浙江大学学报（医学版）
浙江大學學報（醫學版）
절강대학학보（의학판）
JOURNAL OF ZHEJIANG UNIVERSITY MEDICAL SCIENCES
2014年
2期
200-206
,共7页
徐倩%金梦媚%郑文文%朱丽%徐水凌
徐倩%金夢媚%鄭文文%硃麗%徐水凌
서천%금몽미%정문문%주려%서수릉
分枝杆菌, 结核%Toll样受体%信号传导%树突细胞%核因子-κB%肿瘤坏死因子α%细胞,培养的%小鼠
分枝桿菌, 結覈%Toll樣受體%信號傳導%樹突細胞%覈因子-κB%腫瘤壞死因子α%細胞,培養的%小鼠
분지간균, 결핵%Toll양수체%신호전도%수돌세포%핵인자-κB%종류배사인자α%세포,배양적%소서
Mycobacterium tuberculosis%Toll-like receptors%Signal transduction%Dendritic cells%Nuclear factor-κB%Tumor necrosis factor-alpha%Cells,cultured%Mice
目的：研究人结核分枝杆菌侵入抗原提呈细胞后诱导树突细胞（ DC ）成熟的机制。方法：选择小鼠DC2．4细胞株为抗原提呈细胞的效应细胞，建立人结核分枝杆菌H37 Rv株的细胞混合培养模型。在不同混合培养时段，采用实时荧光定量PCR检测DC2．4细胞Toll样受体2（TLR2）、TLR4 mRNA的表达量；蛋白质印迹法检测DC的核因子κB （ NF-κB ） p65蛋白的表达；免疫酶联吸附试验定量检测DC产生α肿瘤坏死因子（ TNF-α）的水平；采用间接免疫荧光法和流式细胞术检测DC2．4表面CD80和CD86的表达情况。结果：H37 Rv与DC2．4共培养2 h后开始有细菌侵入；共培养6、8、10、12 h后，细菌侵入率分别为（37．9±5．6）％、（51.2±7．6）％、（57．2±8．9）％、（63.9±12．4）％；TLR2、TLR4 mRNA也开始增量，共培养10 h时达高峰，之后开始下降；共培养6 h时，NF-κB p65的表达量明显增高；TNF-α的表达量在共培养6 h开始上调，8 h时达高峰，随后逐渐下降；共培养6 h时，DC2．4表面CD80、CD86表达量明显增高。结论：人结核分枝杆菌侵入DC2．4时，通过激活TLR2/4-NF-κB 信号通路，诱导 DC 成熟，提高其抗原提呈能力。
目的：研究人結覈分枝桿菌侵入抗原提呈細胞後誘導樹突細胞（ DC ）成熟的機製。方法：選擇小鼠DC2．4細胞株為抗原提呈細胞的效應細胞，建立人結覈分枝桿菌H37 Rv株的細胞混閤培養模型。在不同混閤培養時段，採用實時熒光定量PCR檢測DC2．4細胞Toll樣受體2（TLR2）、TLR4 mRNA的錶達量；蛋白質印跡法檢測DC的覈因子κB （ NF-κB ） p65蛋白的錶達；免疫酶聯吸附試驗定量檢測DC產生α腫瘤壞死因子（ TNF-α）的水平；採用間接免疫熒光法和流式細胞術檢測DC2．4錶麵CD80和CD86的錶達情況。結果：H37 Rv與DC2．4共培養2 h後開始有細菌侵入；共培養6、8、10、12 h後，細菌侵入率分彆為（37．9±5．6）％、（51.2±7．6）％、（57．2±8．9）％、（63.9±12．4）％；TLR2、TLR4 mRNA也開始增量，共培養10 h時達高峰，之後開始下降；共培養6 h時，NF-κB p65的錶達量明顯增高；TNF-α的錶達量在共培養6 h開始上調，8 h時達高峰，隨後逐漸下降；共培養6 h時，DC2．4錶麵CD80、CD86錶達量明顯增高。結論：人結覈分枝桿菌侵入DC2．4時，通過激活TLR2/4-NF-κB 信號通路，誘導 DC 成熟，提高其抗原提呈能力。
목적：연구인결핵분지간균침입항원제정세포후유도수돌세포（ DC ）성숙적궤제。방법：선택소서DC2．4세포주위항원제정세포적효응세포，건립인결핵분지간균H37 Rv주적세포혼합배양모형。재불동혼합배양시단，채용실시형광정량PCR검측DC2．4세포Toll양수체2（TLR2）、TLR4 mRNA적표체량；단백질인적법검측DC적핵인자κB （ NF-κB ） p65단백적표체；면역매련흡부시험정량검측DC산생α종류배사인자（ TNF-α）적수평；채용간접면역형광법화류식세포술검측DC2．4표면CD80화CD86적표체정황。결과：H37 Rv여DC2．4공배양2 h후개시유세균침입；공배양6、8、10、12 h후，세균침입솔분별위（37．9±5．6）％、（51.2±7．6）％、（57．2±8．9）％、（63.9±12．4）％；TLR2、TLR4 mRNA야개시증량，공배양10 h시체고봉，지후개시하강；공배양6 h시，NF-κB p65적표체량명현증고；TNF-α적표체량재공배양6 h개시상조，8 h시체고봉，수후축점하강；공배양6 h시，DC2．4표면CD80、CD86표체량명현증고。결론：인결핵분지간균침입DC2．4시，통과격활TLR2/4-NF-κB 신호통로，유도 DC 성숙，제고기항원제정능력。
Objective: To investigate the mechanism of Mycobacterium tuberculosis invasion to mouse dendritic cells ( DC ) .Methods: Mycobacterium tuberculosis strain H37Rv was co-cultured with mouse DC2.4 cells.The mRNA expression of Toll-like receptor 2/4(TLR2/4) in DC2.4 cells was detected by fluorescent quantitative real-time PCR and the protein expression of nuclear factor κB ( NF-κB ) was assessed by Western blotting .The extracellular concentration of tumor necrosis factor α( TNF-α) was measured by ELISA methods during Mycobacterium Tuberculosis invasion .Indirect immunofluorescent staining and flow cytometry assay were used to detect the expression of CD80 and CD86 on DC2 .4 cells before and after invasion .Results:The invasion of Mycobacterium tuberculosis in DC2.4 cells was observed after 2 h of co-incubation.The rates of invasion were (37.9 ±5.6)%,(51.2 ±7.6)%,(57.2 ±8.9)%and(63.9 ± 6.8)% at 6,8,10 and 12 h after co-incubation,respectively.The mRNA expression level of TLR2 /4 was significantly increased at 6 h but decreased at 10 h after co-incubation.The expressions of NF-κB p65 and TNF-αwere higher in DC2.4 cells after being invaded by 6 , 8 , and 10 h and then gradually decreased .CD80 and CD86 expression were increased on DC 2 .4 at 6 h after co-incubation .Conclusion:Invasion of Mycobacterium tuberculosis strain H37 Rv to DC might enhance its antigen-presenting function through activation of TLR 2/4-NF-kB signaling pathway .
