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目的:在体外细胞水平,初步探讨TLR2/4在MTB Hsp16.3刺激小鼠骨髓来源巨噬细胞的表达及作用。方法:从BALB/c小鼠的胫腓骨中取骨髓细胞,与GM-CSF共培养获得骨髓来源的M0型巨噬细胞,流式检测F4/80和CD11b的表达并观察其形态;100 ng/ml MTB Hsp16.3刺激M0巨噬细胞,分别培养0、12、24、36、48、72 h,Real-time PCR检测不同时间点TLR2/4的表达水平;利用RNAi干扰技术沉默巨噬细胞表面的TLR2/4受体,MTB Hsp16.3刺激沉默TLR2/4的M0巨噬细胞,分别培养0、12、24、36、48、72 h,Real-time PCR检测不同时间点TLR2/4及Ym-1、Fizz1、IL-10、TNF-α、i NOS、TGF-β细胞因子的表达水平。结果:M0型巨噬细胞在MTB Hsp16.3刺激后出现了较短的伪足,沉默TLR2/4的M0型巨噬细胞在MTB Hsp16.3刺激后出现了较长伪足;M0巨噬细胞在MTB Hsp16.3刺激后高表达TLR2/4。MTB Hsp16.3刺激沉默TLR2/4的M0型巨噬细胞,高表达M1型巨噬细胞相关的细胞因子IL-6、TNF-α、i NOS,低表达M2型巨噬细胞相关的细胞因子IL-10、TGF-β、Ym-1、Fizz1。结论:MTB Hsp16.3通过TLR2/4促进M0型巨噬细胞向M2型转化,抑制M1型巨噬细胞,这可能参与了MTB躲避巨噬细胞的吞噬过程。
OBJECTIVE: To investigate the expression and effect of TLR2 / 4 on murine bone marrow-derived macrophages stimulated by MTB Hsp16.3 in vitro. Methods: Bone marrow cells were obtained from the tibia and fibula of BALB / c mice and co-cultured with GM-CSF to obtain bone marrow-derived M0 macrophages. The expression of F4 / 80 and CD11b was detected by flow cytometry. ml MTB Hsp16.3 stimulated M0 macrophages were cultured 0,12,24,36,48,72 h, Real-time PCR detection of TLR2 / 4 expression at different time points; the use of RNA interference technology to silence the surface of macrophages TLR2 / 4 and MTB Hsp16.3 stimulated TLR2 / 4 M0 macrophages were cultured 0,12,24,36,48,72 h, Real-time PCR detection of TLR2 / 4 at different time points and Ym -1, Fizz1, IL-10, TNF-α, i NOS and TGF-β cytokines. RESULTS: M0-type macrophages showed short pseudopodia after stimulation with MTB Hsp16.3. M0-type macrophages with TLR2 / 4 silencing showed longer pseudopodia after stimulation with MTB Hsp16.3. M0 macrophages TLR2 / 4 is overexpressed after MTB Hsp16.3 stimulation. MTB Hsp16.3 stimulated TLR2 / 4-overexpressing M0 macrophages, which expressed high levels of IL-6, TNF-α, and iNOS associated with M1 macrophages, cytokines IL associated with low expression of M2 macrophages -10, TGF-β, Ym-1, Fizz1. CONCLUSION: MTB Hsp16.3 can promote the transformation of M0 macrophages into M2 by TLR2 / 4 and inhibit M1 macrophages, which may be involved in the phagocytosis of MTB by macrophages.