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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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Abstract  

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.

URL:

http://www.zjujournals.com/med/10.3785/j.issn.1008-9292.2013.02.009     OR     http://www.zjujournals.com/med/Y2013/V42/I2/184


磷脂酶C分子在结核分枝杆菌触发树突状细胞细胞骨架重排中的作用

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。
方法:建立人结核分枝杆菌H37Rv株DC2.4细胞混合培养模型。采用罗丹明标记的鬼笔环肽(Palloidin-TRITC)染细胞F-actin,用抗微管蛋白β亚单位的小鼠一抗和荧光素标记的兔抗小鼠二抗染细胞微管,检测H37Rv株侵入DC2.4细胞时细胞骨架的变化情况,并计算F-actin重排率。采用酶联免疫吸附法(ELISA)检测DC2.4细胞浆和细胞膜中PLC分子的表达。采用PLC分子抑制剂U73122预处理DC2.4细胞,观察H37Rv株侵入率变化以及对细胞骨架变化的影响。
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。
结论:结核分枝杆菌可通过激活DC2.4细胞PLC分子触发F-actin细胞骨架重排,从而侵入DC2.4细胞,且PLC分子的表达主要存在于DC2.4细胞膜上。


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 

[1]RAJA A.Immunology of tuberculosis
[J].Indian J Med Res,2004,120:213-232.

[2]KAUFMANN S H.Tuberculosis: back on the immunologists′ agenda
[J].Immunity,2006,24:351-357.

[3]REECC S T,KAUFMANN S H.Rational design of vaccines against tuberculosis directed by basic immunology
[J].Int J Med Microbiol,2008,298:143-150.

[4]PHILPOTT D J,CANTEY,SHERMAN P M,et al.Increased adherence of Escherichia coli RDEC-1 to human tissue culture cells results in the activation of host signaling pathways
[J].J Infect Dis,1995,172:136-143.

[5]LIN M,ZHU M X,RIKISA Y.Rapid activation of protein tyrosine kinase and phospholipase C-  2 and increase in cytosolic free calcium are required by Ehrlichia chaffeensis for internalization and growth in THP-1 cells
[J].Infect Immun,2002,70:889- 898.

[6]ELENA G,ELISABETTA I,LUCIETTA F,et al.Infection of human macrophages and dendritic cells with Mycobacterium tuberculosis induces a differential cytokine gene expression that modulates T cell response
[J].J Immunol,2001,166:7033-7041.

[7]ISABELLE P,ALAIN L,SERVIN,et al.Piracy of decay-Accelerating factor(CD55) signal transduction by the diffusely adhering strain Escherichia coli C1845 promotes cytoskeletal F-actin rearrangements in cultured human intestinal INT407 cells
[J].Infect Immun,1998,66:4036-4042.

[8]AKOMPONG T,SPENCER R L,MCEWEN B S.Glucocorticoids inhibit soluble phospholipase C activity and cytosolic guanine nucleotide regulatory protein-α1 immunoreactivity in spleen
[J].Endocrinology,1993,133:1963 - 1970.

[9]MIZUSHIMA N,LEVINE B,CUERVO A M,et al.Autophagy fights disease through cellular selfdigestion
[J].Nature,2008,451:1069-1075.

[10]WADSWORTH S J,GOLDFINE H. Listeria monocytogenes phospholipase C-dependent calcium signaling modulates bacterial entry into J774 macrophage-like cells
[J].Infect Immun,1999, 67:1770-1778.
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