钙离子及信号转导和转录激活因子3信号分子在创伤弧菌临床分离株 B2侵入树突状细胞中的作用

The effects of intracellular calcium concentration and signal transducers and activators of transcription 3 on the invasion of Vibrio vulnificus clinical isolates B2 into dendritic cells

目的：探讨创伤弧菌临床分离株 B2侵入树突状细胞（DC）过程中细胞内钙离子[Ca2＋]i浓度的改变以及信号转导和转录激活因子3（STAT3）信号分子的作用。方法建立创伤弧菌 B2株与DC 2．4细胞的混合培养模型，流式细胞术检测不同侵入时间段内细胞凋亡率和坏死率，荧光探针 Fluo-8-AM 观察[Ca2＋]i 的改变，Western 印迹和免疫荧光技术检测 STAT3和磷酸化 STAT3（p-STAT3，705位点）相对分子表达量和细胞内分布。数据分析采用 t 检验。结果创伤弧菌 B2株与 DC 2．4细胞混合培养1、2、3和4 h 后，其细胞凋亡率分别为（13．10±4．72）％、（30．10±3．52）％、（46．20±15．61）％和（31．00±19．10）％，创伤弧菌1．1758株与创伤弧菌 B2株细胞凋亡率比较差异有统计学意义（t 值分别为4．30、22．33、4．30和4．30，P 值分别为0．040、0．002、0．040和0．040）；同时细胞坏死率分别为（19．70±3．50）％、（39．20±4．60）％、（40．90±13．80）％和（62．10±8．20）％，创伤弧菌1．1758株与创伤弧菌 B2株细胞坏死率比较差异有统计学意义（t 值分别为9．93、14．09、4．30和14．09，P 值分别为0．010、0．005、0．049和0．005）。与创伤弧菌1．1758株相比，创伤弧菌 B2株能在相同时间内引起更为明显的细胞凋亡和坏死。荧光显微镜下观察到[Ca2＋]i 的荧光强度随着细胞凋亡的增加而增强。STAT3分子的总蛋白量有所增加，但 p-STAT3（705位点）相对分子表达量则呈下降趋势，与对照组相比 p-STAT3（705位点）分子在细胞内的分布未见明显改变。结论创伤弧菌 B2株在侵入 DC 过程中，升高[Ca2＋]i 的同时抑制 STAT3（705位点）磷酸化，有可能是创伤弧菌 B2株诱导细胞快速凋亡和坏死的重要机制。

Objective To explore the effects of intracellular calcium concentration （[Ca2 ＋ ]i ）and signal transducers and activators of transcription 3 （STAT3 ）signaling pathway on the invasion of Vibrio vuknificus （Vv ）clinical isolates B2 into dendritic cells （DC）.Methods The co-culture model of Vv B2 and DC 2.4 was constructed.Apoptosis and necrosis rates in different invasion phases were detected by flow cytometry.The calcium fluorescence probe Fluo-8-AM was used to detect the change of [Ca2 ＋ ]i ,and Western blot and immunofluorescent techniques were used to explore the relative molecular expressions and intracellular distributions of STAT3 and phosphorylation STAT3 [p-STAT3 （705 ）].Data were analyzed by t-test.Results After co-culture ofVv B2 and DC 2.4,the apoptosis rates of 1 ,2,3 and 4 h were （13.10±4.72）%,（30.10±3.52）%,（46.20±15 .61）% and（31 .00 ±19.10）%,respectively.The differences were statistical significant between Vv standard strain （1 .1758）and Vv B2 （t=4.30,22.33, 4.30 and 4.30,respectively;P =0.040,0.002,0.040 and 0.040,respectively）.The necrosis rates of 1 , 2,3 and 4 h in co-culture of Vv B2 and DC 2.4 were（19.70 ±3.50 ）%,（39.20±4.60 ）%,（40.90 ± 13.80）% and （62.10 ± 8.20 ）%,respectively.The differences were statistical significant between Vv 1 .1758 andVv B2 （t=9.93,14.09,4.30 and 14.09,respectively;P =0.010,0.005 ,0.049 and 0.005 , respectively）.Compared to Vv 1 .1758,Vv B2 induced more apparent cell apoptosis and necrosis within the same time.By fluorescence microscope,intensity of calcium was enhanced with the rising of apoptosis. Although the total expression of STAT3 increased,the relative molecular expression of p-STAT3 （705 ） decreased.There was no significant difference in its distribution in cells compared to the blank group. Conclusion The rapid apoptosis and necrosis of DC during Vv B2 invasion into DC may be caused by increasing [Ca2 ＋ ]i and inhibiting phosphorylation of p-STAT3 （705）simultaneously.