CAJ | 학술논문

目的：我们通过体外诱导、扩增并分选出CD4+CD25+ T-reg回输入受体大鼠，并协同低剂量西罗莫司去诱导大鼠肝移植免疫耐受，研究探讨CD4+CD25+ T-reg细胞亚群在移植免疫耐受过程中扮演的角色。方法将实验动物分为急性排斥组（DA→LEW）、免疫耐受组（LEW→DA）、低剂量西罗莫司（0.1 mg·kg-1·d-1）和CD4+CD25+ T-reg协同作用组（实验组）。每组8只实验动物。各组肝移植大鼠的存活率比较，应用HE染色法观察移植肝术后7 d组织病理学变化，检测CD4+CD25+Foxp3+ T-reg细胞亚群在各组大鼠移植肝脏、外周血总单核细胞数中所占的百分比。RT-PCR检测术后7 d移植肝脏Foxp3 mRNA的表达水平。用ELISA检测血浆中细胞因子IL-10、TGF-β的水平。结果实验组对比排斥组，能够长期存活，获得免疫耐受（P＜0.05）。实验组移植肝脏中CD4+CD25+Foxp3+ T-reg细胞亚群所占的比例明显高于排斥组（P＜0.001），且在移植肝脏中，实验组Foxp3 mRNA表达含量明显增加（P＜0.001）。实验组血浆中细胞因子IL-10、TGF-β的表达水平高于排斥组（P＜0.001）。结论我们通过流式细胞分选技术将体外扩增诱导成熟的受体大鼠CD4+CD25+T-reg细胞分离收集，然后回输受体协同低剂量的西罗莫司能够成功地诱导了长期肝移植免疫耐受。从而为临床应用CD4+CD25+ T-reg细胞抑制免疫排斥反应，诱导移植免疫耐受提供了一条新思路和理论实验依据。
목적：아문통과체외유도、확증병분선출CD4+CD25+ T-reg회수입수체대서，병협동저제량서라막사거유도대서간이식면역내수，연구탐토CD4+CD25+ T-reg세포아군재이식면역내수과정중분연적각색。방법장실험동물분위급성배척조（DA→LEW）、면역내수조（LEW→DA）、저제량서라막사（0.1 mg·kg-1·d-1）화CD4+CD25+ T-reg협동작용조（실험조）。매조8지실험동물。각조간이식대서적존활솔비교，응용HE염색법관찰이식간술후7 d조직병이학변화，검측CD4+CD25+Foxp3+ T-reg세포아군재각조대서이식간장、외주혈총단핵세포수중소점적백분비。RT-PCR검측술후7 d이식간장Foxp3 mRNA적표체수평。용ELISA검측혈장중세포인자IL-10、TGF-β적수평。결과실험조대비배척조，능구장기존활，획득면역내수（P＜0.05）。실험조이식간장중CD4+CD25+Foxp3+ T-reg세포아군소점적비례명현고우배척조（P＜0.001），차재이식간장중，실험조Foxp3 mRNA표체함량명현증가（P＜0.001）。실험조혈장중세포인자IL-10、TGF-β적표체수평고우배척조（P＜0.001）。결론아문통과류식세포분선기술장체외확증유도성숙적수체대서CD4+CD25+T-reg세포분리수집，연후회수수체협동저제량적서라막사능구성공지유도료장기간이식면역내수。종이위림상응용CD4+CD25+ T-reg세포억제면역배척반응，유도이식면역내수제공료일조신사로화이론실험의거。
Objective CD4+CD25+ regulatory T cells were induced, amplified, sorted in vitro, and then they were transfused back to recipient and cooperated with low dose sirolimus to induce immune tolerance in rat liver transplantation. At the same time, the role of CD4+CD25+ regulatory T cells would been clarified in the process of transplantation immune tolerance. Methods Experimental animals were randomly divided into acute rejection group (DA→LEW); immune tolerance group (LEW→DA);experimental group (sirolimus+CD4+CD25+ T-reg). There were 8 pairs rats in each group. We observed the survival rate of rats and liver graft pathological changes in every group. CD4+CD25+Foxp3+T-reg cells were respectively extracted and detected in liver and PBMC by flow cytometry. The expression level of Foxp3 mRNA in every group was monitored by RT-PCR. Simultaneously, the level of cytokine IL-10 and TGF-β was detected by ELISA. Results Compared with rejection group, rat in experimental group could acquire long-time survival(P<0.05). The percent of CD4+CD25+Foxp3+T-reg cells in the graft and PBMC significantly increased in experimental group (P<0.001). The expression level of Foxp3 mRNA and cytokine IL-10 and TGF-β in experimental group was significantly more higher than rejection group (P<0.001). Conclusions CD4+CD25+ regulatory T cells were induced, amplified and sorted in vitro, we could successfully induce long-term liver allograft survival by transfusing them back to recipient and being on the low dose sirolimus. A new sight was brought to us that we could expand CD4+CD25+ T-reg and transfuse back to recipient to induce transplantation tolerance and suppress graft rejection in clinic.