摘要
为检测吲哚胺2,3-双加氧酶(IDO)基因修饰的原代培养软骨细胞对T淋巴细胞的免疫抑制作用。将pEGFP-N1-IDO真核表达载体转染至C57小鼠原代培养的软骨细胞,体外检测IDO修饰的软骨细胞培养液中色氨酸水平的变化;使用CFSE标记技术,检测IDO修饰的软骨细胞体外混合淋巴细胞反应中对同种异体T淋巴细胞增殖的影响。结果:pEGFP-N1-IDO成功被导入原代培养的小鼠软骨细胞,荧光显微镜和流式细胞术均检测到EGFP的表达;体外实验表明,原代培养的软骨细胞上清可检测到一定量的色氨酸水平,而转染IDO 24 h后的软骨细胞上清中未检测到色氨酸,提示IDO转染的软骨细胞表达了具有活性IDO;使用CFSE标记技术,检测IDO修饰的软骨细胞体外混合淋巴细胞反应中对同种异体T淋巴细胞增殖的影响,发现IDO基因转染组96 h后总体增殖指数为1.122±0.017,单纯淋巴细胞阴性对照组为1.026±0.016,阳性单纯软骨细胞组为1.201±0.026,较阳性对照组,IDO基因组同种异体T淋巴细胞增殖得到明显抑制。IDO基因修饰的软骨细胞可以抑制T淋巴细胞的增殖。
The immunosuppressive role of the indoleamine 2,3-dioxygenase(IDO) gene-modified chondrocytes on the proliferation of T lymphocytes was investigated in order to improve the efficiency of the in vivo transplantation with cartilage cells by prolonging the survival time of transplants and inducing the immuno-tolerance to the grafts,in which the eukaryotic expression vector pEGFP-N1-IDO was transfected to the primarily cultured chondrocytes of C57 mice through lipofectamine,and the content of tryptophan in the supernatants of cell cultures was determined.Meanwhile,the proliferation of the allogeneic T cells in mixed lymphocyte reaction(MLR) was analyzed by CFSE labeling technique.In these ways,plasmid pEGFP-N1-IDO was successively transfected to the primarily cultured chondrocytes and tryptophan could be detected in the supernatants of cultured cells after 24 hours cultivation of the gene-modified cells,indicating that IDO gene-transfected chondrocytes produced active IDO in cultural supernatants.As demonstrated by CFSE labeling technique,the total proliferation indeces of the IDO gene-modified chondroncytes transfected,negative control and positive control after 96 hrs cultivation in MLR were 1.122±0.017,1.026±0.016 and 1.201±0.026 respectively.It is concluded that the proliferation of T lymphocytes can be inhibited by the chondrocytes modified by IDO gene.
出处
《现代免疫学》
CAS
CSCD
北大核心
2008年第5期353-357,共5页
Current Immunology
基金
国家自然科学基金资助项目(30472007)
广东省自然科学基金资助项目(04009337)
广东省科技计划社会发展计划资助项目(2004B34001002)
西安市科技计划资助项目(2006)
作者简介
段小红,医师,硕士,主要从事组织工程学研究. 崔鹏程,E-mail:cuipc@fmmu.edu.cn.通讯作者
