抗RANKL多克隆抗体抑制T细胞介导的破骨细胞形成的体外研究  

In vitro study on the amelioration of T cell-mediated osteoclastogenesis by anti-RANKL-polyclonal antibody

作　　者：林晓萍[1,2] 韩晓哲 魏巍[1] Martin A.Taubman 

机构地区：[1]中国医科大学附属盛京医院口腔科 [2]Department of Immunology,The Forsyth Institute.Boston,MA 02115,USA

出　　处：《上海口腔医学》2009年第6期604-608,共5页Shanghai Journal of Stomatology

基　　金：辽宁省自然科学基金(20042062);辽宁省教育厅科研基金(202013124)~~

摘　　要：目的:探讨抗核因子-КB受体活化因子配体(RANKL)多克隆抗体对T细胞介导的破骨细胞形成的影响。方法:制备兔抗鼠RANKL多克隆抗体,使用不同浓度的抗体(1、5及10μg/mL),观察小鼠重组RANKL诱导的破骨细胞形成,显微镜下计数抗酒石酸酸性磷酸酶(TRAP)阳性多核细胞数/孔;体外分离、纯化、扩增大鼠的颈T淋巴细胞,给予不同浓度的兔抗鼠RANKL特异性抗体进行干预,取其上清和T细胞,分别与RAW264.7细胞共同培养,TRAP法测定T细胞介导的破骨细胞形成,显微镜下计数TRAP(+)多核细胞数/孔;ELISA测定细胞上清中的可溶性RANKL浓度(sRANKL,pg/mL)。实验结果采用SPSS11.5软件包进行统计学分析。结果:兔抗鼠RANKL抗体既可减少RANKL诱导的TRAP(+)多核细胞数,也可减少T细胞介导的TRAP(+)多核细胞数,5μg/mL及10μg/mL抗体组差异显著(P<0.05),且呈浓度依赖性;上清中检出sRANKL的浓度与不加抗体对照组相比明显降低,具有统计学意义(P<0.05)。结论:抗RANKL特异性抗体可通过减少sRANKL的表达水平,直接阻断RANKL与核因子-КB受体活化因子(RANK)的结合,降低T细胞介导的破骨样细胞吸收。PURPOSE：To characterize the effect of receptor activator of NF-kB ligand （RANKL） polyclonal antibody on T cell-mediated osteoclasogenesis.METHODS：Rabbit anti-rat RANKL polyclonal IgG antibody was performed,performing recombinated mouse RANKL-induced osteoclastogenesis in different antibody doses （1μg/mL、5μg/mL and 10μg/mL）,the number of TRAP positive multinuclear cells （cells/well） was calculated under microscopy.The cervical lymph nodes in rats were enriched T-cells for in vitro experiments.The T cell-mediated osteoclastogenesis was tested by co-culture assay of RAW 264.7 cells with formalin-fixed T cell or T cell supernatant,the number of TRAP positive multinuclear cells （cells/well） was calculated under microscopy.In some experiments,the concentration of sRANKL （soluble RANKL） was measured in supernatant by ELISA （pg/mL）.SPSS 11.5 software package was used for statistical analysis.RESULTS：Rabbit anti-rat RANKL antibody inhibited osteoclastogenesis by recombined mouse RANKLinduced,and dose-dependent;Anti-RANKL antibody inhibited T cell and culture supernatant induced osteoclastogensis,significantly different from that in the group without antibody （P0.05）,and dose-dependent;Anti-RANKL specific antibody also markedly reduced sRANKL concentration in supernatant,significantly different from that in the control group without antibody （P〈0.05）.CONCLUSIONS：Anti-RANKL specific antibody blocks direct effect of RANKL and interferes with immune T cell-mediated bone resorption by reduction of sRANKL level.

关 键 词：核因子-КB受体活化因子配体 抗酒石酸酸性磷酸酶染色 破骨细胞形成 伴放线放线杆菌 T淋巴细胞 

分 类 号：R78[医药卫生—口腔医学]