中华器官移植杂志
中華器官移植雜誌
중화기관이식잡지
CHINESE JOURNAL OF ORGAN TRANSPLANTATION
2009年
5期
295-299
,共5页
薛利军%尹路%陈春球%张贵阳%孙广涛%杜海磊%倪俊声%陈雪华%林谋斌%彭承宏%李宏为
薛利軍%尹路%陳春毬%張貴暘%孫廣濤%杜海磊%倪俊聲%陳雪華%林謀斌%彭承宏%李宏為
설리군%윤로%진춘구%장귀양%손엄도%두해뢰%예준성%진설화%림모빈%팽승굉%리굉위
RNA干扰%T淋巴细胞,抑制效应%免疫抑制法
RNA榦擾%T淋巴細胞,抑製效應%免疫抑製法
RNA간우%T림파세포,억제효응%면역억제법
RNA interference%T-lymphocytes,suppressor-effector%Immunosuppression
目的 探讨通过RNA干扰技术诱导产生的CD8+ CD28-抑制性T淋巴细胞(Ts细胞)的免疫学特性.方法 取SD大鼠骨髓,培养分离树突状细胞(DC),设计、合成主要组织相容性复合物(MHC)Ⅰ类小片段干扰RNA(siRNA),以MHC Ⅰ siRNA转染DC.先以Wistar大鼠肠系膜淋巴组织液刺激转染MHCI siRNA的DC,然后将DC与从SD大鼠脾脏分离得到的CD8+T淋巴细胞共同培养,通过磁珠法分离出Ts细胞.分别在由SD大鼠脾脏淋巴细胞(反应细胞)和Wistar大鼠肠系膜淋巴组织细胞(刺激细胞)组成的混合淋巴细胞培养体系中加入数量不等的Ts细胞,检测反应细胞增殖情况;分别以Wistar大鼠肠系膜淋巴组织细胞和卵白蛋白(OVA)刺激SD大鼠脾脏淋巴细胞,然后再按不同比例加入Ts细胞,检测各组脾脏淋巴细胞的增殖情况;在由SD大鼠脾脏淋巴细胞、Wistar大鼠肠系膜淋巴组织液和Ts细胞组成的混合淋巴细胞培养体系中加入可溶性重组白细胞介素2(rrIL-2),观察IL-2对Ts细胞功能的影响;采用实时定量聚合酶链反应(PCR)测定Ts细胞中转化生长因子β(TGF-β和γ干扰素(IFN-γ)mRNA的表达,流式细胞仪和实时PCR检测Ts细胞上CD25分子的表达.结果 Ts细胞对SD大鼠脾脏淋巴细胞和Wistar大鼠肠系膜淋巴组织细胞之问的混合淋巴细胞反应(MLR)具有抑制作用,但对于SD大鼠脾脏淋巴细胞和OVA之间的MLR则无抑制作用.在SD大鼠脾脏淋巴细胞、Wistar大鼠肠系膜淋巴组织液和Ts细胞组成的混合淋巴细胞培养体系中加入rrIL-2后,SD大鼠脾脏细胞的增殖并无明显增加(P＞0.05).与CD8+CD28+T淋巴细胞和CD8+ T淋巴细胞比较,Ts细胞的TGF-β和IFN-γ mRNA的表达量明显升高(P＜0.01,P＜0.05),而CD25的表达量明显降低(P＜0.05).结论 采用经MHC I siRNA干扰的DC能够诱导CD8+T淋巴细胞产生CD8+ CD28-Ts细胞;Ts细胞在体外具有免疫抑制特性,其免疫抑制作用不被外源性IL-2所逆转,且其免疫调节作用具有抗原特异性.
目的 探討通過RNA榦擾技術誘導產生的CD8+ CD28-抑製性T淋巴細胞(Ts細胞)的免疫學特性.方法 取SD大鼠骨髓,培養分離樹突狀細胞(DC),設計、閤成主要組織相容性複閤物(MHC)Ⅰ類小片段榦擾RNA(siRNA),以MHC Ⅰ siRNA轉染DC.先以Wistar大鼠腸繫膜淋巴組織液刺激轉染MHCI siRNA的DC,然後將DC與從SD大鼠脾髒分離得到的CD8+T淋巴細胞共同培養,通過磁珠法分離齣Ts細胞.分彆在由SD大鼠脾髒淋巴細胞(反應細胞)和Wistar大鼠腸繫膜淋巴組織細胞(刺激細胞)組成的混閤淋巴細胞培養體繫中加入數量不等的Ts細胞,檢測反應細胞增殖情況;分彆以Wistar大鼠腸繫膜淋巴組織細胞和卵白蛋白(OVA)刺激SD大鼠脾髒淋巴細胞,然後再按不同比例加入Ts細胞,檢測各組脾髒淋巴細胞的增殖情況;在由SD大鼠脾髒淋巴細胞、Wistar大鼠腸繫膜淋巴組織液和Ts細胞組成的混閤淋巴細胞培養體繫中加入可溶性重組白細胞介素2(rrIL-2),觀察IL-2對Ts細胞功能的影響;採用實時定量聚閤酶鏈反應(PCR)測定Ts細胞中轉化生長因子β(TGF-β和γ榦擾素(IFN-γ)mRNA的錶達,流式細胞儀和實時PCR檢測Ts細胞上CD25分子的錶達.結果 Ts細胞對SD大鼠脾髒淋巴細胞和Wistar大鼠腸繫膜淋巴組織細胞之問的混閤淋巴細胞反應(MLR)具有抑製作用,但對于SD大鼠脾髒淋巴細胞和OVA之間的MLR則無抑製作用.在SD大鼠脾髒淋巴細胞、Wistar大鼠腸繫膜淋巴組織液和Ts細胞組成的混閤淋巴細胞培養體繫中加入rrIL-2後,SD大鼠脾髒細胞的增殖併無明顯增加(P＞0.05).與CD8+CD28+T淋巴細胞和CD8+ T淋巴細胞比較,Ts細胞的TGF-β和IFN-γ mRNA的錶達量明顯升高(P＜0.01,P＜0.05),而CD25的錶達量明顯降低(P＜0.05).結論 採用經MHC I siRNA榦擾的DC能夠誘導CD8+T淋巴細胞產生CD8+ CD28-Ts細胞;Ts細胞在體外具有免疫抑製特性,其免疫抑製作用不被外源性IL-2所逆轉,且其免疫調節作用具有抗原特異性.
