慢性应激老年抑郁大鼠海马血管新生的变化机制及文拉法辛的干预作用

发布时间：2018-03-04 17:15

本文选题：老年抑郁　切入点：血管新生　出处：《昆明医科大学》2013年博士论文　论文类型：学位论文

【摘要】：[目的] 建立慢性应激老年大鼠抑郁模型,评估老年抑郁大鼠海马血管新生的变化及其相关的分子机制,研究抗抑郁药物文拉法辛的干预作用。 [方法] 1.SPF级雄性老年SD大鼠(20月龄)共72只。根据随机数字表将大鼠随机分为模型组、文拉法辛组、生理盐水组、对照组各18只。模型组、文拉法辛组、生理盐水组均给予经改良的慢性不可预测的温和应激结合孤养共5w,文拉法辛组于应激后第3w末开始同时给予文拉法辛(5mg.kg-1.d-1)腹腔注射持续14d；生理盐水组于应激后第3w末开始同时给予生理盐水腹腔注射持续14d；对照组不给予任何处理,3只／笼正常喂养共5w。采用敞箱实验和蔗糖消耗实验评估大鼠抑郁模型建立情况,并观察老年抑郁大鼠及文拉法辛治疗后的行为学特征。 2.对上述大鼠标本,采用HE染色观察各组大鼠海马的形态学变化。应用免疫组织化学技术测定各组大鼠海马的微血管密度(microvessels density, MVD)以评估老年抑郁大鼠及文拉法辛干预后的海马血管新生的变化。 3.采用免疫组织化学技术测定各组大鼠海马血管内皮生长因子(vascular endothelial growth factor, VEGF)、血管生成素1(angiopoietin1, ANG1)、碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)的蛋白表达变化。采用免疫印迹(western blot)和逆转录聚合酶链反应(reverse transcript polymerase chain reaction, RT-PCR)技术测定各组大鼠海马VEGF及其受体血管内皮生长因子受体2(vascular endothelial growth factor receptor2, VEGFR2)、ANG1及其受体内皮细胞特异性酪氨酸受体2(tyrosine kinase with immunoglobulin and epide growth factor homology domain2, TIE2)、bFGF及其受体成纤维细胞生长因子受体1(fibroblast growth factor receptor1, FGFR1)的蛋白及mRNA表达水平。以评估老年抑郁大鼠及文拉法辛干预后的海马促血管新生因子表达的变化。 4.采用Western Blot和RT-PCR测定各组大鼠海马的磷脂酰肌醇-3-激酶(phosphoinositide3-kinase, PI3K)、蛋白激酶B (protein kinase B, Akt)、Janus蛋白酪氨酸激酶1(janus protein tyrosine kinase1, JAK1)、信号转录与转导活化因子3(signal transducer and activator of transcription3, STAT3)的蛋白及mRNA表达水平。并采用Western Blot测定磷酸化-Akt (phospho-Akt, p-Akt)蛋白表达水平。以评估老年抑郁大鼠及文拉法辛干预后的与海马血管新生作用相关的细胞内信号转导分子的变化。 5.采用多元线性回归分析,评估MVD与抑郁行为学指标敞箱实验的水平运动得分、垂直运动得分、清洁时间和蔗糖消耗实验的蔗糖消耗量之间的关系。采用Bivariate相关分析,评估VEGF蛋白、ANG1蛋白、bFGF蛋白相互之间的关系；并评估MVD与VEGF蛋白、MVD与ANG1蛋白、MVD与bFGF蛋白之间的关系。采用一元线性回归分析,评估MVD与VEGF蛋白、MVD与bFGF蛋白之间的关系。 [结果] 1.敞箱实验和蔗糖消耗实验显示模型组、文拉法辛组和生理盐水组抑郁模型均建立成功。敞箱实验结果：组内比较：模型组、文拉法辛组、生理盐水组于应激后3w、应激后5w分别与同组应激前1d比较,模型组和生理盐水组应激后5w与同组应激后3w比较,水平运动得分、垂直运动得分均降低(P均0.05),清洁时间缩短(P0.05)。组间比较：应激后5w,与对照组比较,文拉法辛组的水平运动得分、垂直运动得分降低(P0.05),清洁时间缩短(P0.05)；模型组和生理盐水组的水平运动得分、垂直运动得分明显降低(P0.01),清洁时间显著缩短(P0.01)；与生理盐水组比较,文拉法辛组垂直运动得分增加(P0.05)。蔗糖消耗实验结果：组内比较：模型组和生理盐水组应激后5w分别与同组应激前1d、同组应激后3w比较,蔗糖消耗、蔗糖偏爱减少(P均0.05)。组间比较：应激后5w,与对照组比较,模型组和生理盐水组的蔗糖消耗显著减少(P0.01)；与生理盐水组比较,文拉法辛组的蔗糖消耗增加(P0.05)。 