本文選題：表觀遺傳學(xué) + 慢性應(yīng)激��； 參考：《北京中醫(yī)藥大學(xué)》2016年博士論文

【摘要】：目的本研究采用慢性應(yīng)激大鼠模型,從表觀遺傳學(xué)(DNA甲基化和組蛋白乙�；�)和細(xì)胞信號(hào)通路(PKA信號(hào)通路)的角度,探討針刺對(duì)慢性應(yīng)激模型(CUMS)大鼠行為學(xué)和腦源性神經(jīng)營(yíng)養(yǎng)因子(BDNF)蛋白及基因表達(dá)的影響,為闡明針刺的抗抑郁作用機(jī)制提供一定的依據(jù)。方法1：40只雄性SD大鼠,體重(220～240)g,隨機(jī)分為空白對(duì)照組(空白組)、模型組、模型+氟西汀組(氟西汀組)、模型+針刺組(針刺組)。除空白組,其余大鼠均接受28 d慢性溫和不可預(yù)知刺激,氟西汀組在應(yīng)激前30 min灌胃給藥(1.8mg/kg/d),針刺組在應(yīng)激前30 min針刺“百會(huì)”和“印堂”穴,每天一次。在實(shí)驗(yàn)第0d、7d、14d、21d、 28 d分別進(jìn)行曠場(chǎng)、糖水實(shí)驗(yàn),稱取體重。實(shí)驗(yàn)結(jié)束后取海馬組織和血清。ELISA法檢測(cè)血清BDNF蛋白,RT-PCR法檢測(cè)海馬BDNF mRNA, Western-blot法檢測(cè)海馬BDNF、乙�；M蛋白H3K9 (acH3K9)、脫乙�；�2(HDAC2)蛋白,甲基化PCR法檢測(cè)海馬BDNF基因啟動(dòng)子I甲基化。2：60只雄性SD大鼠,體重(220～240)g,隨機(jī)分為空白對(duì)照組(空白組)、模型組、模型+氟西汀組(氟西汀組)、模型+針刺組(針刺組)、模型+氟西汀+H89組(氟西汀+H89組)、模型+針刺+H89組(針刺+H89組)。除空白組,其余大鼠均接受21d慢性溫和不可預(yù)知刺激。針刺組和氟西汀組的造模和治療干預(yù)方法同上。氟西汀+H89組和針刺+H89組在造模前1周進(jìn)行側(cè)腦室套管埋置手術(shù),在應(yīng)激前60 min給予側(cè)腦室注射H89溶液(10μm,5μL),隔天一次。兩個(gè)組在應(yīng)激前30 min分別進(jìn)行灌胃和針刺,每天一次。所有大鼠在實(shí)驗(yàn)第0d和第21d進(jìn)行曠場(chǎng)、糖水實(shí)驗(yàn),稱取體重。實(shí)驗(yàn)結(jié)束后取海馬組織。Western-blot法檢測(cè)海馬中蛋白激酶A (PKA)、 cAMP反應(yīng)元件結(jié)合蛋白(CREB)和磷酸化CREB (p-CREB)的蛋白含量。結(jié)果1.針刺和氟西汀對(duì)模型大鼠行為學(xué)的影響實(shí)驗(yàn)前,各組大鼠行為學(xué)檢測(cè)均無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)。造模后,與空白組相比,模型組的運(yùn)動(dòng)次數(shù)、糖水偏好百分比和體質(zhì)量明顯降低(P0.01,P0.01,P0.01)。與模型組相比,針刺和氟西汀均能提高模型大鼠的運(yùn)動(dòng)次數(shù)(P0.05,P0.05)、糖水偏好百分比(P0.01,P0.01)和體質(zhì)量(P0.05,P0.01),但針刺與氟西汀對(duì)各項(xiàng)行為學(xué)的作用趨勢(shì)不同。2.針刺和氟西汀對(duì)血清、海馬中BDNF蛋白和海馬BDNF mRNA的影響與空白組相比,模型組大鼠血清中和海馬中BDNF蛋白含量均顯著降低(P0.01,P0.01),海馬中BDNF mRNA也明顯降低(P0.01)；與模型組相比,氟西汀能顯著增加血清BDNF蛋白表達(dá)(P0.01)和海馬BDNF mRNA表達(dá)(P0.01),針刺能顯著增加血清和海馬BDNF蛋白表達(dá)(P0.01,P0.01)和海馬BDNF mRNA表達(dá)(P0.01),但針刺增加血清BDNF蛋白和海馬BDNF mRNA作用不如氟西汀(P0.05,P0.01)。3.血清BDNF蛋白、海馬BDNF蛋白與行為學(xué)的相關(guān)性分析血清BDNF蛋白表達(dá)與海馬BDNF蛋白表達(dá)存在正相關(guān)性(P0.05)。曠場(chǎng)運(yùn)動(dòng)次數(shù)與血清BDNF蛋白存在正相關(guān)性(P0.05),但糖水偏好百分比與血清BDNF蛋白相關(guān)性不顯著(P0.05)；海馬BDNF蛋白與行為學(xué)(曠場(chǎng)運(yùn)動(dòng)次數(shù)、糖水偏好百分比)均存在正相關(guān)性(P0.05,P0.01)。4.海馬BDNF蛋白、海馬BDNF mRNA與行為學(xué)的相關(guān)性分析海馬BDNF蛋白與海馬BDNF mRNA相關(guān)性不顯著(P0.05)。但曠場(chǎng)運(yùn)動(dòng)次數(shù)和糖水偏好百分比與海馬BDNF mRNA均有顯著正相關(guān)性(P0.000,P0.005)。5.針刺和氟西汀對(duì)海馬BDNF DNA啟動(dòng)子I甲基化的影響各組大鼠海馬BDNF DNA啟動(dòng)子I甲基化差異均沒(méi)有統(tǒng)計(jì)學(xué)意義(B 0.05)。6.針刺和氟西汀對(duì)海馬acH3K9和HDAC2蛋白的影響與空白組相比,模型組海馬內(nèi)acH3K9蛋白含量明顯降低(P0.05)；與模型組相比,氟西汀組和針刺組的acH3K9蛋白含量均有上升趨勢(shì),但是沒(méi)有統(tǒng)計(jì)學(xué)意義(P0.05,P0.05)。氟西汀組與針刺組相比差異也不顯著(P0.05)。與空白組相比,模型組海馬內(nèi)HDAC2蛋白含量顯著升高(P0.01)；與模型組相比,氟西汀和針刺均可降低海馬內(nèi)HDAC2的蛋白表達(dá)(P0.01)。針刺組和氟西汀組之間的差異不顯著(P0.05)。7.海馬acH3K9蛋白、HDAC2蛋白與BDNF mRNA的相關(guān)性分析海馬acH3K9蛋白表達(dá)與海馬HDAC2蛋白表達(dá)有顯著的負(fù)相關(guān)性(P0.005)。