供体特异性体内免疫状态定量检测方法的研究

目的 建立具有供体抗原特异性的体内免疫状态定量检测方法,探讨该方法 的流式检测技术参数、荧光染色条件、输注细胞数及确定最佳测试时间点.方法 取C57及BALB/C小鼠脾脏制备脾细胞单细胞悬液,分别用不同浓度羧基荧光素乙酰乙酸(CFSE)染色后制备1∶1混合细胞悬液,按混合细胞输注C57-BALB/C小鼠皮肤移植排斥模型,不同时间点(分别为输注后1、2、4、10 h、1、2、3、6 d),流式细胞仪检测外周血两种荧光细胞的比例变化,同时设未移植BALB/C小鼠为对照组,探索CFSE荧光染色条件、输注细胞数及确定最佳测试时间点,按公式计算供体特异性细胞杀伤率:%specific lysis=1-donor cells/recipient cells×100%.结果 CFSE染色时需要较大浓度差才能完全把两群脾细胞完全分离开,在浓度相差20倍时流式直方图才显示为两个完全独立的峰;即CFSE低浓度用0.3 μmol/L,高浓度用6 μmol/L.C57-BALB/C皮肤移植排斥模型在皮肤移植后两周即可建立,混合细胞悬液输注后,模型组与对照组输注混合细胞后1、2、4、10 h、1、2 d特异性杀伤率差异有统计学意义(t=39.5、41.1、26.2、27.4、15.8、5.5,P均＜0.01).模型组输注后1 h和2 h杀伤率分别高达(87.4±4.5)%、(92.2±3.9)%.结论　供体特异性体内免疫状态定量检测方法 流式检测设定在淋巴细胞门,两种细胞CFSE的染色浓度差控制在20倍左右,输注细胞总数在一定范围内对结果　没有影响,输注细胞后2～4h为最佳检测时间点,体内细胞毒性实验是一种简单、准确、稳定的免疫状态体内定量监测方法.

Abstract：

Objective To establish a method of donor specific cytotoxicity assay in vivo for quantitatively detecting the immune status of the recipient using flow cytometry. Methods C57BL/6j (H-2b)and BALB/C(H-2d) splenocytes were harvested, labelled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) at a final concentration of 0. 3 and 6 μ moL/L, respectively. A skin transplantation model was established with the recipients BALB/C mice and the donors C57BL/6 mice. The transplantation medels were divided into 8 groups. Similarly, BALB/C mice without skin transplantation served as negative controls. After intravenous transfusion of the CFSE-labelled donor/recipient splenocytes mixture, the following examinations were carried out at different time points ( 1, 2, 4, 10 h, 1, 2, 3, 6 days). The ratio of donor/recipient splenocytes labelled with CFSE in host peripheral blood was quantitatively estimated.The percent specific lysis was calculated using the equation: % specific lysis = 1 - donor cells/recipient cells × 100%. Results Compared with the control groups, the percent specific lysis of donor cells in the rejection model mice 1 h, 2 h, 4 h, 10 h, 1 day, 2 days after transfer was significantly higher than in normal BALB/C mice (t =39.5, 41.1, 26.2, 27.4, 15.8 and 5.5, all P＜0. 01). In the control groups,the percent of specific lysis was rapidly increased (87.4 ± 4. 5 ) % at 1 st h after transfer, and at 2nd h the percent was (92. 2 ± 3.9) %. Conclusion The method is simple, precise, reliable and well suited for quantitatively detecting the immune status of skin transplant recipients in vivo. Surveillance of the percent of specific lysis is helpful in the early detection of acute rejections, and in judgment of the therapeutic effect of immunosuppressive drugs and possibility of infection.

Research of donor specific quantitative immunological assay to monitor the immune status in vivo

Objective To establish a method of donor specific cytotoxicity assay in vivo for quantitatively detecting the immune status of the recipient using flow cytometry. Methods C57BL/6j (H-2b)and BALB/C(H-2d) splenocytes were harvested, labelled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) at a final concentration of 0. 3 and 6 μ moL/L, respectively. A skin transplantation model was established with the recipients BALB/C mice and the donors C57BL/6 mice. The transplantation medels were divided into 8 groups. Similarly, BALB/C mice without skin transplantation served as negative controls. After intravenous transfusion of the CFSE-labelled donor/recipient splenocytes mixture, the following examinations were carried out at different time points ( 1, 2, 4, 10 h, 1, 2, 3, 6 days). The ratio of donor/recipient splenocytes labelled with CFSE in host peripheral blood was quantitatively estimated.The percent specific lysis was calculated using the equation: % specific lysis = 1 - donor cells/recipient cells × 100%. Results Compared with the control groups, the percent specific lysis of donor cells in the rejection model mice 1 h, 2 h, 4 h, 10 h, 1 day, 2 days after transfer was significantly higher than in normal BALB/C mice (t =39.5, 41.1, 26.2, 27.4, 15.8 and 5.5, all P＜0. 01). In the control groups,the percent of specific lysis was rapidly increased (87.4 ± 4. 5 ) % at 1 st h after transfer, and at 2nd h the percent was (92. 2 ± 3.9) %. Conclusion The method is simple, precise, reliable and well suited for quantitatively detecting the immune status of skin transplant recipients in vivo. Surveillance of the percent of specific lysis is helpful in the early detection of acute rejections, and in judgment of the therapeutic effect of immunosuppressive drugs and possibility of infection.