CTLA4Ig基因修饰的树突状细胞抑制角膜移植排斥反应的机制研究

Inhibitory mechanism of CTLA4Ig-transfected DC in corneal transplantation

目的研究CTLA4Ig基因修饰的树突状细胞(DC)抑制角膜移植排斥反应的机制。方法采用脂质体转染法将质粒CTLA4Ig转入DC,用RT-PCR检测转染前后DC中吲哚胺2,3二氧化酶(IDO)的变化,CCK8检测CTLA4Ig-DC对同种异体T细胞的增生的影响,用AnnexinV/PI双染色流式细胞术检测L-色氨酸对CTLA4-DC功能的影响,建立大鼠同种异体角膜移植模型,分别在术后注入DC和DC+L-色氨酸于受体体内,观察疗效。结果转染CTLA4Ig后促进DC上IDO的表达,CTLA4-DC在体外能诱导同种异体T细胞凋亡,而L-色氨酸能有效拮抗CTLA4-DC的功能,动物实验中DC+L-色氨酸组大鼠角膜植片的存活时间较DC组缩短(P<0.01),且颌下淋巴结内未见明显T细胞凋亡。结论CTLA4-DC可能通过降低局部色氨酸浓度,从而延长角膜植片的存活时间。

Objective The aim of this paper was to study the inhibitory mechanism of CTLA4-dendritic cells （ CTLA4- DC） in corneal transplantation. Methods The plasmid PG/CTLA41g was transferred into the dendritic cells of F344 rat mediated by LipofectamineTM 2000. The change of the expression of indolamine 2,3 dioxgenase （IDO） mRNA in dendritic cells was measured by using semiquantutative reverse transcription polymerase chain reaction. The proliferative responses of T cells in Lewis rat by the effect of the transfected dendritic cells were determined using CCK8. The dendritic cells （48 hours after transfer） were divided into two groups, and mixed lymphocyte reaction （MLR） was performed between dendritic cells and T cell. Ltryptophan was put into the experiment group,and T cell apoptosis was determined by the flow cytometry assay in AnnexinV/Pl. Corneal transplantation was performed from F344 rats to Lewis rat,and the dendritic cells of above two groups were injected into the Lewis rat after the operation. Survival of corneal allograft was evaluated by Holland criterion. T cell apoptosis in the submandibular lymph node （SLMN） was determined by TUNEL assay. Results The expression of IDOmRNA was increased in dendritic cells after the transfer（ P 〈 0. 05）. CTLA41g-transferred dendritic cells could induce allogeneic T cell apoptosis,and L-tryptophan could decrease the apoptosis rate （ P 〈 0. 05 ）. The survival time of corneal graft in dendritic cells ＋ L-tryptophan group was shorter than that of dendritic cells group （ P 〈 0.01 ） , and the apoptosis cells in SLMN was significantly reduced in comparison with dendritic cells group. Conclusion The overexpression of IDO in CTLA41g-transferred dendritic cells indicate CTLA41g-DC prolong the survival time of corneal allografts.