海馬BDNF mRNA表達(dá)與海馬acH3K9蛋白表達(dá)有一定的正相關(guān)性(P0.05),與海馬HDAC2蛋白表達(dá)有顯著的負(fù)相關(guān)性(P0.000)。8.海馬acH3K9蛋白、HDAC2蛋白與行為學(xué)的相關(guān)性分析曠場(chǎng)運(yùn)動(dòng)次數(shù)與海馬acH3K9蛋白表達(dá)有一定正相關(guān)性(P0.05),與海馬HDAC2蛋白表達(dá)有顯著的負(fù)相關(guān)性(P0.000)。糖水偏好百分比與海馬acH3K9蛋白表達(dá)沒(méi)有顯著相關(guān)性(P0.05),與海馬HDAC2蛋白表達(dá)有顯著的負(fù)相關(guān)性(P0.000)。9.針刺和氟西汀對(duì)海馬PKA-CREB信號(hào)通路的影響PKA特異性阻斷劑H89能阻斷氟西汀和針刺對(duì)模型大鼠行為學(xué)(水平穿越格數(shù)、豎立次數(shù)、糖水消耗量)的改善作用,且對(duì)氟西汀治療作用的阻斷更加顯著。各組海馬內(nèi)CREB蛋白表達(dá)差異不顯著(P0.05)。與空白組相比,模型組海馬內(nèi)PKA-α和p-CREB蛋白表達(dá)明顯降低(P0.01,P0.01)。與模型組相比,氟西汀組的PKA-α蛋白顯著增高(P0.05),p-CREB蛋白表達(dá)有上升趨勢(shì),但是沒(méi)有統(tǒng)計(jì)學(xué)意義(P0.05),針刺組的PKA-α和p-CREB蛋白表達(dá)均顯著增高(P0.01,P0.05),氟西汀+H89組和針刺+H89組的PKA-α蛋白表達(dá)也增高(P0.05,P0.05)。與氟西汀組相比,針刺組的PKA-α和p-CREB蛋白表達(dá)均無(wú)顯著差異(P0.05,P0.05),氟西汀+H89組的PKA-α蛋白表達(dá)無(wú)顯著差異(P0.05),但p-CREB蛋白表達(dá)顯著下降(P0.01)。與針刺組相比,針剌+H89組的PKA蛋白表達(dá)也無(wú)顯著差異(P0.05),但針刺+H89組的p-CREB蛋白含量顯著降低(P0.01)。結(jié)論1.慢性應(yīng)激可以降低大鼠的自主探究行為,抑制獎(jiǎng)賞系統(tǒng)和抑制體重增長(zhǎng)。針刺能改善慢性應(yīng)激誘導(dǎo)的抑郁樣行為。2.慢性應(yīng)激誘導(dǎo)的糖水偏好百分比和曠場(chǎng)運(yùn)動(dòng)次數(shù)下降與海馬BDNF蛋白和BDNF mRNA含量降低有關(guān),針刺可以通過(guò)提高海馬BDNF蛋白和BDNF mRNA表達(dá)改善模型大鼠的抑郁樣行為。3.慢性應(yīng)激誘導(dǎo)的大鼠抑郁樣行為及海馬BDNF mRNA降低與海馬內(nèi)HDAC2蛋白含量增加有關(guān),與組蛋白H3K9乙�；�(acH3K9)含量降低。針刺可以通過(guò)下調(diào)海馬HDAC2蛋白表達(dá),促進(jìn)組蛋白H3K9乙�；�,增加BDNF mRNA表達(dá),緩解模型大鼠的抑郁樣行為。4.慢性應(yīng)激誘導(dǎo)的大鼠抑郁樣行為與海馬內(nèi)PKA信號(hào)通路受到抑制有關(guān),針刺可通過(guò)激活海馬內(nèi)PKA信號(hào)通路發(fā)揮抗抑郁作用。5.海馬內(nèi)BDNF、 acH3K9、 HDAC2及PKA信號(hào)通路在抑郁癥的病理生理學(xué)中均起重要作用,本研究證實(shí)針刺可以通過(guò)調(diào)節(jié)上述指標(biāo)緩解模型大鼠的抑郁樣行為,為闡明針刺的抗抑郁作用機(jī)制提供了科學(xué)依據(jù)。
[Abstract]:Objective to explore the effects of acupuncture on behavior and brain derived neurotrophic factor (BDNF) protein and gene expression in chronic stress model (CUMS) rats from the angle of epigenetics (DNA methylation and histone acetylation) and cell signaling pathway (PKA signaling pathway), and to elucidate the antidepressant effect of acupuncture in order to clarify the antidepressant effect of acupuncture. Methods at 1:40 male SD rats, weight (220~240) g, were randomly divided into blank control group (blank group), model group, model + fluoxetine group (fluoxetine group), model + acupuncture group (acupuncture group). Except for blank group, the other rats were treated with 28 d chronic mild unpredictable stimulation, and fluoxetine group was given 30 min before stress. Drug (1.8mg/kg/d), acupuncture group at 30 min before stress "Baihui" and "printing hall" point, once a day. In the experiment, 0d, 7d, 14d, 21d, 28 d respectively to carry out the open field, sugar water experiment, weighed. After the experiment, the hippocampus and serum.ELISA method were taken to detect serum BDNF protein, RT-PCR method was used to detect hippocampus BDNF mRNA, hippocampus detection method was