甲基转移酶抑制剂延长小鼠心脏移植物存活期的作用研究

Prolongation of cardiac allograft survival by methyltransferase inhibitor

探讨甲基转移酶抑制剂5-氮杂-2′-脱氧胞苷调控体内幼稚T细胞转化对同种心脏移植物存活期的影响及其机制。将C57BL/6小鼠分为实验与对照2组,实验组每日腹腔注射5-氮杂-2′-脱氧胞苷(0.25mg/kg体质量),对照组接受等体积的二甲亚砜腹腔注射。然后,提取小鼠脾细胞,抗CD3与抗CD28抗体协同共刺激后,检测CD4+Foxp3+T细胞占CD4+T细胞比例。以BALB/c小鼠为供鼠,两组C57BL/6小鼠为受鼠,建立腹部异位心脏移植模型,监测两组移植心存活时间,检测受鼠脾细胞中CD4+T细胞凋亡率,观察移植心脏组织病理学变化。结果显示,实验组移植前脾细胞中CD4+Foxp3+T细胞占CD4+T细胞比例为(22.7±2.6)%,明显高于对照组的(12.6±1.1)%,差异有统计学意义(P<0.05);实验组移植物存活(22.8±2.8)d,明显长于对照组(8±1.8)d,P<0.05);实验组移植后脾细胞中CD4+T细胞凋亡率(11.5±1.22)%,高于对照组的(6.6±0.91)%,差异有统计学意义(P<0.05)。实验组移植物淋巴细胞浸润数量少于对照组。以上结果提示甲基转移酶抑制剂5-氮杂-2′-脱氧胞苷受体腹腔注射可延长同种移植心脏存活期,其机制可能与调控幼稚T细胞转化为CD4+Foxp3+T细胞,诱导CD4+T细胞凋亡有关。

To explore the impact on cardiac allograft survival and its possible mechanism of the naive T cell conversion regulated by DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine（5aza）,male C57BL/6mouse were divided into 2groups,the experimental group received intraperitoneal injection of 5aza[dissolved in dimethyl sulfoxide（DMSO）],and the control group received the same amount of DMSO.Mouse splenocytes were extracted after 7days’injection,the percentage of CD4＋Foxp3＋T cell were detected by Flow Cytometry after costimulated by anti-CD3 and anti-CD28 antibodies.Abdominal heterotopic heart transplantation model was established by using male BALB/c mice as donors,and C57BL/6（from the experimental and control group）as recipients respectively.The survival time of cardiac allograft was compared between the 2groups;the apoptosis rate of CD4＋T cell in recipients,splenocytes was also compared between the 2groups at day 14post-operation.The graft tissues were examined for lymphocyte infiltration by HE staining.In splenocytes of the experimental group,the CD4＋Foxp3＋T cell accounted for（22.7±2.6）% of CD4＋T cell,which was significantly higher than that in the control group [（12.6±1.1）%]（P〈0.05）.The survival time of the cardiac grafts in the experimental group[（22.8±2.8）d]was longer than that in the control group[（8±1.8）d]（P〈0.05）.The apoptosis rate of CD4＋T cell in recipients’splenocytes of the experimental group at day14post-operation was（11.5±1.22）%,significantly higher than that of the control group [（6.6±0.91）%]（P〈0.05）.Pathological examination showed that the graft lymphocyte infiltration of the experimental group was obviously less serious than that of the control groups.We conclude that intraperitonealy injected methyltransferase inhibitor 5aza could significantly prolong allograft survival time and the mechanism could be related to the induction of regulatory T cells transformed by naive T cells as well as induction of apoptosis of CD4＋T cell.