免疫研究中的热点问题。曾有实验表明定位于溶酶体的Rab7b，能够通过转运TLR4受体到溶酶体进行降解，从而负相调控TLR4的信号转导。而Rab7和Rab7b在分子水平上，有超过50％的一致性和65％的相似性，因此我们推测Rab7和Rab7b可能有相似的功能。目的：本实验通过建立稳定表达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细胞因子的影响。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Poly I:C刺激稳定表达细胞系不同时间后检测细胞因子的表达量变化。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刺激后分泌的细胞因子显著降低，而Rab7T22N过表达后其的分泌又恢复。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，负向调控了TLR信号通路。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。 Research of Rab7 negative regulation of TLR3/4 pathway Abstract Toll-like receptor(TLR) family can identify almost all s some of the structural components and metabolites, induced cytokines and chemokines start the bodys natural immunity through the signal transduction pathways, constitute the first line of defense of a body to resist pathogenic micro-organisms , in immunological research has been a popular concern in the family.However, different TLRs have different ligand, such as TLR3 primarily identify viral dsRNA and Poly I:C,TLR4 major recognition LPS .However, the activation of TLR is a double edged sword.on the one hand, activat TLRs in innate immune responses and adaptive immune responses play an important role, on the other hand, the activation transition and activation anomalism may cause acute and chronic diseases. Therefore，negative regulation on the TLR signaling pathway bacome a hot issue .Reports have shown that lysosme-associated Rab7b negatively regulate TLR4 signal transduction by promoting lososomal degradation of TLR4. Since Rab7 and Rab7b have more than 50% identity and 65% simila