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

Role of phospholipase C in cytoskeleton rearrangements of dendritic cells invaded by Mycobacterium tuberculosis

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。

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 ceils 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 〈 O. 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 eytomembrane of DC2. 4 cells, but not in cytoplasm. Conclusions： Mycobacterium tuberculosis can provoke to F-aetin rearrangements through PLC molecule, which would further lead to Mycobacterium tuberculosis invasion of DC2.4 cells.