2.HE染色显示：对照组海马锥体细胞层厚,排列整齐、紧密,毛细血管数量正常；模型组和生理盐水组海马锥体细胞层变薄,细胞间隙增大,排列紊乱、疏松,毛细血管数量减少；文拉法辛组海马锥体细胞层薄,但细胞排列整齐、紧密,毛细血管数量明显增多,管腔不规则。免疫组织化学染色结果显示：与对照组比较,模型组和生理盐水组海马微血管密度降低(P0.05)；与生理盐水组比较,文拉法辛组海马微血管密度增加(P0.05)。 3.与对照组比较,模型组和生理盐水组海马VEGF蛋白及VEGFR2蛋白、VEGFmRNA及VEGFR2mRNA表达降低(P0.05)：与生理盐水组比较,文拉法辛组海马VEGF蛋白及VEGFR2蛋白、VEGFmRNA及VEGFR2mRNA表达增加(P0.05)。 4.与对照组比较,模型组和生理盐水组海马ANG1蛋白及TIE2蛋白、ANG1mRNA及TIE2mRNA表达增加(P0.05)；与生理盐水组比较,文拉法辛组海马ANG1蛋白及TIE2蛋白、ANG1mRNA及TIE2mRNA表达增加(P0.05)。 5.与对照组比较,模型组和生理盐水组海马bFGF蛋白及FGFR1蛋白、bFGFmRNA及FGFR1mRNA表达降低(P0.05)；与生理盐水组比较,文拉法辛组海马bFGF蛋白及FGFR1蛋白、bFGFmRNA及FGFR1mRNA表达明显增加(P0.01)。 6.与对照组比较,模型组和生理盐水组海马P13K蛋白及Akt蛋白、p-Akt蛋白、PI3KmRNA及AktmRNA表达均下降(P0.05)；与生理盐水组比较,文拉法辛组海马P13K蛋白及Akt蛋白、p-Akt蛋白、PI3KmRNA及AktmRNA表达明显增加(P0.01)。 7.四组海马JAK1蛋白及STAT3蛋白、JAK1mRNA及STAT3mRNA表达差异无统计学意义(P0.05)。 8.在MVD与行为学结果的回归分析中,敞箱实验的垂直运动得分系数有统计学意义(P0.05),回归方程为MVD=5.056+0.580垂直运动得分。在VEGF蛋白、ANG1蛋白、bFGF蛋白的相关分析中,VEGF蛋白与ANG1蛋白、VEGF蛋白与bFGF蛋白均显著相关(r分别为0.573、0.742,P分别为0.003、0.000)。在MVD与VEGF蛋白的回归分析中,VEGF蛋白系数有显著统计学意义(P0.01),回归方程为MVD=3.391+11.507VEGF。在MVD与bFGF蛋白的回归分析中,bFGF蛋白系数有显著统计学意义(P0.01),回归方程MVD=3.047+10.884bFGF。 [结论] 1.本实验成功建立了老年大鼠抑郁模型。老年抑郁大鼠有其抑郁样症状特征：精神运动性迟滞症状突出,并随应激时间延长而加重；兴趣下降症状较晚才出现。相同剂量的文拉法辛针对不同症状疗效存有差异：可遏制活动迟滞症状的加重,但不能恢复到正常水平；可防治兴趣下降症状出现。 2.慢性应激老年抑郁大鼠的海马微血管密度减少,血管新生可能受抑。文拉法辛干预后海马微血管密度增加,对海马血管新生可能有促进作用。老年抑郁大鼠的海马微血管密度与老年大鼠的抑郁样症状有关,提示老年抑郁障碍可能与血管损伤有关。 3.经过持续5周的经改良的慢性不可预测的温和应激结合孤养,老年抑郁大鼠海马VEGF及受体VEGFR2、ANG1及受体TIE2、bFGF及受体FGFR1的蛋白及mRNA表达可发生改变。经文拉法辛14天抗抑郁治疗可促进老年抑郁大鼠海马VEGF及受体VEGFR2、ANG1及受体TIE2、bFGF及受体FGFR1的蛋白及mRNA的表达。海马VEGF蛋白与ANG1蛋白、VEGF蛋白与bFGF蛋白有关。海马微血管密度与VEGF蛋白有关,海马微血管密度与bFGF蛋白有关。提示VEGF、ANG1、bFGF在血管新生的分子机制中可能参与了老年抑郁障碍的发病和文拉法辛的抗抑郁作用。 4.在与血管新生作用相关的细胞内信号通路中,慢性应激老年抑郁大鼠的海马PI3K/Akt信号通路可能受到影响,文拉法辛可促进PI3K/Akt信号通路的活化。JAK1/STAT3信号通路未发生明显变化。提示PI3K/Akt信号通路在血管新生的分子机制中可能参与了老年抑郁障碍的发病及文拉法辛的抗抑郁作用。
[Abstract]:[Objective]
Objective to establish a chronic stress depression model in aged rats, evaluate the changes of hippocampus angiogenesis and related molecular mechanisms in elderly depression rats, and study the intervention effect of antidepressant venlafaxine.
[method]
