雷帕霉素对重型再生障碍性贫血模型小鼠脾脏CD4＋CD25＋ T细胞凋亡的影响

Rapamycin affect the apoptosis of splenic CD4＋CD25＋ regulatory T cells of mouse severe aplasticanemia model

目的探讨雷帕霉素（Rapamycin, RAPA）对重型再生障碍性贫血（SAA）模型小鼠CD4＋CD25＋ Treg细胞凋亡情况的影响及可能的机制。 方法以近交系雌性BALB/c小鼠作为对照（对照组），应用IFN-γ腹腔注射联合白消安灌胃的方法建立SAA小鼠模型（SAA组），并使用RAPA腹腔注射5 d治疗SAA小鼠（RAPA组）。骨髓活检病理学检查观察各组小鼠的骨髓造血组织变化；免疫磁珠分选出各组小鼠脾脏Treg细胞，用流式细胞术检测其凋亡率；Western blot法检测各组小鼠脾脏Treg细胞的Akt、磷酸化（p）-Akt、Stat3、p-Stat3的表达水平；收集各组小鼠外周血和脾脏中单个核细胞，用流式细胞术检测CD4＋CD25＋Foxp3＋ Treg细胞的比例变化。 结果与对照组相比，SAA组造血细胞明显减少，且造血细胞被大量的脂肪组织所代替，RAPA组造血细胞亦明显减少，可见大量脂肪细胞；对照组、SAA组、RAPA组小鼠胫骨骨髓造血组织面积分别为（94.25±4.20）%、（7.00±2.00）%、（9.75±1.83）%，差异有统计学意义（Welch F=1 441.822，P〈0.001）；RAPA组略高于SAA组[Δx=2.15%（95%CI 0.15%~5.35%），P=0.037]。对照组、SAA组、RAPA组脾脏Treg细胞凋亡率分别为（19.84±1.39）%、（29.85±2.72）%、（22.39±3.71）%，差异有统计学意义（F=18.338，P〈0.001）；RAPA组Treg细胞凋亡率明显低于SAA组。RAPA组脾脏Treg细胞中Akt、Stat3的表达水平高于SAA组，而p-Akt和p-Stat3表达水平低于SAA组（P值均〈0.05）。SAA组、RAPA组的脾脏CD4＋CD25＋Foxp3＋/CD4＋CD25＋、CD4＋CD25＋Foxp3＋/CD4＋细胞比例均低于对照组，且RAPA组明显高于SAA组（P值均〈0.017）。 结论IFN-γ联合白消安诱导的SAA模型小鼠脾脏Treg细胞存在过度凋亡现象，而RAPA可能通过抑制Treg细胞内Akt及Stat3的磷酸化上调Foxp3的表达，从而抑制Treg细胞的凋亡。

Objective To explore the effects and possible mechanism of rapamycin （RAPA） on apoptosis of CD4 ＋ CD25＋ Tregs from the mouse severe aplastic anemia （SAA） model. Methods The BALB/c female SAA model mice were induced by interferon-gamma in combination with busulphan. The SAA model mice were intraperitoneal injection with RAPA at daily dose of 0.5 mg/kg for 5 days （the RAPA-treated group, n = 15） in the SAA group （n = 15） and the un-treated group （n = 15） were control. Bone marrow hematopoiesis changes were observed by the patho-morphological examination of femurs. The mononuclear cells of the peripheral blood and spleen were subjected to assess the intracellular Foxp3 expression in CD4 ＋ CD25 ＋ Tregs by flow cytometry （FCM）. In addition, after being pured by immunomagnetic beads, the splenic CD4＋CD25＋ Tregs was subjected to assess apoptosis by FCM and the Akt and Stat3 phosphorylation by using of western blot. Results The patho-morphological examination offemurs showed normal marrow cell proliferation in un-treated group and hypocellularity in both SAA group and RAPA-treat group, with an increase in the number of fat cells. The bone marrow hematopoietic tissue ratio in RAPA-treat group was higher than SAA group [ （9.75±1.83）% vs （7.00±2.00）%, b2 = 2.15% （95% CI 0.15%-5.35%）, P = 0.037]. In the SAA group, FCM analysis showed down-expression of Foxp3 in CD4＋ CD25＋ Tregs compared with the un-treated group. However, after treatment with RAPA, the expression of Foxp3 in CD4＋CD25＋ Tregs was increased （P〈 0.017）. Compared with the un-treated group, increased CD4 ＋ CD25 ＋ Tregs apoptosis [（19.84 ±1.39）% vs （29.85 ± 2.72）% ] with increased Akt phosphorylation accompanied by increased Stat3 phosphorylation was found in SAA group （P 〈 0.05, respectively）. On the contrary, RAPA-treated group exhibited CD4 ＋ CD25 ＋ Tregs with a reduction in apoptosis rate [（22.39 ± 3.71）% ], Akt phosphorylation and Star3 phosphorylation compared with the SAA group （P〈0.05, respectively）. Conclusion These results indicate that RAPA may increase the expression of Foxp3 by down-regulation the levels of Akt and Stag phosphorylation and reduce apoptosis in splenic CD4＋CD25＋ Tre＋s from the mice model of SAA.