氯氮平对雄性C57BL/6小鼠胰腺β细胞凋亡的影响

Effects of clozapine on apoptosis of pancreatic beta cells in the male C57BL/6 mice

目的:探讨氯氮平对雄性C57BL/6小鼠胰腺β细胞凋亡的影响。方法:实验于2004-03/09在南京医科大学第一附属医院中心实验室完成。选择清洁级雄性C57BL/6小鼠63只,体质量(20±2)g,由上海斯莱克实验动物有限责任公司提供[许可证号:SCXK(沪)2003-0003]。随机分为空白组21只、氯氮平4mg/kg组21只、氯氮平20mg/kg组21只,各组小鼠的体质量和基础血糖值无显著差异(P>0.05)。所有动物每7只分笼饲养于光照12h(7Am～7Pm),相对湿度45%的普通设施中,普通全价鼠饲料喂养,自由进食及饮水。①模型制作:氯氮平充分研磨后加入蒸馏水,配成浓度为2g/L和0.4g/L的悬浊液,灌胃前充分匀。氯氮平4mg/kg组灌胃0.4g/L的悬浊液0.2mL,氯氮平20mg/kg组灌胃2g/L的悬浊液0.2mL,空白组灌胃同样剂量的蒸馏水。按发病不同时间每组再分为3组,各7只,超急性期组灌胃1次;急性期组灌胃1次/d,共7次;慢性期组灌胃1次/d,共28次。②标本的处理:禁食12h(8Pm～8Am)后,超急性期组在第1次灌胃后3h,急性期组(1周)和慢性期组(4周)灌胃1次/d,最后一次灌胃后3h,摘眼球取血后颈椎脱臼处死小鼠,立即取小鼠的胰腺置体积分数为0.1的中性甲醛液中固定24h,常规制作石蜡切片,厚4μm。采用苏木精-伊红染色,光镜下观察胰岛炎的情况。应用原位末端转移酶标记法染凋亡细胞,用链霉亲合素-生物素化过氧化物酶复合物免疫组织化学法染β细胞,进行β细胞凋亡计数。结果:纳入动物63只,均进入结果分析。①苏木精-伊红染色显微镜观察显示:空白对照组、氯氮平4mg/kg组、氯氮平20mg/kg组小鼠胰岛均位于胰腺外分泌部之中,其岛形丰满,轮廓清晰,胰岛细胞分布均匀,均未见淋巴细胞浸润。②免疫组织化学染色显微镜观察显示:空白对照组、氯氮平4mg/kg组、氯氮平20mg/kg组小鼠胰岛轮廓清晰,胰岛细胞分布均匀,内外分泌部界限清楚,大量的β细胞胞浆呈棕黄色的胰岛素阳性表达,未见β细胞凋亡,可见少量散在的胰腺腺泡细胞凋亡。结论:氯氮平对胰腺β细胞没有明显的毒害作用,不诱导β细胞的凋亡。

AIM： To investigate the effects of clozapine on apoptosis of pancreatic β cells in male C57BL/6 mice, METHODS： The study was carried out in the central laboratory of the First Affiliated Hospital of Nanjing Medical University from March to September 2004. Sixty-three male C57BL/6 mice of clean grade weighed 18 to 22 g were provided by Shanghai laboratory animal center [certification number： SCXK （Shanghai） 2003-0003], The mice were randomly divided into blank group （n=21）, clozapine 4 mg/kg group （n=21） and clozapine 20 mg/kg group （n=21）, and there were insignificant differences in the body mass and basic blood glucose among the groups （P 〉 0.05）. The animals were housed （7 mice per cage） with free access to food and water in a temperature-regulated room in which the relative humidity was 45% with a 12-hour light/dark cycle （lights on 7：00-19：00）. The mice were randomly divided into three groups, which administrated with distilled water, clozapine 4 mg/kg group, clozapine 20 mg/kg group, respectively. The weight and basic blood sugar were not different between the three groups. ①Model establishment： Clozapine was completely grinded and diluted with distilled water to prepare suspensions of 2 and 0.4 g/L respectively, which should be shaken to be even completely. The mice in the clozapine 4 mg/kg group were treated with gastric perfusion of 0.2 mL clozapine of 0.4 g/L）, those in the clozapine 20 mg/kg group were treated with gastric perfusion of 0.2 mL clozapine of 2 g/L, and those in the blank group were given gastric perfusion of distilled water of the same dosage. According to the different time of attack, each group was subdivided into 3 groups with 7 mice in each： super-acute group were given gastric perfusion once, the acute group were treated with gastric perfusion once a day for 7 times, and the chronic group were treated with gastric perfusion once a day for 28 times. ② Sample treatment： Mice were fasted for 12 hours （8 Pm-8 Am）, and they were killed by means of dislocation of cervical vertebra after blood collection by excising eyeballs at 3 hours after gastric perfusion in the super-acute group and 3 hours after the last gastric perfusion in the acute group （1 week） and chronic group （4 weeks）, and then the pancreas was fixated in neutral formalin with the volume fraction of 0.1 for 24 hours and made into paraffin section with the thickness of 4μm. Insulitis was observed under light microscope by hematoxylin-eosin （HE） staining. The apoptotic cells were marked by in situ nick end-labeling （TUNEL） and β cells were labeled by streptavidin-biotin-peroxidase complex （SABC）, and the apoptosis of βcells was counted. RESULTS： All the 63 animals were involved in the analysis of results. ① HE staining results： The edges of the pancreatic islets were clear and the islets cells showed proportional distribution in the blank control group, elozapine 4 mg/kg group and elozapine 20 mg/kg group. There were no infiltration of lymph cells in the islets of pancreas. ② SABC results： Exterior and interior secretory portions were distinct. The β cells whose cytoplasm was buffy stained positively with insulin and there were no pancreatic apoptotic β cells in the blank control group, clozapine 4 mg/kg group and clozapine 20 mg/kg group. CONCLUSION： The effects of clozapine on apoptosis of pancreatic β cells in the male mice may be not conspicuous. Clozapine may not induce β cells apoptosis.