1.SPF male aged SD rats (20 month old) and 72 in total. According to the random number table, the rats were randomly divided into model group, Vin Rafa Sin group, saline group and control group with 18 rats in each group. Model group, Vin Rafa Sin group, saline group were treated by modified chronic unpredictable Wen Heying induced by isolated feed 5W, Vin Rafa Sin group to stress after 3W starts at the end and give Vin Rafa Sin (5mg.kg-1.d-1) intraperitoneal injection duration 14d; saline group on stress after 3W began at the end of saline intraperitoneal injection for 14d; the control group was not given any treatment, 3 per cage normal feeding 5w. by open field test and sucrose consumption experimental evaluation of depressed rats, and observe the elderly depression rats and Vin Rafa Sin after treatment of behavioral characteristics.
2. of the big mouse, using HE staining to observe the morphological changes of hippocampus of rats in each group. The determination of microvessel density in hippocampus of rats by immunohistochemical technique (microvessels density MVD) to assess changes in rats and the geriatric depression venlafaxine treatment and hippocampi angiogenesis prognosis.
3. immunohistochemistry detected in vascular endothelial growth factor of rats (vascular endothelial, growth factor, VEGF), angiopoietin 1 (angiopoietin1, ANG1), basic fibroblast growth factor (basic fibroblast, growth factor, bFGF). The expression changes of protein by immunoblotting (Western blot) and reverse transcriptase polymerase chain (reverse transcript polymerase chain reaction reaction, RT-PCR) technique were used to detect the rat hippocampal VEGF receptor and vascular endothelial growth factor receptor 2 (vascular endothelial growth factor receptor2, VEGFR2, ANG1) and its receptors in endothelial cell specific tyrosine kinase receptor 2 (tyrosine kinase with immunoglobulin and Epide growth factor homology domain2, TIE2, bFGF) and its receptor into fibroblast growth factor receptor 1 (fibroblast growth factor receptor1, FGFR1) and mRNA protein To evaluate the expression of angiogenesis factor in the hippocampus of aged depressive rats and venlafaxine.
