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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。
To examine the immunosuppressive effect of indoleamine 2,3-dioxygenase (IDO) gene-modified primary cultured chondrocytes on T lymphocytes. The pEGFP-N1-IDO eukaryotic expression vector was transfected into primary cultured chondrocytes in C57 mice, and the changes of tryptophan levels in IDO-modified chondrocytes culture medium were detected in vitro. IDO-modified cartilage Effect of in vitro mixed lymphocyte reaction on the proliferation of allogenic T lymphocytes. Results: pEGFP-N1-IDO was successfully transfected into primary cultured mouse chondrocytes, and the expression of EGFP was detected by fluorescence microscopy and flow cytometry. In vitro experiments showed that a certain amount of primary cultured chondrocyte supernatant could be detected Of tryptophan levels, while tryptophan was not detected in chondrocytes supernatant of transfected IDO 24 h after transfection, suggesting IDO transfected chondrocytes expressed active IDO; CFSE labeling technique was used to detect IDO-modified chondrocytes In vitro mixed lymphocyte reaction on allogeneic T lymphocyte proliferation and found that IDO gene transfection 96 h after the overall proliferation index was 1.122 ± 0.017, lymphocyte negative control group was 1.026 ± 0.016, the positive group of pure chondrocytes Was 1.201 ± 0.026. Compared with the positive control group, IDO genomic allogeneic T lymphocyte proliferation was significantly inhibited. IDO gene-modified chondrocytes can inhibit the proliferation of T lymphocytes.