落新妇甙对心脏移植排斥反应中活化T细胞穿孔素和颗粒酶B表达的影响

Effect of Astilbin on expressions of perforin and granzyme B in activated T cells of mouse heart transplantation model with acute rejection

目的用小鼠心脏移植急性排斥反应体外模型,探讨落新妇甙对心脏移植排斥反应中活化T细胞穿孔素和颗粒酶B表达的影响。方法分离BALB/C小鼠心肌细胞(2×105m l-1)和C57BL/6小鼠的脾细胞(1×106m l-1),前者做刺激细胞,后者做反应细胞,进行混合细胞培养,建立小鼠心脏移植急性排斥反应体外模型。实验分2组:对照组,心肌细胞与脾细胞混合培养;落新妇甙组,在对照组基础上加入落新妇甙(15μg/m l)。TUNEL法检测T淋巴细胞凋亡情况。RT-PCR法检测T细胞穿孔素和颗粒酶B表达情况。结果落新妇甙组T细胞凋亡指数明显高于对照组(P<0.01),穿孔素和颗粒酶B表达明显低于对照组(P<0.01)。结论落新妇甙诱导心脏移植排斥反应中活化T细胞凋亡的同时也抑制了穿孔素和颗粒酶B的释放,从而有效拮抗心脏移植排斥反应。

Objective To investigate the effect of astilbin on expressions of perforin and granzyme B in activated T cells of mouse heart transplantation model with acute rejection. Methods Cardiomyocytes of BALB/C mouse and spleen cells of C57BL/6 mouse were harvested and made into single cell suspensions. The cardiomyocytes （2×10^5 ml^-1）as stimulators and spleen cells （ 1×10^6 ml^-1 ） as responsets were mixed and cultured, The model of mouse heart transplantation with acute rejection in vitro was therefore established. There were two groups in the experiment, Control group is the mixed culture of the cardiomyocytes and spleen cells ; Astilbin group is the mixed culture of the cardiomyocytes and spleen cells with astilbin （15μg/ml）. Apoptosis of T cells were analyzed by TUNEL assay. The expressions of perforin and granzyme B were measured by RT-PCR. Results Apoptosis of activated T cells in Astilbin group was significantly increased than that of the control group（ P 〈0.01 ） , and the expressions of perforin and granzyme B were significantly decreased than those of the control group （ P 〈0.01 ）. Conclusion The prevention of heart transplantation rejection by Astilbin maybe partially related to its inhibition of perforin and granzyme B expressions and induction of apoptosis in activated T cells in mouse heart transplantation models in vitro.