4. using Western Blot and RT-PCR were measured in rat hippocampus were phosphatidylinositol -3- kinase (Phosphoinositide3-Kinase, PI3K), protein kinase B (protein kinase B, Akt), Janus protein tyrosine kinase 1 (Janus protein tyrosine kinase1, JAK1), signal transduction and activation of transcription factor 3 (signal transducer and activator of Transcription3, STAT3 the expression levels of protein and mRNA). And the determination of phosphorylation of -Akt by Western Blot (phospho-Akt, p-Akt). The protein expression level in rats and to evaluate the prognosis of geriatric depression changes associated with hippocampal angiogenesis and intracellular signal transduction molecules Rafael Xin.
5. using multiple linear regression analysis, MVD and evaluation of depression behavior score index of horizontal movement of the open field test, vertical motion score, cleaning time and sucrose consumption test of sucrose consumption relation. Bivariate correlation analysis was used to evaluate VEGF protein, ANG1 protein, bFGF protein, the relationship between MVD and VEGF and evaluation; MVD protein and ANG1 protein, the relationship between MVD and bFGF protein. Using linear regression analysis, evaluation of MVD and VEGF protein, the relationship between MVD and bFGF protein.
[results]
1. open field test and sucrose consumption test showed that the model group, Vin Rafa Sin group and saline group and depression model were successfully established. Open field test results: group comparison: model group, Vin Rafa Sin group, saline group in 3W after stress, stress after 5W respectively with the same group before the stress of 1D, 3W and model group the saline group 5W after stress with the same group after stress, horizontal motion score, vertical movement scores were lower (P < 0.05), shorten the cleaning time (P0.05). Comparison between groups: 5W after stress, compared with the control group, the level of sports scores of the Vin Rafa Sin group, vertical motion score (P0.05), reduce the cleaning time shortened (P0.05); model group and saline group level sports scores, vertical motion score decreased significantly (P0.01), cleaning time was significantly shortened (P0.01); compared with the saline group, Vin Rafa Sin group of vertical movement scores increased (P0.05). The sucrose consumption. Results: group comparison: model group and saline group 5W after stress respectively with the same group of 1D before the stress, the same group of 3W after stress, sucrose consumption, reduced sucrose preference (P 0.05). Comparison between groups: 5W after stress, compared with the control group, sucrose model group and saline group. Consumption was significantly reduced (P0.01); compared with the normal saline group, venlafaxine group of sucrose consumption increased (P0.05).
2.HE staining showed that in control group, the hippocampal pyramidal cell layer thick, neat, compact, the number of capillaries in normal saline group; model group and hippocampal pyramidal cell layer thinning, cell gap increased, disorganized, osteoporosis, decrease in the number of capillaries; venlafaxine Zuhai horse pyramidal cell layer is thin, but the cells arranged in neat, close the number of capillaries, increased significantly, the lumen is irregular. Immunohistochemical staining showed that: compared with the control group, model group and saline group hippocampus decreased microvessel density (P0.05); compared with the normal saline group, venlafaxine group hippocampal microvessel density (P0.05).
3. compared with the control group, the expression of VEGF protein and VEGFR2 protein, VEGFmRNA and VEGFR2mRNA in hippocampus of model group and saline group decreased (P0.05): compared with saline group, the expression of VEGF protein and VEGFR2 protein, VEGFmRNA and VEGFR2mRNA increased in hippocampus of venlafaxine group (P0.05).
