布鲁菌外膜蛋白16对成骨细胞的毒力作用  

Toxic effect of Brucella outer membrane protein 16 on osteoblasts

作　　者：任慧 杨恒 胡飞环 易嘉敏 黎诚耀 王文敬 Ren Hui;Yang Heng;Hu Feihuan;Yi Jiamin;Li Chengyao;Wang Wenjing(Department of Transfusion Medicine,College of Laboratory Technology and Biotechnology,Southern Medical University,Guangzhou 510515,China;Department of Blood Transfusion,Zhujiang Hospital,Southern Medical University,Guangzhou 510280,China;Department of Blood Transfusion,Guangdong Provincial People's Hospital,Guangzhou 510080,China;Guangzhou University of Traditional Chinese Medicine,Guangzhou 510400,China)

机构地区：[1]南方医科大学检验与生物技术学院输血医学系,广东广州510515 [2]南方医科大学珠江医院输血科,广东广州510280 [3]广东省人民医院输血科,广州510080 [4]广州中医药大学,510400

出　　处：《中华地方病学杂志》2021年第6期448-453,共6页Chinese Journal of Endemiology

基　　金：国家重点研发计划(2017YFD0500300);南方医科大学2017年度"科研启动计划"(CX2017N007);南方医科大学2018年度省级大学生创新创业训练计划(201812121114)。

摘　　要：目的观察布鲁菌外膜蛋白(OMP)16脂化型(L16)和非脂化型(U16)对成骨细胞的毒力效应。方法利用大肠埃希菌BL21(DE3)原核表达系统制备L16和U16重组蛋白,并使用镍柱纯化。采用成组设计,利用小鼠成骨细胞系(MC3T3细胞)分别与L16和U16重组蛋白共孵育(L16、U16刺激组),以布鲁菌脂多糖(LPS)刺激物为阳性对照(LPS对照组),未加任何刺激的细胞作为阴性对照,孵育时间为24 h。CCK-8法检测MC3T3细胞的活力;离心收集培养细胞上清,生物发光法测定上清中乳酸脱氢酶(LDH)的释放率,评价L16和U16对MC3T3细胞的毒力作用;进一步使用AnnexinⅤ-PE/7-AAD双染流式细胞术检测MC3T3细胞的凋亡率,以及通过免疫印迹法(WB)检测凋亡执行蛋白Caspase-3的活化水平。结果L16刺激组细胞活力[(56.16±1.63)%]显著低于U16刺激组和LPS对照组[(97.02±1.44)%、(98.64±0.90)%,P均<0.01],LDH释放率[(84.64±0.96)%]显著高于U16刺激组和LPS对照组[(34.82±3.41)%、(26.75±1.95)%,P均<0.01]。AnnexinⅤ-PE/7-AAD染色结果显示,L16刺激组细胞凋亡率为(46.45±2.19)%,而其余组均<1%。WB结果显示,L16刺激组细胞内存在活化的Caspase-3,而U16刺激组和LPS对照组未检测到此活化片段。结论L16能诱导成骨细胞的凋亡,使成骨细胞的增殖受到抑制,而U16的作用不明显,具备完整脂化结构的布鲁菌L16是毒力效应所必需的。Objective This study is designed to investigate the toxicity of lipoprotein(L16)and non-lipoprotein(U16)of Brucella outer membrane protein(OMP)16 on osteoblasts.Methods Recombinant L16 and U16 proteins were prepared by using prokaryotic expression system of Escherichia coli(E.coli)BL21(DE3)and purified by Ni column.Using group design,mouse osteoblasts(MC3T3 cells)were co-incubated with L16 and U16,respectively.Brucella lipopolysaccharide(LPS)stimulus was used as the positive control,and cells without any stimulation were used as the negative control.Incubation time was 24 h.The activity of co-incubated MC3T3 cells were detected by CCK-8;the supernatant of cultured cells was collected and the release rate of lactate dehydrogenase(LDH)in the supernatant was detected by bioluminescence,and the virulence of L16 and U16 on MC3T3 cells was evaluated.AnnexinⅤ-PE/7-AAD double staining flow cytometry was further used to analyze the apoptosis rate of MC3T3 cells,and the activation level of apoptosis executive protein Caspase-3 was detected by Western blotting(WB).Results The activity of MC3T3 cells in L16 group[(56.16±1.63)%]was significantly lower than that in U16 and LPS groups[(97.02±1.44)%,(98.64±0.90)%,P<0.01],the LDH release rate[(84.64±0.96)%]was significantly higher than that in U16 and LPS groups[(34.82±3.41)%,(26.75±1.95)%,P<0.01].AnnexinⅤ-PE/7-AAD double staining results showed that the apoptosis rate was(46.45±2.19)%in L16 group,while the remaining groups were all less than 1%.WB results showed that activated Caspase-3(cleaved-Caspase-3)existed in L16 stimulated cells,but not in U16 stimulated cells and LPS control cells.Conclusion L16 can induce the apoptosis of osteoblasts and inhibit the proliferation of osteoblasts,but U16 has no obvious effect indicating that Brucella L16 with complete lipid structure is necessary for virulence effect.

关 键 词：布鲁杆菌属 外膜蛋白 成骨细胞系 细胞活力 乳酸脱氢酶 细胞凋亡 

分 类 号：R378.5[医药卫生—病原生物学]