used for detection hippocampus BDNF, acetylation histone H3K9 (acH3K9), deacetylase 2 (HDAC2) protein, methylation PCR method to detect the I methylation of BDNF gene promoter in the hippocampus of the male SD rats and weight (220~240) g, randomly divided into blank control group (blank group), model group, model + fluoxetine group (fluoxetine group), model + acupuncture group (acupuncture group), model + fluoxetine +H89 Group (group of fluoxetine +H89), model + acupuncture +H89 group (group of acupuncture +H89). Except for blank group, the rest rats were treated with 21d chronic mild unpredictable stimulation. The model and treatment intervention of the acupuncture group and fluoxetine group were same. The lateral ventricle burial operation was performed in the group of fluoxetine +H89 and the acupuncture group +H89 at 1 weeks before the model, and the side brain was given to the side brain before stress. Intravorum injection of H89 solution (10 mu m, 5 u L) one time every other day. Two groups were gavage and acupuncture at 30 min before stress, once a day. All rats were in the open field of experimental 0d and 21d, sugar water experiment, and weighed. After the experiment, the hippocampus tissue.Western-blot method was used to detect the protein kinase A (PKA), cAMP reaction element binding protein (CR). EB and phosphorylated CREB (p-CREB) protein content. Results 1. acupuncture and fluoxetine had no statistical difference (P0.05) before the experiment on behavior of model rats (P0.05). Compared with the blank group, the number of movement times, the percentage of sugar water preference and body mass decreased significantly (P0.01, P0.01, P0.01). And the model was compared with the model group. Compared with the group, acupuncture and fluoxetine could improve the number of exercise times (P0.05, P0.05), P0.01, P0.01 and body mass (P0.05, P0.01) in the model rats, but the effects of acupuncture and fluoxetine on various behaviourology were different from that of.2. acupuncture and fluoxetine on serum, BDNF protein in hippocampus and BDNF mRNA of hippocampus in the blank group. In the model group, the content of BDNF protein in the serum and hippocampus decreased significantly (P0.01, P0.01), and the BDNF mRNA in the hippocampus decreased significantly (P0.01). Compared with the model group, fluoxetine could significantly increase the expression of serum BDNF protein (P0.01) and the expression of BDNF mRNA in the hippocampus (P0.01). The needle spines could significantly increase the expression of the protein and the sea hippocampal BDNF protein. The expression of horse BDNF mRNA (P0.01), but the increase of serum BDNF protein and hippocampal BDNF mRNA effect is not as good as the.3. serum BDNF protein of fluoxetine (P0.05, P0.01), the