梅毒螺旋体诱导巨噬细胞分泌的外泌体特征及其对人脐静脉内皮细胞增殖的影响

Characteristics of Treponema pallidum -induced macrophage- derived exosomes and its effect on proliferation of human umbilical vein endothelial cells

目的探讨梅毒螺旋体（Tp）体外诱导巨噬细胞分泌的外泌体特征及其对人脐静脉内皮细胞（HUVEC）增殖水平的影响。方法自雄兔睾丸分离Tp。将人单核巨噬细胞（THP-1）诱导为巨噬细胞后，分为实验组（Tp刺激）和对照组（不用Tp刺激），12h后继续培养48h，收集巨噬细胞外泌体悬液，利用差速离心法和膜亲和试剂盒提取外泌体，运用透射电镜、Western印迹鉴定外泌体，Nanoparticle tracking analysis（NTA）粒径分析仪定量分析外泌体。将HUVEC分为实验组、对照组和外泌体洗脱液组，分别用外泌体悬液（4．5×10^8颗粒，实验组和对照组）、外泌体洗脱液刺激培养一定时间后，激光共聚焦扫描显微镜观察HUVEC对外泌体的吞噬，CCK8法检测HUVEC增殖活性。结果透射电镜下的外泌体呈杯托样，为直径30—100nm的微小囊泡。Western印迹显示，外泌体丰富表达膜蛋白CD63、CD9和CD81。相同实验条件下，NTA测定显示，实验组与对照组外泌体粒径（u=1．90，P〉0．05）、浓度（z=-1．604，P=0．109）差异均无统计学意义。两组外泌体分别与HUVEC共培养5h后，激光共聚焦显微镜下见胞内散在分布被吞噬的绿色荧光外泌体。实验组、对照组外泌体分别作用于HUVEC12h后细胞增殖水平均高于外泌体洗脱液组，差异均有统计学意义（P〈0．05）；在24h和48h时对照组细胞增殖水平仍高于外泌体洗脱液组，在48h达到峰值（P〈0．05），实验组细胞增殖水平趋于正常；在72h时实验组、对照组细胞增殖水平与外泌体洗脱液组差异无统计学意义（P〉0．05）。结论THP．1来源的巨噬细胞分泌的外泌体在48h内能显著提高HUVEC细胞增殖水平，在48h达到峰值。在Tp刺激下，THP-1来源的巨噬细胞分泌的外泌体在形态、大小、浓度上与无Tp刺激巨噬细胞分泌的外泌体相似，但仅在短时间（12h）内提高HUVEC增殖水平。

Objective To investigate characteristics of Treponema pallidum （Tp）- induced macrophage-derived exosomes and its effect on the proliferation of human umbilical vein endothelial cells （HUVEC）. Methods Tp strains were collected from the testis of male rabbit, which were infected with Tp （Nichols strain）. The human mononuclear macrophages （THP- 1） were induced into macrophages by incubation with propylene glycol methyl ether acetate （PMA）, and then the macrophages were divided into 2 groups： experimental group incubated with Tp for 12 hours followed by 48- hour normal culture, and control group receiving normal culture. After the treatment, exosome suspensions were collected, and exosomes were extracted by differential centrifugation and exoEasy Maxi Kit. Transmission electron microscopy and Western blot analyses were performed to identify the exosomes, and nanoparticle tracking analysis （NTA） was conducted to measure the diameters and concentrations of exosomes. In vitro cultured HUVECs were divided into 3 groups, which were cultured with the 10 μl of suspensions containing exosomes derived from Tp-stimulated macrophages at a concentration of 4.5 ×10^8/ml （experimental group）, 10 μl of suspensions containing exosomes derived from untreated macrophages at a concentration of 4.5×10^8/ml （control group）, and 10 μ1 of exosome eluents （exosome eluent group）, respectively. After the treatment, confocal laser scanning microscopy was performed to observe the phagocytosis of exosomes of HUVECs, and cell counting kit-8 to evaluate the proliferative activity of HUVECs. Results The exosomes were saucer-like microvesicles with diameters of 30 - 100 nm under the transmission electron microscope. Western blot analyses showed that membrane proteins CD63, CD9 and CD81 were abundantly expressed by exosomes. Under the same conditions, NTA revealed that there were no significant differences in the particle diameter （u = 1.90, P 〉 0.05） and concentration of exosomes （Z = -1.604, P = 0.109） between the experimental group and the control group. After co- culture with HUVECs for 5 hours, confocal laser scanning microscopy showed scatteredly distributed exosomes with green fluorescence in the HUVECs in the experimental group and control group. After 12-hour co-culture with the exosome suspensions, the proliferative activity of HUVECs was significantly higher in the experimental group and the control group than in the exosome eluent group （both P 〈 0.05）. After 24- and 48- hour treatment with exosome suspensions, the proliferative activity of HUVECs in the control group was still significantly increased compared with that in the exosome eluent group, and peaked at 48 hours （all P 〈 0.05）. Moreover, there were no significant differences in the proliferative activity of HUVECs between the experimental group and exosome eluent group at 24 and 48 hours （both P 〉 0.05）. At 72 hours, no significant differences in the proliferative activity of HUVECs were observed among the experimental group, the control group and the exosome eluent group （all P 〉 0.05）. Conclusions The exosomes secreted by THP-I cells- derived macrophages evidently increased the proliferative activity of HUVECs within 48 hours, which peaked at 48 hours. After the stimulation with Tp, the exosomes secreted by THP- 1 cells-derived macrophages were similar to those without Tp stimulation in morphology, size and concentration, and only increased the proliferative activity of HUVECs within 12 hours.