【作者】 张华；

【导师】 臧佩林；

【作者基本信息】 辽宁中医学院 ， 中医内科学， 2005， 博士

【摘要】 目的:探究慢性不可预见性应激方法建造的抑郁症模型大鼠的海马神经元及脑源性神经营养因子(BDNF)的损伤情况,并从行为学、病理形态学及分子生物学角度探讨中药复方保神汤抗抑郁作用的机制及临床实际应用价值。 方法:将90只成年Wista雄性大鼠随机分为正常组、模型组和预防及治疗组(分别包括中药高、低剂量组及阳性药对照组),采用孤养及慢性不可预见性中等强度应激的方法制成抑郁症大鼠模型。对模型大鼠采用中医疏肝解郁、健脾益肾、养心安神之中药复方保神汤进行预防和治疗给药,并与阳性药物(氟西丁)对照。 以Open field实验和糖水消耗实验测定动物的行为变化;分别通过光镜(HE染色)和电镜观察大鼠海马神经元形态和超微结构的变化:使用TUNEL检测技术观察大鼠海马神经细胞凋亡情况:运用免疫组化和原位杂交方法检测大鼠海马BDNF、Bcl-2及Bax表达情况;并用逆转录PCR法(RT-PCR)检测BDNF的mRNA的含量。 所有实验数据均采用SPSSl 1.5软件,用方差分析(ANOVA)及t检验法进行统计分析。 结果:在预防和治疗两阶段中,中药保神汤高、低剂量组均有较显著的预防和治疗由慢性应激所致模型大鼠的抑郁状态和海马神经元的损伤的作用,预防和治疗组间无显著性差异。 从行为学改变及1%蔗糖水消耗实验上:中药组和阳性药组均能改善大鼠抑郁行为(反应活动能力下降及兴趣丧失)及糖水摄取量(反应快感缺失),各组间没有显著性差异(P>0.05),但与模型组比有显著差异(P<0.05)。 从海马神经元形态观察,光镜下:可见到中药高剂量组海马区锥体细胞排列整齐,层次紧密,尼氏体深染;低剂量组锥体细胞排列较为紧密,细胞数目有所减少,尼氏体浅染。阳性药组及模型组锥体细胞排列松散,细胞缺失,尼氏体浅染甚至溶解;电镜观察:模型组可见到异染色质增多、聚集,胞质内细胞器结构明显减少,核呈不规则形,偶见凋亡小体的存在;阳性药组亦见细胞核变形,异染色质聚集,细胞器不丰富;中药高低剂量防、治组均能明显改善上述改变,尤以高剂量组为明显,见核呈圆形,核膜清晰,胞质内细胞器丰富,异染色质偏多,呈团块状。 RT-PCR 结果表明:中药保神汤高低剂量组均可增加海马区BDNF的表达,以高剂量组为明显,与阳性药对照组及模型组比,有显著性差异。 免疫组化及原位杂交结果表明:在防治两阶段,中药保神汤组的BDNF及海马神经元抗凋亡基因BcL-2的表达有明显增加,而促凋亡基因Bax的表达明显下降,与阳性药对照组及模型组相比,均有显著性差异。 TUNEL 染色观察:模型组及阳性药组大鼠海马神经元细胞核内可见明显增多的含有棕黄色颗粒的凋亡细胞,而中药组无论是预防还是治疗给药,其凋亡细胞数均明显减少,有显著性差异。 结论:长期慢性应激造成的抑郁模型大鼠可见海马神经元损伤,细胞凋亡增加。中药复更多还原

【Abstract】 Objectives: To investigate hippocampal neuronal damage and Brain-derived Neurotrophic Factor (BDNF) in chronic unpredictable stress-induced depression model rats and pathologically and molecular biologically anti-depressive mechanisms and clinical possibility of Bao Shen Tang , a Chinese traditional herb prescription.Methods: 90 adult male Wista rats were randomly divided into four groups, the control group, the model group, the prevention group and the treatment group. Further, the prevention and the treatment group rats were classified into three groups separately, high concentration, low concentration of Bao Shen Tang and positive control group. Rat depression models were established by lonely raising and chronic unpredictable mild stress method. To prevent and treat neuronal damage, depression model rats were administered by Bao Shen Tang, using fluoxetine hydrochloride capsules as positive control.The changes of rat behavior were determined using open-field test and sucrose solution consumption test. Ultrastructural changes of the rat hippocampal neurons were observed by electron microscope. Apoptosis in the hippocampal neurons were detected employing the TUNEL staining. Immunohistochemistry and situ hybridization were used to determine BDNF , Bcl-2 and Bax expression. Reverse-transcription PCR (RT-PCR) was used to measure BDNF Mrna expression. SPSS11.0 software was used for statistical comparisons (t test and one-way analysis of variance). All data were expressed as means ± SD. And a p value less than 0.05 is considered statistically significant.Results: Both high and low concentrations of Bao Shen Tang have preventive and treatment effect on depressive state and neuronaldamage. There was no significant difference between prevention group and treatment group.Analysis of behavior changes and one percent of sucrose solution consumption test; Administration of Bao Shen Tang and fluoxetine hydrochloride capsules improved depressive behavior (reduced activity ability and stimulation loss) and sucrose solution uptake (reaction anhedonia) in depression model rats (p<0. 05).There is no significant difference among groups (p>0.05).Morphological analysis of the hippocampal neurons: Under the light lens: It is obvious that hippocampi area prick cell arrange tidy , administrative levels is compactness, the Neis body is deep to dye in the group of high concentration of Bao Shen Tang. The low dosage of a prick cell arranges more close, the cell number has decreased, the Neis body is shallow to dye. positive control group and model a prick cells arrange lax, cell imperfection, the Neis body is shallow to dye even fuse, observed from the electricity lens : Model group differ chromatin increasing, coming together, cell organ quality inside the cell afterbirth obviously reducing, nucleus assume irregular form, accidentally seeing apoptotic body exist; positive control group also sees the cell nucleus transforms, the differ chromatin gathering, the cell organ is not abundant; The high and low concentration of Bao Shen Tang in prevent, cures group all can be obviously ameliorative above changing , particularly regard high concentration group as obvious, see the nucleus assume circular, the karyotheca is clear, cell organ inside the afterbirth quality abundant, differ dye the quality is partial to many, assume the conglomeration form.RT-PCRanalysis: mRNA levels of BDNF of the hippocampal neurons in Bao Shen Tang-treated rats, in particular, in high concentration of Bao Shen Tang-treated rats, were significantly increased (p<0. 05) compared with those of the hippocampal neurons in depression model and positive control rats.Iimnunohistochemical and situ hybridization analysis of BDNF and apoptosis-related genes: BDNF and anti-apoptotic Bc1-2 gene expression was markedly increased (p<0.05)In contrast, Bax expression was significantly decreased in the hippocampal neuronsof Bao Shen Tang-treated rats compared with those in positive control and depression model rats.Histological analysis with TUNEL method: The number of TUNEL-positive h更多还原