【作者】 郭磊磊；

【导师】 胡随瑜；

【作者基本信息】 中南大学 ， 中西医结合临床， 2009， 博士

【摘要】 目的观察电针对心肌梗死并抑郁模型大鼠行为学和海马神经元可塑性的影响,探讨心梗并抑郁动物模型建立方法、心梗并抑郁可能的发病机制,及电针作用机理。方法1.将成年雄性Sprague-Dawley大鼠随机分为正常对照组、假手术对照组、心肌梗死并抑郁模型对照组、电针试验组和氟西汀对照组。在急性心肌梗死模型基础上,选用21天慢性轻度不可预见性应激加孤养造模。2.造模后的第1,7,14和21天,分别观察电针对各组大鼠敞箱实验、液体消耗等行为学指标的变化。3.应用TUNEL方法检测海马神经细胞元凋亡。4.采用免疫组化法观察大鼠海马神经元Bc1-2和Bax阳性细胞数和灰度值。5.RT-PCR技术检测大鼠海马BDNFmRNA,CREBmRNA和ERK1/2mRNA表达的差异。6.运用Western-blot检测大鼠海马BDNF,P-CREB和p-ERK1/2蛋白表达量的差异。结果1.心梗后并经21天慢性轻度不可预见性及孤养应激,模型组大鼠的体重增加缓慢,敞箱实验中的水平穿越格数、竖立次数、理毛时间显著减少,中央格停留时间、粪便粒数增加;总液体消耗、糖水消耗明显下降,而纯水消耗显著增多,与正常组比较,均差异有显著性(P<0.05),提示慢性应激能够影响心梗后大鼠的体重和行为学指标;经过3周的治疗后,电针组和氟西汀组各项指标测值与模型组比较均有明显改善,差异有显著性(P<0.05)。2.TUNEL检测结果显示海马CA3区、齿状回均观察到细胞核被染成棕黄色的凋亡细胞,CA1区未见阳性细胞表达。与正常组比较,模型组大鼠海马凋亡细胞数增加,差异有显著性(P<0.05);与模型组比较,电针组和氟西汀组凋亡细胞明显减少,但电针组与氟西汀组差异无显著性(P>0.05)。3.免疫组化结果显示海马CA3区、齿状回均观察到细胞质被染成棕黄色的阳性细胞,CA1区未见阳性细胞表达。与正常组比较,模型组大鼠海马CA3区和齿状回Bax蛋白明显增加,Bcl-2蛋白和Bcl-2/Bax比值明显减少,差异有显著性(P<0.05);与模型组比较,电针组和氟西汀组Bax蛋白明显减少,而Bcl-2蛋白和Bcl-2/Bax比值明显增加,差异有显著性(P<0.05),但电针组与氟西汀组比较差异无显著性(P>0.05)。4.RT-PCR检测结果显示,与正常组比较,模型组大鼠海马BDNFmRNA,CREBmRNA和ERK1/2mRNA蛋白表达量明显减少(P<0.05);与模型组相比,电针组和氟西汀组大鼠海马BDNFmRNA,CREBmRNA和ERK1/2mRNA表达量增加(P<0.05)但电针组与氟西汀组比较差异无显著性(P>0.05)。5.Westen-blot检测结果显示,与正常组比较,模型组大鼠海马BDNF,P-CREB和p-ERK1/2蛋白表达量明显减少(P<0.05);与模型组相比,电针组大鼠海马BDNF,P-CREB和p-ERK1/2蛋白表达量增加(P<0.05)但电针组与氟西汀组比较差异无显著性(P>0.05)。结论1.急性心肌梗死手术后结合慢性不可预见性轻度应激,可成功制作心梗并抑郁大鼠复合模型,该模型具备心肌梗死和抑郁双重特点,符合抑郁动物模型的评定标准。模型制作重复性好、症状稳定、持续时间较长,为心身疾病的基础研究提供了一个思路和方法。2.电针治疗1周后,模型鼠体重和行为学指标无明显改善,经过3周的电针治疗,模型鼠的体重较前增加,行为学指标明显改善,说明电针3周时间才能逐步发挥有效的抗抑郁作用。3.模型鼠海马CA3区、齿状回出现了细胞凋亡,且抗凋亡蛋白Bcl-2表达减少,促凋亡蛋白Bax表达增加,二者比值降低,给予电针干预3周后,Bcl-2表达增加,Bax表达减少,二者比值增高,提示心梗并抑郁的发生与海马神经元凋亡有关,电针通过抗凋亡机制,对海马神经元细胞起保护作用。4.模型鼠海马BDNF表达下调、P-CREB水平降低,推测心梗并抑郁与海马功能异常有关,存在神经可塑性降低,神经元细胞受损的表现。3周的电针治疗可显著上调大鼠海马内P-CREB水平,提高BDNF的水平,可能是电针抗抑郁、保护海马神经元细胞的分子机制之一。5.模型鼠海马p-ERK1/2水平降低,反映出心梗并抑郁存在ERK信号通路异常,本研究中电针治疗3周后,p-ERK1/2水平显著增高,提示电针可以激活ERK信号通路,通过相关ERK信号级联中的关键分子发挥抗抑郁作用。更多还原