correlation between the BDNF protein of the hippocampus and the behaviourology, the positive correlation between the expression of the serum BDNF protein and the expression of hippocampal protein is positive. P0.05, but there was no significant correlation between the percentage of sugar water preference and serum BDNF protein (P0.05), and there was a positive correlation between hippocampal BDNF protein and behavior (the number of open field movements, the percentage of sugar water preference) (P0.05, P0.01).4. hippocampal BDNF protein, and the correlation analysis of hippocampal BDNF mRNA and Study on the relationship between hippocampal BDNF protein and hippocampal BDNF No significant (P0.05). But there was a significant positive correlation between the frequency of open field movement and the percentage of sugar water preference and hippocampal BDNF mRNA (P0.000, P0.005).5. acupuncture and the effect of fluoxetine on the I methylation of the promoter of BDNF DNA in the hippocampus. There was no significant difference in the difference of I methylation of BDNF DNA promoter in hippocampus of rats (0.05) Compared with the blank group, the content of acH3K9 protein in the hippocampus of the model group was significantly lower than that in the blank group (P0.05). Compared with the model group, the content of acH3K9 protein in the fluoxetine group and the acupuncture group increased, but there was no statistical significance (P0.05, P0.05). The difference between the fluoxetine group and the acupuncture group was not significant (P0.05) and the blank group (P0.05, P0.05). The cH3K9 protein content was significantly lower than that in the model group (P0.05, P0.05). Compared with the model group, the content of HDAC2 protein in the hippocampus increased significantly (P0.01). Compared with the model group, fluoxetine and acupuncture could reduce the protein expression of HDAC2 in the hippocampus (P0.01). The difference between the acupuncture group and the fluoxetine group was not significant (P0.05).7. hippocampal acH3K9 protein, the correlation of HDAC2 egg white and BDNF mRNA was analyzed, and the expression of acH3K9 protein in the hippocampus and the sea were in the sea. The expression of horse HDAC2 protein has significant negative correlation (P0.005). There is a positive correlation between the expression of hippocampal BDNF mRNA and the expression of acH3K9 protein in the hippocampus (P0.05), and there is a significant negative correlation with the expression of HDAC2 protein in the hippocampus (P0.000).8. hippocampal acH3K9 protein, the correlation of HDAC2 protein and behavioral studies analysis of the frequency of open field and the expression of the hippocampal acH3K9 protein There was a positive correlation (P0.05), and there was a