目的:探讨供体来源低剂量未成熟树突状细胞外来体(immature dendritic cells exosome,im Dex)联合供体抗原特异型调节性T细胞(regulatory t cells,Tregs)对于肝脏移植物的保护作用。方法:以BN-〉Lewis大鼠为供受体,建立大鼠原位肝移植模型。利用超高速梯度离心法获得im Dex,采用流式细胞术鉴定该im Dex的表型。不同剂量im Dex用于本移植模型,生存分析探究其对肝脏移植物的保护作用及该作用与剂量的相关关系。利用磁珠分选技术获得Treg,混合淋巴细胞培养获得供体抗原特异性Treg,将不同性质的Treg用于移植受体,验证抗原特异性Treg保护肝脏移植物。进一步探究两者联合使用的效果,应用免疫荧光、流式细胞术研究Treg在移植受体中的分布。结果:超高速梯度离心法分选出的im Dex具有im DC表型,其保护肝脏移植物的最佳作用剂量为20μg,Treg发挥保护移植物作用具有抗原特异性。两者联合应用组生存时间长于对照组(P〈0.05)。病理显示,输注的Treg分布于受体移植物。结论:Im Dex联合抗原特异性Treg可以有效保护大鼠肝脏移植物。
Objective: To investigate the effect of donor derived imDex and donor antigen special Treg on the rat liver transplantation. Methods: The orthotopic liver transplantation model in rats was established by using the BN/Lewis rats as donors and recipients. Ultra high speed gradient centrifugation was utilized to obtain the imDex, and flow cytometry was used to identify the phenotype of imDex. Different doses of imDex were used for the recipients, to explore the correlation between the doses of imDex and the protective effect on the liver allograft through survival analysis. Treg was obtained with magnetic bead separation technology, then using mixed lymphocyte culture, the donor antigen specially reactive Treg was obtained. Treg of different natures was used in transplant recipients, to verify the protective effects of antigen specific Treg on liver grafts. Then combined use of both in recipients was carried out. With the technologies of immunofluorescence and flow cytometry, the distribution of Treg in transplant recipients was determined. Results: Ultra high speed gradient centrifugation could isolate imDex and maintain the phenotype of imDC. The best effective dose of imDex was 20 μg in rat liver transplantation. Treg had a protective role in the graft with antigen specificity. The survival time in the combined application group was longer than that of the control group (P〈0.05). Pathology of rat liver grafts showed the infused Treg was distributed into the liver graft. Conclusion: ImDex combined with antigen specific Treg can effectively protect the rat liver graft, and both have synergistic effect.