国际免疫学杂志
國際免疫學雜誌
국제면역학잡지
INTERNATIONAL JOURNAL OF IMMUNOLOGY
2014年
6期
526-530
,共5页
王雪野%高鹏%韩梅%王新梅
王雪野%高鵬%韓梅%王新梅
왕설야%고붕%한매%왕신매
CD4+ CD25+免疫调节T细胞%体外扩增
CD4+ CD25+免疫調節T細胞%體外擴增
CD4+ CD25+면역조절T세포%체외확증
CD4+ CD25+ regulatory T cells%In vitro amplification
目的 探讨小鼠CD4+ CD25+调节性T细胞(Tregs)的体外扩增方法及扩增后Tregs的免疫功能.方法 利用免疫磁珠法分离小鼠Tregs;采用anti-CD3mAb+rIL-2和Allo-APC+rIL-2两种方法进行体外扩增,通过TRANSWELL培养系统、细胞因子表达及CFSE染色标记等方法检测Tregs免疫抑制作用的机制.结果 体外分离的Tregs纯度为89.5％;活细胞率约为98％.Tregs扩增的倍数在两组各时间点差异无统计学意义.扩增后CD4+ CD25+T细胞、CD4+T细胞和CD4+ CD25-T细胞的每分钟计数(CPM)分别为1 470、12 700和14 300.混合淋巴细胞反应(MLR)中当CD4+:CD25+T细胞的比例为1∶1时,抑制率为79％,比例为8∶1时,抑制率为33％.CFSE标记后,进入分裂周期的CD4+T细胞的百分比在未加入CD4+ CD25+T细胞组为83.7％,加入CD4+ CD25+T细胞组为31.7％,差异具有统计学意义(P=0.0006).CD4+ CD25+T细胞对CD4+T细胞的增殖抑制率在Transwell和Non-Transwell的培养系统中分别为＜5％和95％.Transwell培养组中IL-2含量高于Non-Transwell组,差异具有统计学意义[Transwell培养组为(158.33±2.08)pg/mL,Non-Transwell组为(23.00±2.00) pg/mL,P ＜0.0001].结论 采用免疫磁珠法分离小鼠Tregs可行,Tregs可有效扩增;Tregs的作用机制为抑制CD4+T细胞的增殖及抑制CD4+T细胞分泌IL-2;其抑制作用需要细胞-细胞间接触;体外扩增后的Tregs仍具有免疫特性,其体外免疫抑制作用增强.
目的 探討小鼠CD4+ CD25+調節性T細胞(Tregs)的體外擴增方法及擴增後Tregs的免疫功能.方法 利用免疫磁珠法分離小鼠Tregs;採用anti-CD3mAb+rIL-2和Allo-APC+rIL-2兩種方法進行體外擴增,通過TRANSWELL培養繫統、細胞因子錶達及CFSE染色標記等方法檢測Tregs免疫抑製作用的機製.結果 體外分離的Tregs純度為89.5％;活細胞率約為98％.Tregs擴增的倍數在兩組各時間點差異無統計學意義.擴增後CD4+ CD25+T細胞、CD4+T細胞和CD4+ CD25-T細胞的每分鐘計數(CPM)分彆為1 470、12 700和14 300.混閤淋巴細胞反應(MLR)中噹CD4+:CD25+T細胞的比例為1∶1時,抑製率為79％,比例為8∶1時,抑製率為33％.CFSE標記後,進入分裂週期的CD4+T細胞的百分比在未加入CD4+ CD25+T細胞組為83.7％,加入CD4+ CD25+T細胞組為31.7％,差異具有統計學意義(P=0.0006).CD4+ CD25+T細胞對CD4+T細胞的增殖抑製率在Transwell和Non-Transwell的培養繫統中分彆為＜5％和95％.Transwell培養組中IL-2含量高于Non-Transwell組,差異具有統計學意義[Transwell培養組為(158.33±2.08)pg/mL,Non-Transwell組為(23.00±2.00) pg/mL,P ＜0.0001].結論 採用免疫磁珠法分離小鼠Tregs可行,Tregs可有效擴增;Tregs的作用機製為抑製CD4+T細胞的增殖及抑製CD4+T細胞分泌IL-2;其抑製作用需要細胞-細胞間接觸;體外擴增後的Tregs仍具有免疫特性,其體外免疫抑製作用增彊.
목적 탐토소서CD4+ CD25+조절성T세포(Tregs)적체외확증방법급확증후Tregs적면역공능.방법 이용면역자주법분리소서Tregs;채용anti-CD3mAb+rIL-2화Allo-APC+rIL-2량충방법진행체외확증,통과TRANSWELL배양계통、세포인자표체급CFSE염색표기등방법검측Tregs면역억제작용적궤제.결과 체외분리적Tregs순도위89.5％;활세포솔약위98％.Tregs확증적배수재량조각시간점차이무통계학의의.확증후CD4+ CD25+T세포、CD4+T세포화CD4+ CD25-T세포적매분종계수(CPM)분별위1 470、12 700화14 300.혼합림파세포반응(MLR)중당CD4+:CD25+T세포적비례위1∶1시,억제솔위79％,비례위8∶1시,억제솔위33％.CFSE표기후,진입분렬주기적CD4+T세포적백분비재미가입CD4+ CD25+T세포조위83.7％,가입CD4+ CD25+T세포조위31.7％,차이구유통계학의의(P=0.0006).CD4+ CD25+T세포대CD4+T세포적증식억제솔재Transwell화Non-Transwell적배양계통중분별위＜5％화95％.Transwell배양조중IL-2함량고우Non-Transwell조,차이구유통계학의의[Transwell배양조위(158.33±2.08)pg/mL,Non-Transwell조위(23.00±2.00) pg/mL,P ＜0.0001].결론 채용면역자주법분리소서Tregs가행,Tregs가유효확증;Tregs적작용궤제위억제CD4+T세포적증식급억제CD4+T세포분비IL-2;기억제작용수요세포-세포간접촉;체외확증후적Tregs잉구유면역특성,기체외면역억제작용증강.
Objective To detect the immune function of CD4 + CD25 +T cells (Tregs),amplified in vitro.Methods We used the micro-magnetic beads to isolate Tregs.Two means were adopted to amplify Tregs (anti-CD3mAb group and allo-APC group).The function of Tregs was detected with the Transwell system,cytokine detection and CFSE marker.Results The purity of Tregs is 89.5％.The amplification times were not significantly deferent between the two groups.The counts per minute(CPM) of CD4 + CD25 +T cell,CD4 + T cell and CD4 + CD25T cell is 1 470,12 700 and 14 300 respectived.In mixed lymphocyte reaction (MLR),when the ratio of CD4/CD25+ T cell is 1∶ 1 and 8∶1,suppression ratio is 79％ and 33％.We labeled CD4+T cells with CFSE,the percent is 83.7 ％ without Tregs,and 31.7 ％ with Tregs (P =0.0006).The proliferation inhibition ratio inducedby is 5％ in Transwell system and 95％ in Non-Transwell system.The concentration of IL-2 in Transwell system is significantly higher than that in Non-Transwell(158.33 ± 2.08) pg/mL vs (23.00 ± 2.00) pg/mL,P ＜ 0.0001).Conclusion The method of micro-magnetic beads can be used to isolate Tregs.Tregs can be amplified effectively and inhibit CD4 +T cells proliferation.Tregs amplified in vitro still have immune with function with enhanced immune-suppressive effect.
