术前输注供者脾细胞联合应用西罗莫司延长小鼠移植心存活时间的机理

Donor specific transfusion combined with sirolimus treatment induced long-term survival of cardiac grafts in mice

目的 探讨移植术前输注供者脾细胞（DST）联合应用西罗莫司（SRL）延长小鼠移植心存活时间的机理。方法 单向混合淋巴细胞培养中的刺激细胞及静脉输注的脾细胞均采用经丝裂霉素C处理后失去增殖活性的Balb／c（H-2^d）小鼠脾细胞（包括体外实验和体内实验）。（1）体外实验：分为3组，空白对照组：取正常C57BL／6（H-2^b）小鼠（136小鼠）的脾细胞作为反应细胞；SRL组：取用SRL（1．5μ·g^-1·d^-1）灌胃7d后的B6小鼠脾细胞作为反应细胞；DST＋SRL组：取行脾细胞静脉输注8d、次日予SRL（1．5μl·g^-1·d^-1）灌胃7d后的B6小鼠的脾细胞作为反应细胞。观察各组在单向混合培养中的脾细胞增殖能力。（2）体内实验：Balb／c小鼠为供者，B6小鼠为受者，建立颈部异位心脏移植模型。实验分为3组，空白对照组：单纯行心脏移植；DST组：受者移植前8d给予供者脾细胞静脉输注；DST＋SRL组：受者移植前8d给予供者脾细胞静脉输注，次日予以SRL灌胃7d（1．5μl·g^-1·d^-1）。术后观察各组移植心的存活时间、病理表现、受者淋巴细胞中CD4^＋CD25^＋调节性T细胞（Tr细胞）和凋亡细胞比例的改变及同种抗原刺激后增殖活性变化。结果 体外实验中，DST＋SRL组反应细胞增殖活性平均为（13．76±2．81）％，明显低于空白对照组（28．14％±5．53％，P〈0．05）。体内实验中，空白对照组、DST＋SRL组以及DST组移植心平均存活时间分别为（8．6±0．48）d、（35±14．4）d和（4．83±0．52）d，DST＋SRL组存活时间显著延长（P〈0．05）；流式细胞术（FACS）分析显示DST＋SRL组脾细胞中CD4^＋CD25^＋Tr细胞比例平均为（3．39±0．21）％，明显高于空白对照组（1．57％±0．3％，P〈0．05）。结论移植术前输注供者脾细胞联合应用西罗莫司可通过清除同种反应性T细胞克隆，增加受者体内CD4^＋CD25^＋Tr细胞的比例，减低对同种抗原的应答能力，从而延长同种小鼠移植心存活时间。

Objective To investigate the effects of donor specific transfusion （DST） combined with Sirolimus （SRL） on engraftment and demonstrate the mechanisms. Methods （1） 136 was treated with DST （on day -8） combined with SRL （from day -7 to -1,1.5 μl.g^-1 .d^-1 ）. On day 0,136 splenocytes were harvested to co-culture with Balb/c splenocytes pretreated with mitomycin C in one way mixed lymphocyte cultures （one way-MLC）. After 48 h, the proliferation, the apoptosis and the CD4^＋ CD25^＋ T cells ratio of cultured cells were detected. （2） The heterotopic cardiac transplantation model was built （from Balb/c to 136）. The recipients were pretreated with the protocol as above mentioned. On day 0, SRL was withdrawn and the transplantations were performed. The mean survival time （MST） of the grafts and the detections as in vitro were measured. Result Both in vitro and in vivo, the protocol suppressed the proliferation of splenocytes as well as enhanced the apoptosis and the CD4^＋ CD25^＋ T cells ratio. The MST of graft was （35. 0 ± 14. 4） days. Conclusions DST combined with SRL could prolong cardiac allograft survival even if withdrawing immunosuppressive after trans- plantation. This result may be associated with inhibition of T cell proliferation. It demonstrated that SRL could expand the CD4^＋ CD25^＋ T cells and increase the apoptosis of splenocytes.