结核分枝杆菌Hsp16.3刺激小鼠巨噬细胞向M2分化  

Mycobacterium tuberculosis Hsp16. 3 induces murine macrophages derived from M2

作　　者：李姗姗[1] 秦欢[1] 丁陈波 李龙梅[1] 刘梅[1] 李娜娜[1,2] 张继东[1] 徐林[1] 罗军敏(指导)[1] 

机构地区：[1]遵义医学院免疫学教研室,贵州省生物治疗人才基地,遵义563099 [2]遵义医学院附属医院呼吸二科,遵义563099

出　　处：《中国免疫学杂志》2015年第12期1595-1600,共6页Chinese Journal of Immunology

基　　金：国家自然科学基金地区项目(81460249)资助

摘　　要：目的:探讨结核分枝杆菌热休克蛋白16.3(Mycobacterium tuberculosis heat shock proteins 16.3,MTB Hsp16.3)在体外细胞水平对小鼠骨髓来源M1型巨噬细胞的影响作用。方法:从BALB/c小鼠的胫股骨中取骨髓细胞,与GM-CSF共培养获取骨髓来源的M0型巨噬细胞,流式检测CD11b和F4/80的表达;分别用IFN-γ、IL-4诱导M0型巨噬细胞,光镜下观察细胞形态,荧光定量PCR、ELISA检测各组细胞IL-12、TNF-α、i NOS、IL-10、TGF-β、Arg-1的表达情况,构建小鼠骨髓来源M1/M2型巨噬细胞的技术平台;用MTB Hsp16.3刺激M0、M1型巨噬细胞,分别孵育24、48、72、96 h,采用ELISA、qRT-PCR检测不同时间点细胞培养液上清和细胞内的IL-12、TNF-α、i NOS、IL-10、IL-4、TGF-β、Arg-1的表达情况。结果:光镜下观察M0型巨噬细胞形态不规则,细胞伪足较短;M1型巨噬细胞形状呈长梭形,并有伪足伸出现,突触很长;M2型巨噬细胞伪足较短,细胞整体形态较为收拢。FCM检测有90%以上的骨髓细胞细胞具有CD11b和F4/80双阳性的特征。ELISA和qRT-PCR检测发现,M1型巨噬细胞高表达IL-12、TNF-α、i NOS;M2型巨噬细胞高表达IL-4、TGF-β、IL-10、Arg-1。MTB Hsp16.3刺激巨噬细胞后,M0和M1型巨噬细胞均高表达IL-10、TGF-β,低表达TNF-α、i NOS,且在48-72 h增加或降低的水平达到峰值。结论:成功诱导了小鼠骨髓来源的M1、M2型巨噬细胞;MTB Hsp16.3促进M1型巨噬细胞高表达M2型相关的细胞因子,可促进M1型巨噬细胞向M2型样巨噬细胞转换。Objective： To investigate mycobacterium tuberculosis heat shock proteins 16. 3（ Mycobacterium tuberculosis heat shock proteins 16. 3,MTB Hsp16. 3） effect on mouse bone marrow-derived M1 macrophages in vitro. Methods： Bone marrow cells were isolated from tibia and femurs of BALB / c mice and incubated with GM-CSF. Then detects the expression of CD11 b and F4 /80 with flow cytometry; M0 macrophages were treated with IFN-γ or IL-4,respectively. The expression of IL-12,TNF-α,i NOS,IL-10,TGF-β,Arg-1were analyzed by qRT-PCR and ELISA. The light microscope observed morphology of M0 macrophages,constructing technology platform of bone marrow-derived M1 / M2 macrophages. The M0 / M1 macrophages were stimulated with MTB Hsp16. 3 for 24 h,48 h,72 h and96 h. ELISA and qRT-PCR detected the expression of IL-12,TNF-α,i NOS,IL-10,IL-4,TGF-β and Arg-1 in cell culture supernatant and intracellular at different time point. Results： Morphology analysis showed that M0 macrophages were irregular and had shorter pseudopodia,M1 macrophages had elongated fibroblastoid morphology,whereas M2 macrophages were round cells stretching out pseudopodia. There were more than 90% of F4 /80 positive cells expressing CD11 in bone marrow cells by FCM. ELISA and qRT-PCR results indicated that M1 macrophage upregulated the expression of IL-12,TNF-α,i NOS,whereas M2 macrophage upregulated the expression of IL-4,TGF-β,IL-10,Arg-1. After MTB Hsp16. 3 stimulated macrophages,the expression of IL-10,TGF-β were upregulated,the expression of TNF-α and i NOS were downregulated both by M0 and M1 macrophages at the 48-72 h to the peak. Conclusion： Together,these data indicated that we successfully derived M1 and M2 macrophages from murine bone marrow-derived macrophage. Mycobacterium tuberculosis Hsp16. 3 promoted M1 macrophages expression of the M2 phenotype markers and the M1 phenotype macrophages to become M2 macrophage-like.

关 键 词：结核分枝杆菌 巨噬细胞 结核分枝杆菌热休克蛋白16.3 

分 类 号：R392.11[医药卫生—免疫学]