摘要
为了构建猪IL 10真核表达载体并在猪软骨细胞中表达。将目的基因酶切连入真核表达载体pcDNA3 1,以FuGENE 6介导转入猪软骨细胞中表达 ,RT PCR检测IL 10在软骨细胞中mRNA表达水平 ,ELISA检测细胞培养上清中IL 10蛋白含量。观察其对效应细胞PBMC分泌IFN γ和软骨细胞MHCII类分子表达的影响。结果显示 ,成功构建了猪IL 10真核表达载体 ,并转入软骨细胞 ,检测到细胞内IL 10mRNA转录 ,细胞培养 2 4、 4 8和 72h后 ,经ELISA检测上清中IL 10蛋白含量分别为 196 1 9、 2 92 5 9和 2 95 3 4pg/ml,能明显抑制经PHA刺激的PBMC分泌IFN γ (抑制率达 6 8 1% )和软骨细胞MHCII类分子的表达。猪IL 10真核表达载体的成功构建并在软骨细胞中表达 ,发挥其免疫调节功能 ,为研究IL 10诱导软骨细胞体内同种异体移植耐受奠定了基础。
To observe the expression of porcine IL-10 on the chondrocytes of pigs,eukaryotic expression vector for porcine IL-10 was constructed by inserting the target gene into vector pcDNA3.1 with endonuclease digestion and ligation. The recombinant vector was then transfected into chondrocytes of pigs by FuGENE6. The IL-10 mRNA was detected by RT-PCR and the contents of IL-10 in the supernatants of cell cultures were assayed by ELISA. Its influence on the secretion of IFN-γ by PBMC and the expression of the MHC II molecule on chondrocytes were observed by using the mixed lymphocyte culture and FACS analysis. The experimental results showed that the eukaryotic expression vector for porcine IL-10 had been successfully constructed and had been transfected into chondrocytes of pigs. Contents of porcine IL-10 in the supernatants of cell cultures as determined by ELISA in 24,48 and 72 hours of cultivations were 1 961.9 pg/ml, 2 925.9 pg/ml and 2 953.4 pg/ml respectively. The porcine IL-10 was showed to have a distinctly inhibitory effects on the level of IFN-γ secretion by PBMC activated with PHA (inhibition rate:68.1%) as well as the expression of MHC II molecules on chondrocytes. It concludes that the successful construction of eukaryotic expression vector for porcine IL-10 and its expression in chondrocytes of pigs may provide a foundation for the induction of immune tolerance of allogenic chondrocyte transplantation in vivo.
出处
《现代免疫学》
CAS
CSCD
北大核心
2004年第4期272-275,共4页
Current Immunology
基金
国家"973"基础研究资助项目 (G19990 5 43 0 0 )
上海市高等学校科学技术发展基金资助项目 ( 0 3BK2 0 )
关键词
猪软骨
真核表达
猪自介素10
porcine chondrocyte
eukaryotic expression
porcine IL-10
