小鼠CD4^＋记忆性T淋巴细胞的产生、纯化、鉴定及皮肤移植的检测

Production, purification, identification and function of mouse CD4^＋ memory T lymphocytes

目的建立CD4^＋记忆T细胞（memory T lymphocytes，Tm）的免疫磁珠分离方法，并检测其体外部分功能。方法借助小鼠皮肤移植模型，制备受鼠脾细胞悬液，运用免疫磁珠方法分离CD4^＋Tm，用台盼蓝染色法和流式细胞仪检测分选所得细胞的活性和纯度，并在体外用ELISPOT技术检测其不同抗原刺激时IFN-γ活性分泌频数。结果小鼠皮肤移植皮片平均存活时间为（13．6±0．9）d；分离所得CD4^＋Tm的活细胞百分率为（98．4±0．7）％；流式细胞术检测表型，其中CD4^＋CD8^-细胞比例占（96．06±2．49）％，CD44^＋CD62L^-细胞比例占（94．13±2．36）％，CD4^＋CCR7^-细胞比例占（96．39±1．93）％；在无刺激物作用或者大鼠脾细胞、供体脾细胞、刀豆素A等刺激物作用时，IFN-γ活性分泌频数分别为3±1、6±3、339±14、108±9个／10^4，差异有统计学意义。结论通过小鼠皮肤移植模型及免疫磁性分离技术，可制备高纯度无菌的CD4^＋Tm，其细胞活力不受影响，作用具有良好的供体特异性。这为深入研究Tm在移植物免疫方面的作用提供了技术资料。

Objective To establish a method for isolating CD4^＋ memory T lymphocytes（Tm）, and to identify partial functions of these cells in vitro. Methods To draw assistance from mouse skin transplantation model and separate the splenocytes of recipients. The Tm were isolated by magnetic activated cell sorting（ MACS）. The activity and the purity of the isolated cells were detected by trypan blue staining and fluorescence activated cell sorting（FACS）, respectively. With ELISPOT assay, IFN-γ-secreting lymphocytes among these cells were detected and counted in vitro. Results Mean survival time of skin allografts was 13.6 ±0.9 d. The activity of the CD4^＋ Tm was 98% approximately. The proportion of CD4^＋ CD8^- , CD44^＋ CD62L^- , CD4^＋ CCR7^- lymphocytes were 96.06% ± 2.49%, 94.13% ± 2.36%, 96.39% ± 1.93%, respectively. When these cells were not stimulated or stimulated by different antigens such as rice splenocytes, donor splenocytes, Con A, the numbers of IFN-γ-secreting lymphocytes were 3 ± 1, 6±3, 339±14, 108±9 per 10^4 cells. Conclusion Magnetic activated cell sorting isolation kit can isolate CD4^＋ Tm aiming at skin allograft effectively, which facilitates us to investigate the role of memory T cells in transplantation immunity.