急性排斥期静脉注射供体脾脏细胞对异体小鼠皮片存活的影响

Effects of intravenous infusion of allogeneic donor spleen cells in acute rejection period on mouse skin allograft survival

【目的】研究急性排斥期静脉注射供体脾细胞对异体小鼠皮片存活的影响,并初步探讨其机制。【方法】96只受体C57BL/6小鼠随机分为A组(阳性对照组)、B组(实验组)、C组(对照组)、D组(阴性对照组),每组24只。各组小鼠均进行同种异体皮片移植,A组移植后腹腔注射环孢素A 30 mg·(kg·d)-1,B组移植后腹腔注射环孢素A 30 mg·(kg·d)-1,第7天球后静脉注射供体脾细胞,第10天环孢素A减量至15 mg·(kg·d)-1;C组移植后腹腔注射环孢素A 30 mg·(kg·d)-1,第7天球后静脉注射生理盐水,第10天环孢素A减量至15 mg·(kg·d)-1;D组只进行同种异体皮片移植。大体观察移植皮片存活情况并绘制生存曲线,术后第11天取各组小鼠移植皮片行病理切片染色以评估免疫排斥病理分级,荧光定量PCR检测术后第1、4、7、8、10天小鼠脾脏白细胞介素-2(interleukin-2,IL-2)m RNA变化及第11天脾脏IL-10、转化生长因子β(transforming growth factor,TGF-β)m RNA水平,第8、10、15天行流式检测各组小鼠脾细胞调节性T细胞(Treg)比例变化。【结果】B组的移植皮片平均存活时间较C组延长3.18天,差异具有统计学意义(P<0.01)。对照皮肤排斥病理分级,第11天病理HE染色示A、B组病理分级为0级,而C、D组为3级。B组小鼠脾脏调节性T细胞比例在第8天到第10天有明显升高的变化,其余各组无明显变化。对比第10天脾脏调节性T细胞比例,B组较C组高,差异具有统计学意义(P<0.05)。B组小鼠脾脏IL-2 m RNA在第7到8天有明显的升高,其余各组无明显变化。对比第11天脾脏IL-10、TGF-βm RNA的比值,B组较C组高,差异具有统计学意义(P<0.05)。【结论】急性排斥期静脉注射供体脾脏细胞,能增加脾脏调节性T细胞数量,提高抑制性细胞因子IL-10、TGF-βm RNA水平,从而显著延长同种异体皮片移植物存活时间。

【Objective】To study the effects of intravenous infusion of allogeneic donor spleen cells in acute rejection period on mouse skin allograft survival and preliminarily explore its mechanism.【Methods】A total of 96 recipient C57BL/6 mice were randomly divided into group A（positive control group）, group B（experimental group）, group C（control group） and group D（negative control group）, 24 per group. All mice received allogeneic skin grafts. After the transplantation, mice in group A, B and C received intraperitoneal injection of cyclosporine A（Cs A） 30 mg·（kg·d）- 1. Mice in group B received retrobulbar intravenous injection of donor spleen cells on day 7, and the dosage of Cs A reduced to 15 mg·（kg·d）-1on day 10. Mice in group C received retrobulbar intravenous injection of normal saline solution on day 7, then the dosage of Cs A also reduced to 15 mg·（kg·d）-1on day 10.Mice in group D received no treatment after the transplantation. The changes of skin allografts were observed and demonstrated by survival curves. On day 11 after the operation, mouse skin allograft biopsy samples in all groups were prepared for pathological slice andHE staining to evaluate the levels of immune rejection. The IL-2 m RNA levels of recipient splenocytes on postoperative day 1,4,7,8and 10, the IL- 10 and TGF- β m RNA levels of recipient splenocytes on postoperative day 11 were determined by fluorescence quantitative PCR. The ratios of regulatory T cells in mouse spleen cells on day 8, 10 and 15 were determined by flow cytometry.【Conclusion】Intravenous infusion of allogeneic donor spleen cells in acute rejection period can increase the quantity of spleen regulatory T cells and the m RNA levels of inhibitory cytokines IL-10 and TGF-β, thereby significantly prolonging the survival time of skin allografts.