促红细胞生成素对大鼠脑组织缺血再灌注海马CA1区神经元的作用

Effects of erythropoietin on neurons in CA1 region of hippocampus in rats with cerebral ischermia reperfusion injury

目的:观察促红细胞生成素对缺血再灌注大鼠脑组织海马CA1区神经元数量以及凋亡细胞数量变化、热休克蛋白70表达的影响。方法:实验于2003-03/2004-12在解放军第三军医大学新桥医院中心实验室完成。选择健康清洁级Wistar大鼠66只,随机分为正常对照组6只、缺血再灌注生理盐水组30只(生理盐水对照组)、缺血再灌注促红细胞生成素组30只(促红细胞生成素治疗组)。线栓法制备大鼠大脑中动脉阻断局灶性脑缺血模型,缺血2h后再灌注,促红细胞生成素治疗组经腹腔按200U/kg注入生理盐水稀释的重组人红细胞生成素(200U/mL),生理盐水对照组经腹腔注入等量的生理盐水。再灌注后观察时相点为2,6,12,24,48h,应用苏木精-伊红染色观察脑海马CA1区细胞数量情况,应用末端脱氧核苷酸转移酶缺口标记法检测海马CA1区神经元凋亡情况,应用免疫组织化学法测定热休克蛋白70表达情况。结果:66只大鼠全部进入结果分析。①脑海马CA1区细胞的计数:生理盐水对照组再灌注后12,24,48h比正常对照组细胞数明显减少犤(268.6±44.5)个/视野,(240.8±22.5)个/视野,(201.8±30.7)个/视野,(337.3±45.3)个/视野,t=2.845～5.587,P<0.01犦,促红细胞生成素治疗组再灌注后12,24,48h比生理盐水对照组细胞数明显增加犤(286.7±33.8)个/视野,(271.9±30.4)

AIM: To observe the effect of erythropoietin (EPO) on the numbers of apoptosis and neurons, and the expression of heat shock proteins 70 in CA1 region of hippocampus in rats with ischermia reperfusion injury. METHODS: The experiment was finished in the Center Laboratory,Xinqiao Hospital of Third Military Medical University of Chinese PLA from March 2003 to December 2004.Totally 66 Wistar rats were divided randomly into normal control group (n=6), ischermia reperfusion saline group (saline control group, n=30) and ischermia reperfusion EPO group (EPO treated group, n=30).Models of ischemia reperfusion were established after middle cerebral artery occlusion.After ischemia for two hours, rats in EPO treated group were injected with recombinant human erythropoietin(200 U/mL) diluted with saline into abdominal cavity, and the saline group received the same dose of saline. At the time points of 2, 6, 12, 24 and 48 hours following reperfusion,the number of cells in CA1 region of hippocampus was detected with Hematoxylin Eosin staining, the neuronal apoptosis was detected by using terminal deoxynucleotidyl transferase mediated nick end labeling staining,and the expression of HSP70 was detected by using immunohischemistry staining. RESULTS: All 66 rats were involved in the result.①After reperfusion for 12, 24 and 48 hours, the cellular number in saline control group(268.6±44.5, 240.8±22.5, 201.8±30.7) reduced significantly as compared with normal control group(337.3±45.3)(t=2.845-5.587,P< 0.01), which increased significantly in EPO treated group (286.7±33.8, 271.9±30.4, 258.3±29.5) as compared with saline control group(t=3.189-5.589, P< 0.01).②After reperfusion for 2, 6, 12, 24 and 48 hours, the TUNEL positive cellular number of saline control group (16.5±3.5, 28.4±6.5, 48.8±7.6, 60.7±10.2, 81.6±12.3) increased obviously as compared with normal control group(8.8±2.1) (t=2.985-6.324, P< 0.01), which decreased obviously in EPO treated group (12.7±3.4, 21.5±5.8, 32.7±4.6, 42.8±6.6, 51.8±9.5) as compared with saline control group (t=2.985-6.324,P< 0.01). ③After reperfusion for 2, 6, 12, 24 and 48 hours, the expression of HSP70 in saline control group (0.264±0.027, 0.328±0.053, 0.475±0.067, 0.587±0.095, 0.512±0.063) increased significantly as compared with normal control group (0.225±0.024) (t=3.254-6.028, P< 0.01). After reperfusion for 2, 6, 12 hours, the expression of HSP70 in EPO treated group(0.335±0.033, 0.448±0.055, 0.647±0.078) increased significantly as compared with saline control group(t=3.262-5.483,P< 0.01). CONCLUSION: EPO can alleviate neurons apoptosis in CA1 region of hippocampus in rats with focal ischermia reperfusion injury, and increase the expression of HSP70, so as to protect the nerve cells.