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-aetin 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-aetin 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.%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2h,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2h后,DC2.4细胞膜中的PLC分子表达即开始升高,至8h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.
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