IL-10基因在大鼠骨髓树突状细胞的表达

The expression of IL-10 gene in dendritic cells from bone marrow stem cells of rats

目的检测人白细胞介素10(hIL-10)基因在大鼠骨髓来源的未成熟树突状细胞(immature dendritic cell,imDC)中的表达。方法实验分4组:mDC组、imDC组、空载体组及hIL-10基因转染组。用hIL-10真核表达载体(pIRES2-EGFP-hIL-10)转染大鼠骨髓来源的imDC,应用ELISA、Western blot、流式细胞术及混合淋巴细胞反应(MLR)检测hIL-10在各组中的表达。结果荧光显微镜观察显示pIRES2-EGFP-hIL-10质粒转染imDC后可见绿色荧光;ELISA检测hIL-10基因转染组的hIL-10表达明显高于mDC组、imDC组及空载体组;经Westen blot检测hIL-10基因转染组有hIL-10蛋白表达;流式细胞术显示hIL-10基因转染组hIL-10基因转染后imDC表面分子CD86、CD80与其他三组相比略呈低表达;转染后混合淋巴细胞反应(MLR)显示,hIL-10转染组的imDC对T细胞的增殖反应均弱于imDC组和空载体组(P=0.024和P=0.033)。结论外源性hIL-10基因成功导入大鼠imDC并表达。

Objective To investigate the expression of hIL-10 gene in dendritic cells from bone marrow stem cells of rats. Methods The experiment was divided into four groups：mDC group, imDC group, empty vector group and IL-10 gene transfection group. The plasmids of pIRES2-EGFP-hIL-10 were transfected into imDC to detect expression of hIL-10 gene by ELISA, Western blot, flow cytometry and MLR. Results After transfection,green fluorescence was observed in imDC under fluorescence microscope. The result of ELISA showed that the expression of hIL-10 in hIL-10 group was significantly higher than that in the other groups（P = 0. 004,0. 004 and 0.003）. Western blot result showed the positive expression of IL-10 protein in IL-10 gene transfection group. CD86 and CD80 expression on the imDC surface in IL-10 gene transfection group was lower than in the other groups by flow cytometry. The result of MLR demonstrated that the imDC induced lower T-cell in hIL-10 group than in imDC group and empty vector group（ P = 0.024,P = 0.033 ）. Conclusion The extrinsic source gene is successfully transfected into imDC and could be expressed.