【Abstract】 AIMTo observe the effects on the change of behavior and hippocampal neurone in model rats of depressed post-MI with electro-acupuncture. And to investigate the measure on model rats of depressed post-MI,in accord with the mechanism on post-MI depression and treatment with electro-acupunctureMETHODS1.All adult male Sprague-Dawley rats were divided into four groups randomly including normal control group,sham operated group,model group,electro-acupuncture(EA) group and fluoxetine(FXT)group.The model was established with the measure of chronic stress depression and singly housed in 21 days after acute myocardial infarction.2.The behaviors of rats have been detected by open-field test and sucrose preference test on the beginning,the 7th,the 14th and the 21 st day by treated with EA.3.TUNEL method was used to measure the hippocampal apoptosis.4.Immunohistochemistry method was used to measure the expression of Bcl-2 and Bax in the hippocampus.5.The contents of BDNF,P-CREB and p-ERK protein expression in rat hippocampus were investigated with Western-blot technique.6.The contents of BDNFmRNA,CREBmRNA and ERK1/2mRNA expression in rat hippocampus were investigated with RT-PCR technique.RESULTS1.Compared with normal control group,rats in model group,which were exposed to chronic unpredicted mild stressors and were singly housed after 21 days in accordance with decreased weight and fluid intake significantly.And the behavior of model rats including reduced Squaresc rossed,rears,grooming and increased central time stopped in open-field test were observed.It also showed that a significant reduction of consumption of sucrose solutions,and an increased pure water.(p＜0.05).It suggested that chronic stress could affect weight and behavior of rats.After 3 weeks of antidepressant treatment,all the records of the behavioral testing of the rats in EA and FTX groups have significantly improved(p＜0.05).It suggested that both EA and FXT could improve body weight and behavior of depressive rats.2.The positive cells in the CA3 and dentate gyrus subregion of hippocampus were observed with the TUNEL.But,the positive cells were not observed in the CA1 subregion of hippocampus.Compared to the normal group,the positive cells were increased in the model group (P＜0.05),while the positive cells were decreased in the EA and FXT group compared to the model group(P＜0.05).And there weren’t difference between the EA group and FXT group(P＞0.05).3.The positive cells in the CA3 and dentate gyrus subregion of hippocampus were observed with the inununohistochemistry method.But, the positive cells were not observed in the CA1 subregion of hippocampus.Compared to the normal group,the Bax protein was increased in the model group(P＜0.05)in accord with decreased in Bcl-2 protein and Bcl-2/Bax(P＜0.05).But,compared to the model group,the decreased Bax protein and increased Bcl-2 protein,Bcl-2/Bax in the EA and FXT group(P＜0.05).And there weren’t significant difference between the EA group and FXT group(P＞0.05) 4.The observation of Western-blot showed that the contents of BDNF,P-CREB和p-ERK1/2 protein expression in hippocampus of rats in depression model group were significantly decreased compared to control group(P＜0.05).While,the contents of BDNF,P-CREB和p-ERK1/2 protein expression in hippocampus of rats were increased in EA and FXT group(P＜0.05) in accordance with no significant difference between EA group and FXT group(P＞0.05)5.The observation of RT-PCR showed that the expression of BDNFmRNA,CREBmRNA and ERK1/2mRNAin hippocampus of rats in depression model group were significantly decreased compared to control group(P＜0.05).While,the expression of BDNFmRNA, CREBmRNA and ERK1/2mRNA were increased in EA and FXT group(P＜0.05).There weren’t significant difference between the EA group and FXT group(P＞0.05)CONCLUSION1.The model of depressed post-MI in rat could be established successfully with chronic unpredicted mild stressors after AMI,which own the feature of MI and depression in accordance with the standard of evaluation in animal model of depression.The reproducibility of this model is safe which the symptom of depression is stable and exist in longer time.It provides a possible measure on the psychophysiological disorder to research.2.It suggested that treatment of EA couldn’t affect weight and behavior of model rats in a week.After 3 weeks of antidepressant treatment,all the records of the behavioral testing in rats with EA were improved significantly.It showed that treatment of EA could improve weight and behavior of depressive rats in 3 weeks.3.The positive cells in the CA3 and dentate gyrus subregion of hippocampus were observed in the model rats in accordance with the increased Bax protein and decreased Bcl-2 protein and Bcl-2/Bax. But,there were opposite phenomenon in the EA group.It suggested that neuron apoptosis of hippocampus was related to the depression after MI.Treatment of EA could protect the hippocampus neuron by reducing the neuron apoptosis.4.The decreased BDNF and P-CREB in hippocampus of model rats showed that the depressed post-MI maybe related to the disfunction in hippocampus neurone.The BDNF and P-CREB could be increased in 3 weeks,which was the possible mechanism on the treatment with EA to resist depression and protect the hippocampus neurone.5.The decreased p-ERK1/2 in hippocampus of model rats suggested that there was the abnormal signal pathway in the post-MI depression. The treatment with EA in 3 weeks could play a role in acting on the ERK1/2 signal pathway through the key factors.更多还原