人CCL25基因慢病毒表达载体的构建及其对TEC黏附分子表达的影响

Construction of lentiviral vector over-expressing CCL25 and effect of CCL25 on TEC adhesion molecules

通过构建人趋化因子CCL25基因过表达慢病毒载体并转染人原代胸腺上皮细胞(TEC),探讨过表达CCL25对人TEC黏附分子表达的影响,为研究CCL25在重症肌无力患者胸腺异常表达的作用奠定基础。PCR和DNA测序鉴定携带CCL25基因的慢病毒构建成功,病毒滴度约为4×108 Tu/ml,慢病毒对TEC感染效率达80%。Western blotting结果显示感染组细胞CCL25蛋白表达量显著增高。RT-PCR结果显示,与对照组相比,感染组HLA-A、HLA-DR表达无显著变化,P-selectin、VCAM-1和ICAM-1表达显著增高,提示我们人胸腺上皮细胞CCL25基因的过表达可一定程度影响TEC的功能特性,可能以通过增加P-selectin、VCAM-1和ICAM-1的表达量来影响淋巴祖细胞的胸腺归巢或胸腺内胸腺细胞的阳性选择过程,进而引起胸腺细胞发育异常,细胞亚群比例失调。

Our aims are to construct a lentiviral vector over-expressing human CCL25 gene, and to examine its ability to infect human thymus epithelial cell （TEC） in vitro. The primers for PCR amplification of CCL25 gene fragments were designed. The pCDS13B-1 vectors were digested by XbaI and EcoRI enzyme, followed by ligase mediated ligation to CCL25 fragments. Cor- rectly connected cloning pCD513B-1-CCL25 was identified by PCR and DNA sequencing and was co-transfected into HEK 293T cells with plasmid pMD2.0G and plasmid psPAX2. The supernatant containing lentivirus lenti-CCL25 gene was harvested, and the titer value of lentiviral Vector was about 4× 108 Tu/ml. The recombinant lentivirus were transfected into the original gener- ation of human TEC（TEC obtained from thymic tissure of patients with congenital heart diseases）. Expression rate of GFP was observed about 80%. Western blotting showed that the infected TEC had significantly higher expression of CCL25 protein. RT-PCR showed that the infected TEC had significantly higher expression of P-selectin, VCAM-1 and ICAM-1 mRNA, but there was no difference in HLA-A and HLA-DR expression.