创伤弧菌经细胞Toll样受体2、4途径导致树突状细胞急性坏死的研究

Vibrio vulnificus induces acute necrosis of dendritic cells via toll-like receptor 2 and 4 pathways

目的探索创伤弧菌（Vv）感染树突状细胞（DC），经Toll样受体（TLR）2、4途径致DC急性坏死的实验研究。方法建立Vv1．1758株与DC2．4混合培养感染模型，用光学显微镜观察细胞感染率，电子显微镜观察Vv定位与细胞结构变化，实时定量反转录PCR测定TLR2、4mRNA表达量，ELISA法检测TNF-α表达量，DNA梯度电泳定性检测细胞凋亡，流式细胞术定量检测细胞凋亡及坏死率。率的比较采用X^2检验，多组间数据比较采用方差分析。结果混合培养0．5、1、2、4、6h时的细胞感染率分别为（7．8±0．8）％、（13．9±1．1）％、（34．6±4．9）％、（77．8±10．2）％和（95．8±13．1）％，混合培养2h后的感染率与培养0．5h相比差异有统计学意义（均P〈0．05）。细菌定位于DC2．4细胞内侧，2h核染色质明显活跃，核内出现凋亡小体；4h胞质内出现空泡，染色质聚集，胞膜毁损严重；6h线粒体高度肿胀变形，细胞坏死。TLR2、4mRNA表达于0．5h已达峰值。TNF-α在1h时开始增高（P〈0．05），2h达峰值。DNA梯度电泳2h呈冲刷状坏死，4-5h出现720bp与900bp凋亡带。2、4、6h时细胞早期凋亡率分别为（3．1±3．8）％、（7．8±4．7）％和（12．7±8．2）％，显著高于对照组的（0．5±0．7）％（均P〈0．05）。细胞坏死率分别为（16．7±12．5）％、（41．6±25．9）％和（75．5±33．6）％，显著高于对照组的（2．3±0．8）％（均P〈0．05）。结论Vv感染DC可通过TLR2、4表达上调，TNF-α增加导致DNA降解，期间以细胞凋亡与坏死同时并存，但以坏死方式降解为主。

Objective To explore how Vibrio vulnificus （Vv） invades dendritic cells （DC） and induces acute necrosis of DC via toll-like receptor （TLR） 2 and 4 pathways. Methods Vv 1. 1758 strain and DC 2.4 mixed culture model was established, observed the infection rates of DC with optical microscope, the location of Vv and structural changes of DC by transmission electron microscope. The expression levels of TLR2 and TLR4 mRNA were determined by real-time quantitative reverse transcription polymerase chain reaction （RT-qPCR） and tumor necrosis factor-a （TNF a） protein titers were measured by enzyme-linked immunosorbent assay （ELISA）. DNA ladder qualitative test was used to detect cell apoptosis, while flow cytometry was used to quantify cell apoptosis and necrosis rates. Statistical analysis was done by chi-square test and one-way ANOVE. Results The infectionrates of DCafter0.5, 1, 2, 4 and6 h of mixed culture were （7.8±0.8）%, （13.9±1.1）%, （34.6±4.9）%, （77.8±10.2）% and （95.8 ±13.1）%, respectively. Vv was generally located in the internal cell membrane of DC 2. 4. After 2 h co culture, nuclear chromatins of DC became active and intranuelear apoptosis bodies appeared. After 4 h, cytoplasmic vacuoles appeared, chromatin gathered, and cell membranes were seriously damaged. After 6 h, mitochondria was highly swelled and distorted, and cell apoptosis and necrosis occurred. TLR2 and TLR4 mRNA levels reached peak values after co culture for 0.5 h; TNF-α level began to increase at 1 h （P〈0.05） and reached peak values at 2 h. DNA Ladder electrophoresis presented scouring necrosis after 2 h culture and apoptotic bands appeared between 720 bp and 900 bp after 4 to 5 h culture. Early apoptosis rates of DC after 2, 4 and 6 h culture were （3.1±3. 8）%, （7.8±4. 7）% and （12. 7±8.2）%, and necrosis rates of DC were （16.7±12.5）%, （41.6±25.9）% and （75.5±33.6）%, higher than that of control group （all P〈0.05）. Conclusions Vv infects DC and induce DNA degradation through up regulated expression of TLR2 and TLR4 and increasing of TNF-α inflammatory mediators. During cell degradation, apoptosis and necrosis coexist, while necrosis is predominant.