硫芥诱导大鼠脾脏淋巴细胞凋亡的研究

Lymphocyte apoptosis induced by sulfur mustard in rat spleen

目的探讨硫芥诱导脾脏组织淋巴细胞凋亡以及caspase-3在其中的作用。方法大鼠腹腔注射硫芥,各组动物分别于中毒后1、3、5d后麻醉,开胸取脾。HE染色法观察脾脏组织的病理改变,RT-PCR法检测大鼠脾脏组织caspase-3基因表达,West-ernblot法检测caspase-3的蛋白表达。分离脾脏淋巴细胞,制备硫芥染毒细胞模型。DNA琼脂糖凝胶电泳观察caspase-3抑制剂Ac-DEVD-CHO对细胞DNA降解的影响;流式细胞仪检测Ac-DEVD-CHO对染毒细胞凋亡峰(即亚G1峰)及线粒体跨膜电位(ΔΨm)的影响。结果硫芥中毒大鼠脾脏组织形态发生病理改变,部分淋巴细胞出现了凋亡的特征;脾脏组织caspase-3的mRNA与蛋白表达均增加:mRNA在中毒后1d的表达量与对照组比较具有统计学意义(P<0.05);蛋白表达在中毒后3d和5d与对照组比较具有统计学意义(P<0.05,P<0.01)。caspase-3的特异性抑制剂Ac-DEVD-CHO对体外培养的硫芥染毒淋巴细胞的DNA降解和细胞G1峰左侧凋亡峰的出现有抑制作用。硫芥可导致细胞线粒体跨膜电位降低,随着中毒时间延长,线粒体膜电位的降低更明显。结论硫芥中毒可导致大鼠脾脏组织损伤,细胞凋亡是其作用机制之一,caspase-3可能参与了这一过程并在其中发挥重要的作用。

Objective To investigate the apoptotic effect induced by sulfur mustard on the lymphocytes of the rat spleen,and define the role of caspase-3 during the process.Methods Sulfur mustard was given by intraperitoneal injection in a dose of 3.5mg/kg.The animals were anesthetized and the spleens were harvested at different timepoints after intoxication.The histopathogy of spleen was studied with hematoxylin-eosin staining.Caspase-3 mRNA was detected with RT-PCR.Protein expression of caspase-3 was assayed with Western blotting,lymphocytes of rat spleen were isolated and cultured in vitro and they were challenged with sulfur mustard（100μmol/L）.The effect of Ac-DEVD-CHO（a specific inhibitor of caspase-3）on sulfur mustard-induced apoptosis of the lymphocytes cultured in vitro was evaluated.Fluorescent probe labeled with Rhodamine 123 was used to study mitochondrial potential.Results The histology of rat spleen was affected after sulfur mustard intoxication,as evidenced by apoptosis of a part of lymphocytes.Protein and mRNA expressions of caspase-3 were increased significantly in the spleens of intoxicated rats as compared with that in control group.DNA ladder and ＇sub-G1＇ peak of lymphocytes which were treated with sulfur mustard in vitro were partially improved by Ac-DEVD-CHO（the specific inhibitor of caspase-3）.In addition,mitochondrial potential decreased in a time-dependent manner in the lymphocytes intoxicated by sulfur mustard in vitro compared with that in the control group.Conclusions The spleen is injured in the rat which is intoxicated by sulfur mustard.Lymphocyte apoptosis is one of the mechanisms splenic injury.Caspase-3 may be involved in the process of lymphocyte apoptosis induced by sulfur mustard.