阻断TIM-4分子对M1/M2型巨噬细胞极化及同种异体免疫排斥反应的影响

目的: 运用特异性单克隆抗体阻断抗原T细胞免疫球蛋白及黏蛋白结构域蛋白-4(T-cell immunoglobulin and mucin domain containing protein-4，TIM-4)，探讨其对巨噬细胞(M1/M2)极化及同种异体免疫排斥反应的影响。方法: 构建同种异体免疫排斥模型，将BALB/c小鼠脾细胞悬液注入C57BL/6小鼠腹腔，分为同型对照组(n=6)和抗TIM 4组(n=6)；分别腹腔注射同型对照免疫球蛋白和抗TIM-4单克隆抗体， 300 μg/只；第5天，重复注射1次；第10天，处死两组小鼠，采用流式细胞术检测受体鼠脾和腹腔中M1和M2型巨噬细胞百分比；用实时荧光定量PCR法检测受体鼠脾和腹腔细胞中微小RNA-21(miR-21)、IL-10、p40、p35和EB病毒诱导基因3 (Epstein-Barr virus-induced gene 3，EBi-3)等mRNA表达水平。结果： 在脾细胞中，与同型对照组相比，抗TIM-4组M1和M2型巨噬细胞百分比、miR-21和p40 mRNA表达水平无明显变化(P均>0.05)，但IL-10和p35 mRNA表达水平明显升高(P均<0.05)，EBi-3 mRNA表达水平明显下降(P<0.05)；在腹腔中，与同型对照组相比，抗TIM-4组M1型巨噬细胞百分比和p35 mRNA表达水平无明显变化(P均>0.05)，而M2型巨噬细胞百分比、IL-10、p40和EBi-3 mRNA表达水平均显著升高(P均<0.05)，miR-21表达水平显著降低（P<0.05）。结论： 阻断TIM-4分子促进小鼠腹腔M2增殖分化，上调脾和腹腔细胞中抑炎因子和促炎因子mRNA表达，减轻同种异体免疫排斥反应。

Effect of TIM-4 blockade on the polarization of M1/M2 macrophages and allograft immune rejection

Objective: To explore the effect of T-cell immunoglobulin and mucin domain containing protein-4（TIM-4） on macrophage (M1/M2) polarization and allograft immune rejection by applying specific monoclonal antibodies to block the TIM-4 molecules on the surface of antigen presenting cells. Methods: Balb/c splenocytes were administered into C57BL/6 mice via intraperitoneal injection to set up the alloimmunization model. The immunized mice were divided into anti-TIM-4 group (n=6) and isotype control group (n=6); mice were injected with isotype control immunoglobulin or anti-TIM-4 monoclonal antibody via intraperitoneal, 300 μg per mouse; on the 5th day, mice were received another injection; on the 10th day, the two group mice were sacrificed for subsequent related experiments. The proportion of M1 and M2 macrophages from spleen and peritoneal cavity were detected by flow cytometry (FCM), and the mRNA expression of miR-21, IL-10, p40, p35 and EBi-3 in splenocytes and peritoneal cavity cells were detected by fluorescence quantitative RT-PCR. Results: Compared with the isotype control group, there was no significant difference in the proportion of M1 and M2 of anti-TIM-4 group in the spleen (P>0.05) and the mRNA expression of miR-21 and p40(P>0.05), and the mRNA expression of IL-10 and p35 in the spleen of anti-TIM-4 group was significantly up-regulated(P<0.05), while EBi-3 mRNA expression was greatly down-regulated(P<0.05). Compared with the isotype control group, the proportion of peritoneal M1 macrophages and the p35 mRNA expression in the anti-TIM-4 group was not markedly changed (P>0.05), the proportion of M2 and the mRNA expression of IL-10, p40 and EBi-3 in peritoneal cavity were significantly increased, while miR-21 expression was remarkably decreased(P<0.05). Conclusion: Blockade of TIM-4 molecule may promote the proliferation and differentiation of M2 and up-regulate the mRNA expression levels of anti-inflammatory cytokines and pro-inflammatory cytokines in peritoneal and splenic cells, and reduce the allogeneic immune rejection response. ［Key words］T-cell immunoglobulin and mucin domain containing protein-4; M1/M2 macrophage; microRNA-21; cytokines; polarization; immune rejection