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目的应用供体抗原特异性CD4+CD25+Treg细胞诱导移植免疫耐受,抑制大鼠肾移植慢性排斥反应。方法以SD、Wister大鼠分别为供、受体建立同种异体肾移植慢性排斥反应动物模型;受体脾细胞悬液同供体肾组织抗原孵育培养,诱导获得对供体抗原的特异性;采用MACS法分选具有供体抗原特异性的CD4+ CD25+T细胞,FACS流式细胞法检测分选的CD4+CD25+T细胞及CD4+CD25+Treg细胞纯度;获得之供体抗原特异性CD4+CD25+Treg细胞于同种异体肾移植术后2、4、6、8、10周,分别经尾静脉注射入受者体内(实验组,n=12),选取未注射组为对照(n=12)。观察两组移植肾脏存活时间;术后第10、40、80天MTT法检测比较两组受体脾细胞对供体抗原刺激反应程度;术后第10、20、40、60、80天分别监测各组血肌酐水平。结果FACS流式细胞法检测分选的CD4+CD25+T细胞得率为4.13%,分离纯度为73.34%;CD4+CD25+Treg细胞纯度为65.22%。所分选之CD4+CD25+Treg细胞成功获得供体抗原特异性,对供体抗原的抑制率(IR)为85.4%。移植肾存活时间:治疗组移植肾存活时间为(98.83±6.66)d,显著长于对照组的(78.25±4.71)d,组间差异有统计学意义(P<0.05);术后第40、80天治疗组脾细胞对供体抗原刺激反应程度均明显低于对照组,两组间差异有统计学意义(P<0.05)。术后第20、40、60、80天对照组血肌酐指标明显高于治疗组,同治疗组间差异有统计学意义(P<0.05)。结论供体抗原特异性CD4+CD25+Treg细胞可特异性抑制受体对供体抗原的免疫反应,有效抑制了移植肾慢性排斥反应的发生。
OBJECTIVE: To use donor antigen-specific CD4 + CD25 + Treg cells to induce transplantation tolerance and to inhibit the chronic rejection of renal allograft in rats. Methods SD and Wister rats were used as recipients and recipients to establish animal model of allograft rejection. The spleen cell suspension was incubated with donor kidney tissue antigen to induce the specificity of the donor antigen. The CD4 + CD25 + T cells with donor antigen specificity were sorted by MACS and the purity of sorted CD4 + CD25 + T cells and CD4 + CD25 + Treg cells were detected by FACS; the donor antigen-specific CD4 + CD25 + Treg cells were injected into the recipients via tail vein at 2, 4, 6, 8 and 10 weeks after allo-renal transplantation (experimental group, n = 12) = 12). The survival time of the grafted kidneys in both groups was observed. The levels of donor antigen stimulated by donor spleen cells in the two groups were detected by MTT assay on the 10th, 40th and 80th days after operation. The 10th, 20th, 40th, 60th, Serum creatinine levels in each group. Results The yield of sorted CD4 + CD25 + T cells by FACS was 4.13% with the purity of 73.34%. The purity of CD4 + CD25 + Treg cells was 65.22%. The sorted CD4 + CD25 + Treg cells successfully obtained the donor antigen specificity, the inhibition rate (IR) to the donor antigen was 85.4%. Survival time of renal allografts: The survival time of the grafted kidney in the treatment group was (98.83 ± 6.66) days, which was significantly longer than that in the control group (78.25 ± 4.71) d, the difference was statistically significant (P <0.05) The response of spleen cells to donor antigen in the day treatment group was significantly lower than that in the control group, with a significant difference between the two groups (P <0.05). The index of serum creatinine in the control group was significantly higher than that of the treatment group on the 20th, 40th, 60th and 80th days after operation. The difference was statistically significant (P <0.05). Conclusion Donor antigen-specific CD4 + CD25 + Treg cells can specifically inhibit the immune response of the donor to the donor antigen and effectively suppress the chronic rejection of the graft.