不同毒力马耳他种布鲁氏菌体内外诱导细胞凋亡的研究

摘要

To explore whether differ virulent strains of B.melitensis could induce apoptosis and the differences in inducing apoptosis in vivo and in vitro,Murine macrophage cell line RAW264.7 was infected with virulent strain M28 and vaccine strain M5-90 at MOI=100,the apoptosis of cell line was observed via confocal microscope and measured on flow cytometer by detecting of the apoptotic products caspase3/7 expressed in process of the cell cultivating.Results showed that M5-90 and M28 could induce apoptosis of RAW264.7 after infection,and M5-90 had the highen ability of inducing cell apoptosis than that of M28.Then,mice were intraperitoneally infected with M28 and M5-90 in a dose of 1 ×106 cfu,and killed by cervical dislocation at 3 days,7 days and 42 days post inoculation.The spleens sample were collected aseptically to made sections followed by stain with HE.Apoptosis induced by Brucella M5-90 and M28 in vivo were further observed morphologically by electron microscopy and verified by Tunel assay.Results showed that both M28 and M5-90 could induce apoptosis in mice spleen,and the majority of apoptotic cell was T cells.This work provids an important basis for further investigating the mechanism of apoptosis induced by Brucella and the pathogenic mechanism of Brucells.%为探究不同毒力马耳他种布鲁氏菌能否在体内外诱导凋亡以及诱导凋亡程度的差异,本研究将M28(强毒株)和M5-90(疫苗株)以MOI=100感染小鼠巨噬细胞系RAW264.7,利用激光共聚焦显微镜观察和流式细胞术检测凋亡产物caspase3/7.结果显示:M5-90和M28在体外细胞水平均能够诱导RAW264.7细胞凋亡,且M5-90促进细胞凋亡能力高于M28.然后将M28和M5-90以1×106 cfu的剂量接种BALB/c小鼠,分别在接种后3d、7d和42 d迫杀小鼠,取小鼠脾脏组织制备切片,经HE染色后进行形态学观察,利用透射电镜观察并通过Tunel法检测细胞凋亡.结果显示M5-90和M28感染后均可诱导小鼠脾脏细胞凋亡,凋亡细胞主要为T淋巴细胞.本研究为布鲁氏菌诱导宿主细胞凋亡和布鲁氏菌致病机理研究奠定了基础.