목적 탐토통과RNA간우기술유도산생적CD8+ CD28-억제성T림파세포(Ts세포)적면역학특성.방법 취SD대서골수,배양분리수돌상세포(DC),설계、합성주요조직상용성복합물(MHC)Ⅰ류소편단간우RNA(siRNA),이MHC Ⅰ siRNA전염DC.선이Wistar대서장계막림파조직액자격전염MHCI siRNA적DC,연후장DC여종SD대서비장분리득도적CD8+T림파세포공동배양,통과자주법분리출Ts세포.분별재유SD대서비장림파세포(반응세포)화Wistar대서장계막림파조직세포(자격세포)조성적혼합림파세포배양체계중가입수량불등적Ts세포,검측반응세포증식정황;분별이Wistar대서장계막림파조직세포화란백단백(OVA)자격SD대서비장림파세포,연후재안불동비례가입Ts세포,검측각조비장림파세포적증식정황;재유SD대서비장림파세포、Wistar대서장계막림파조직액화Ts세포조성적혼합림파세포배양체계중가입가용성중조백세포개소2(rrIL-2),관찰IL-2대Ts세포공능적영향;채용실시정량취합매련반응(PCR)측정Ts세포중전화생장인자β(TGF-β화γ간우소(IFN-γ)mRNA적표체,류식세포의화실시PCR검측Ts세포상CD25분자적표체.결과 Ts세포대SD대서비장림파세포화Wistar대서장계막림파조직세포지문적혼합림파세포반응(MLR)구유억제작용,단대우SD대서비장림파세포화OVA지간적MLR칙무억제작용.재SD대서비장림파세포、Wistar대서장계막림파조직액화Ts세포조성적혼합림파세포배양체계중가입rrIL-2후,SD대서비장세포적증식병무명현증가(P＞0.05).여CD8+CD28+T림파세포화CD8+ T림파세포비교,Ts세포적TGF-β화IFN-γ mRNA적표체량명현승고(P＜0.01,P＜0.05),이CD25적표체량명현강저(P＜0.05).결론 채용경MHC I siRNA간우적DC능구유도CD8+T림파세포산생CD8+ CD28-Ts세포;Ts세포재체외구유면역억제특성,기면역억제작용불피외원성IL-2소역전,차기면역조절작용구유항원특이성.
Objective To induce CD8+ CD28- suppressor T cells(CD8+Ts)by DC with MHCⅠ expression by siRNA,under the stimulator of allograft antigen and then investigate the immunological characteristics of Ts.Methods After the isolation and cultivation of DCs from femoral bone of SI)rats.the optimal sequence of MHCⅠ siRNA was designed and constructed,and the MHC Ⅰ siRNA was transfected into DCs.The IX3s were stimulated by mesenteric lymphatic tissue fluid of Wistar rats.and then co-cultured with CD8+ Ts isolated from SD rat spleen.SD rat CD8+T cells were ohtained by magnetic separation method.Cell proliferation was assayed after SD rat spleen cells loading Wistar rat mesenteric lymph node tissue antigen reacted with different amount of Ts.After SD rat spleen cells were stimulated by Wismr rat's mesenteric lymph node and reacted with OVA respectively.different amounts of Ts were added.Gell proliferation of the spleen lymphocytes in different groups was assayed, rrIL-2 was added to the mixed lymphocyte culture system containing Wistar rat's mesenteric lymph node tissue antigen, SD rat's Ts and SD rat spleen lymphocytes, then the influence of the IL-2 on the function of SD rat Ts was observed. The expression of TGF-β and IFN-γ mRNA was examined by Real time PCR, and CD25 was detected by Real time-PCR and flow cytometry in CD8+ CD28- T cells group. Results Ts had inhibitory influence on the mixed lymphocytes proliferation between SD rat' s spleen lymphocytes and Wistar rat's mesenteric lymph node tissue, but exerted no efforts on the mixed lymphocytes proliferation between OVA and SD rat's spleen lymphocytes. After addition of rrIL-2 into the mixed lymphocyte culture system containing Wistar rat mesenterie lymph node tissue antigen and Ts, there was no significant increase in the mixed lymphocytes proliferation of SD rat spleen lymphocytes (P＞0. 05). The expression levels of TGF-βand IFN-γ mRNA were higher in CD8 + CD28- Ts group than in CD8 + CD28+ Ts group and CD8+ Ts group (P＜0. 01, P＜0. 05). The expression of CD25(IL-2R) was lower in CD8+ CD28- Ts group than in CD8 + CD28+ Ts group and CD8 + Ts group (P ＜ 0. 05). Conclusion The CD8+ CD28- suppressor T cells can be induced by DC interfered MHC-I expression under the stimulator of allograft antigen. Ts has characteristics of suppressor-effector in vitro, which cannot be reversed when the rrIL-2 exists, and the immune regulatory effect of Ts has antigen specificity.