4. compared with the control group, the expression of ANG1 protein and TIE2 protein, ANG1mRNA and TIE2mRNA increased in hippocampus of model group and saline group (P0.05). Compared with saline group, the expression of ANG1 protein and TIE2 protein, ANG1mRNA and TIE2mRNA increased in hippocampus of venlafaxine group (P0.05).
5. compared with the control group, the expression of bFGF protein and FGFR1 protein, bFGFmRNA and FGFR1mRNA in hippocampus of model group and saline group decreased (P0.05), compared with the saline group, the expression of bFGF protein and FGFR1 protein, bFGFmRNA and FGFR1mRNA increased significantly in the venlafaxine group (P0.01).
6. compared with the control group, model group and saline group the expression of P13K protein and Akt protein, p-Akt protein, PI3KmRNA and AktmRNA decreased (P0.05); compared with the normal saline group, venlafaxine group the expression of P13K protein and Akt protein, p-Akt protein, PI3KmRNA and AktmRNA expression increased significantly (P0.01).
7. there was no significant difference in the expression of JAK1 and STAT3 protein, JAK1mRNA and STAT3mRNA in the four groups (P0.05).
8. the results in MVD and behavior in regression analysis, significant vertical motion score coefficient of open field experiment (P0.05), the regression equation is MVD=5.056+0.580 vertical motion score. ANG1 protein in VEGF protein, and correlation analysis of bFGF protein, VEGF protein and ANG1 protein, VEGF protein and bFGF protein were significantly correlated (r were 0.573,0.742 and P respectively, 0.003,0.000). In the regression analysis of MVD and VEGF protein, VEGF protein had a statistically significant coefficient (P0.01), the regression equation was MVD=3.391+11.507VEGF. in regression analysis of MVD and bFGF protein, bFGF protein had a statistically significant coefficient (P0.01), the regression equation of MVD=3.047+10.884bFGF.
[Conclusion]
1. this experiment successfully established the model of depression in aged rats. Rats with senile depression depressive like symptoms: psychomotor retardation symptoms, and with the stress time prolonged; the declining interest symptoms appear later. The same dose of venlafaxine for different symptoms curative effect: there are differences between the aggravation of symptoms can curb the activity of hysteresis, but can not be restored to normal levels; prevention and treatment of declining interest symptoms.
In 2. elderly chronic stress depression rats decreased microvessel density and angiogenesis may be inhibited. Venlafaxine intervention of hippocampal microvascular density increased, may contribute to angiogenesis. Hippocampus hippocampus of aged rats with depression and microvessel density in aged rats with depression like symptoms, suggesting that elderly depressive disorder may be related to the damage of blood vessels.
3. after 5 weeks of improved chronic unpredictable mild stress with solitary, VEGF and VEGFR2 receptor in hippocampus in aged depression rats, ANG1 receptor and TIE2 protein, and mRNA and bFGF receptor FGFR1 expression can be changed. 14 days of antidepressant venlafaxine Scripture can promote VEGF and VEGFR2 receptor in hippocampus of elderly depression in rats, ANG1 receptor and TIE2 protein and mRNA expression of bFGF and receptor FGFR1. The expression of VEGF protein and ANG1 protein, VEGF protein and bFGF protein in hippocampus. Microvessel density and VEGF protein, bFGF protein and microvessel density in hippocampus. In VEGF, ANG1, may be involved in depression of old molecules the mechanism of bFGF in angiogenesis in the pathogenesis of venlafaxine and antidepressant effect.
The 4. signal is correlated with angiogenesis in intracellular pathway, pathway of chronic stress depression rats hippocampal PI3K/Akt signal may be affected, venlafaxine can promote the activation of PI3K/Akt signaling pathway.JAK1/STAT3 signaling pathway did not change significantly. The incidence of venlafaxine and may be involved in the elderly depression suggest that PI3K/Akt signal pathway in vascular the new molecular mechanism of antidepressant effect.

【学位授予单位】：昆明医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R749.4

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