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Journal of ZheJiang University(Medical Science)  2013, Vol. 42 Issue (2): 184-191    DOI: 10.3785/j.issn.1008-9292.2013.02.009
Role of phospholipase C in cytoskeleton rearrangements of dendritic cells invaded by Mycobacterium tuberculosis
XU Shui-Ling1, XU Yan2, HUANG Jia1, FAN Hong-Yan1, JIN Meng-Mei1
Medical School of Jiaxing College,Jiaxing 314001,China
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Objective: To investigate the role of phospholipase C(PLC)in cytoskeleton rearrangement of mouse dendritic cells invaded by Mycobacterium tuberculosis.
Methods: Mouse dendritic DC2.4 cells were co-cultured with Mycobacterium tuberculosis H37Rv.F-actin of DC2.4 cells were strained with palloidin-TRITC,the microtubule was stained with anti-β-tubulin monoclonal antibody and FITC-conjugated affnipure anti-mouse IgG.The changes of cytoskeleton in DC2.4 cells induced by Mycobacterium tuberculosis H37Rv were determined by fluorescence microscopy and the rates of F-actin rearrangements were calculated.The expressions of PLC in cytoplasm and cytomemberane of DC2.4 cells were measured by ELISA.DC2.4 cells were pretreated with PLC inhibitor U73122,then F-actin rearrangements induced by invasion of Mycobacterium tuberculosis were observed.
Results: Bacterial invasion was observed while DC2.4 cells were co-incubated with Mycobacterium tuberculosis H37Rv for 2 h.The rates of invasion were(26.1±4.5)%,(39.9±5.6)%,(51.2±5.9)%,(57.9±6.1)% and(63.9±6.8)% at 4,6,8,10 and 12 h of co-culture,respectively; while those were(13.6±3.1)%,(14.2±3.9)%,(15.1±4.3)%,(16.8±4.0)% and(18.3±5.2)% after blocked by PLC,respectively.The rates of the F-actin rearrangements at 2,4,6,8,10 and 12 h after DC2.4 cells were invaded by H37Rv were(26.9±1.5)%,(59.3±2.8)%,(72.7±4.8)%,(78.2±5.9)%,(63.3±2.9)% and (43.2±2.6)%,respectively; while those were(18.5±1.2)%,(22.3±1.7)%,(23.6±2.5)%,(24.8±2.3)%,(22.3±1.3)% and (23.8±1.8)% after blocked by PLC,respectively.There were no changes of the microtubule observed in DC2.4 cells at the same time points.The rates of the F-actin rearrangements before blocked by PLC were higher than those after PLC blockade at 4,6,8 and 10 h(P<0.05).The expressions of PLC in cytomembrane in DC2.4 cells increased after 2 h and reached its highest level at 8 h.The PLC inhibitor U73122 inhibited the expressions of PCL in cytomembrane of DC2.4 cells,but not in cytoplasm.
Conclusions: Mycobacterium tuberculosis can provoke to F-actin rearrangements through PLC molecule,which would further lead to Mycobacterium tuberculosis invasion of DC2.4 cells.

Key wordsMycobacterium tuberculosis      Dendritic cells/cytology      Type C phospholipases      Cytoskeleton/ultrastructure      Gene rearrangement      Mouse dendritic cell      Cytoskeleton      rearrangement      Phospholipase C     
Received: 15 March 2012      Published: 25 March 2013
CLC:  R 378.91  
Cite this article:

XU Shui-Ling, XU Yan, HUANG Jia, FAN Hong-Yan, JIN Meng-Mei. Role of phospholipase C in cytoskeleton rearrangements of dendritic cells invaded by Mycobacterium tuberculosis. Journal of ZheJiang University(Medical Science), 2013, 42(2): 184-191.

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目的:研究结核分枝杆菌(Mycobacterium tuberculosis)侵入小鼠树突状细胞株(DC2.4)时细胞骨架微丝、微管变化及其与磷脂酶C(PLC)分子的关系。
结果:结核分枝杆菌H37Rv株与DC2.4细胞共育2 h,即见有细菌侵入,共育4、6、8、10、12 h后,H37Rv侵入率分别为(26.1±4.5)%、(39.9±5.6)%、(51.2±5.9)%、(57.9±6.1)%和(63.9±6.8)%;H37Rv侵入2、4、6、8、10、12 h时,F-actin重排百分率分别为(26.9±1.5)%、(59.3±2.8)%、(72.7±4.8)%、(78.2±5.9)%、(63.3±2.9)%和(43.2±2.6)%,而PLC信号分子阻断后,DC2.4细胞的侵入率则分别为(13.6±3.1)%、(14.2±3.9)%、(15.1±4.3)%、(16.8±4.0)%和(18.3±5.2)%,F-actin重排率也分别为(18.5±1.2)%、(22.3±1.7)%、(23.6±2.5)%、(24.8±2.3)%、(22.3±1.3)%和(23.8±1.8)%,而微管变化不大;混合培养4、6、8、10 h,PLC信号通路阻断前的侵入率和F-actin重排率明显高于PLC分子阻断后(P<0.05)。侵入2 h后,DC2.4细胞膜中的PLC分子表达即开始升高,至8 h时达最高;U73122可抑制DC2.4细胞膜中PLC分子的表达,而对细胞浆中的PLC分子影响不大。

关键词: 分枝杆菌,  结核,  树突细胞/细胞学,  C型磷脂酶类,  细胞骨架/超微结构,  基因重排,  小鼠树突状细胞,  细胞骨架,  重排,  磷脂酶C 

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