利用RNA干扰诱导产生的CD8+CD28-抑制性T淋巴细胞的免疫学特性

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.

The immunological characteristics of CD8+ CD28- suppressor T cells induced by siRNA technique

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.