关键词: 苍白密螺旋体, 巨噬细胞, 外泌体, 人脐静脉内皮细胞, 细胞增殖

Abstract: Xu Bufang, Wang Qianqiu, Zhang Ruili, Zhang Jinping Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China （Xu BF, Wang QQ）; Department of Dermatology, Wuxi Second Affiliated Hospital of Nanjing Medical University, Wuxi 214002, China （Zhang RL）; Department of Venereology, Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China （Zhang JP） Corresponding authors: Wang Qianqiu, Email: doctorwqq@hotmail.com; Zhang Ruili, Email: reallyvictor@126.com 【Abstract】 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 × 108/ml （experimental group）, 10 μl of suspensions containing exosomes derived from untreated macrophages at a concentration of 4.5 × 108/ml （control group）, and 10 μl 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-1 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.

Key words: Treponema pallidum, Macrophages, Exosomes, Human umbilical vein endothelial cells, Cell proliferation