空瓶刺激心理应激法建立大鼠血管内皮损伤模型的评价

Evaluation of the vascular endothelial cell injury model in rats induced by empty bottle stimulated psychological stress

目的评价空瓶刺激心理应激法建立的大鼠血管内皮损伤模型,为心理应激与血管内皮细胞(VEC)损伤关系的研究提供实验工具。方法将16只雄性SD大鼠随机分为对照组、模型组各8只,两组第1周每天早晚9点定时喂水10 min;第8～21天,对照组(群居饲养)早晚9点依然定时饮水10 min,模型组(孤独饲养)早晚9点按随机表给予空瓶刺激或喂水10 min,两组均按体质量腹腔注射生理盐水(200 g/mL)2次/d。用旷场实验观察大鼠行为学变化;放射免疫法检测血浆皮质醇(COR),ELISA法检测血浆肾上腺素(Adr),硝酸还原酶法检测血浆一氧化氮(NO),放射免疫计数法检测血浆内皮素(ET)、6-酮-前列环素F1α(6-Keto-PGF1α)、血栓素B2(TXB2),化学比色法检测血浆Adr代谢产物H2O2,高效液相色谱法检测血浆甲胺(MA)及甲醛(FA);采用透射电镜观察VEC超微结构。结果与对照组比较,模型组大鼠行为学垂直得分、水平得分及总分均增高(P均<0.05),血浆COR、Adr、ET、TXB2、H2O2、MA、FA水平升高(P均<0.05),NO、6-Keto-PGF1α水平降低(P均<0.05),NO/ET、6-Keto-PGF1α/TXB2值均下降(P均<0.05)。透射电镜显示,模型组VEC的线粒体嵴模糊,部分线粒体出现空泡;对照组VEC线粒体结构完整,嵴清晰、排列紧密。结论空瓶刺激心理应激能够造成VEC功能和结构的损伤,此造模方法可用于心理应激、VEC损伤与保护的相关研究。

Objective To evaluate the animal model of vascular endothelial cell（VEC） damaged by psychological stress in rats,and to provide an experimental tool to study the relationship between psychological stress and the injury of VEC.Methods Sixteen adult male SD rats were randomly divided into two groups,8 in each group： the control group and the model group.During the first week,two groups were fed water regularly at nine o′clock for 10 minutes every morning and evening.In the second and third week,the control group（group feeding） was still fed water regularly for 10 minutes at nine o′clock every morning and evening,but the model group was fed separately with water for ten min or stimulation of empty bottle following the random table at nine o′clock every morning and evening.Both the two groups were injected with NS（200 g/mL） intraperitoneally twice a day.In the end,the depressive-behaviors of rats were evaluated by using an open field test.The plasma cortisol（COR） levels were detected by radioimmunoassay;the plasma levels of Adr were detected by ELISA;the plasma NO levels were detected by nitrate reductase assay;ET,6-Keto-PGF1a and TXB2 were detected by radioimmunoassay;the levels of H2O2 was detected by chemical colorimetry;MA and FA in plasma were detected by HPLC respectively.The ultrastructure of VEC was observed by transmission electron microscope（TEM）.Results Compared with the control group,the vertical,horizontal and total scores were all increased significantly in the model group（P〈0.05）.The plasma levels of COR,Adr,ET,TXB2,H2O2,MA and FA in the model group were significantly higher than those in the control group（P〈0.05）.The plasma levels of NO,6-Keto-PGF1a and the ratio of NO/ET,6-Keto-PGF1a/TXB2 were significantly lower than those in the control group（P〈0.05）.The VEC mitochondria cristae appeared vague and part of them had vacuole in the model group through TEM,while in the control group,VEC mitochondrial cristae were clear,with complete structure and compact arrangement.Conclusion The empty bottle stimulated psychological stress can cause the injury of VEC＇s function and structure,and this method can be used to study the psychological stress,mechanism of protection and injury of VEC.