significant negative correlation with the expression of HDAC2 protein in the hippocampus (P0.000). There was no significant correlation between the percentage of sugar water preference and the expression of acH3K9 protein in the hippocampus (P0.05). There was a significant negative correlation with the expression of HDAC2 protein in the hippocampus (P0.000).9. acupuncture and fluoxetine's effect on the PKA specific resistance of the hippocampus PKA-CREB signaling pathway. H89 can block the improvement of fluoxetine and acupuncture on the behavior of model rats (horizontal crossing number, vertical number, sugar water consumption), and the blocking of fluoxetine treatment is more significant. There is no significant difference in the expression of CREB protein in hippocampus (P0.05). Compared with the blank group, the expression of PKA- alpha and p-CREB protein in the hippocampus of the model group is obvious. Lower (P0.01, P0.01). Compared with the model group, the PKA- alpha protein of the fluoxetine group increased significantly (P0.05), and the expression of p-CREB protein increased, but there was no statistical significance (P0.05). The expression of PKA- alpha and p-CREB protein in the acupuncture group increased significantly (P0.01, P0.05), and the expression of the protein expression in the fluoxetine +H89 group and the needle +H89 group increased. Compared with the fluoxetine group, there was no significant difference in the expression of PKA- alpha and p-CREB protein in the acupuncture group (P0.05, P0.05), and there was no significant difference in the expression of PKA- alpha protein in the fluoxetine +H89 group (P0.05), but the expression of p-CREB protein decreased significantly (P0.01). Compared with the acupuncture group, there was no significant difference in the expression of PKA protein in the needle +H89 group (P0.05). The white content was significantly decreased (P0.01). Conclusion 1. chronic stress can reduce the behavior of autonomic exploration, inhibit reward system and inhibit the growth of body weight. Acupuncture can improve the depression like behavior induced by chronic stress, the percentage of sugar water preference and the decline of the number of open field exercise times induced by chronic stress induced by.2. and the decrease of the content of BDNF protein and BDNF mRNA in the hippocampus are reduced. Acupuncture can improve the depressive behavior of the model rats by improving the expression of BDNF protein and BDNF mRNA in the hippocampus and the depression like behavior induced by.3. chronic stress in rats and the decrease of BDNF mRNA in the hippocampus is related to the increase of the content of HDAC2 protein in the hippocampus, and the content of the histone H3K9 acetylation (acH3K9) decreases. The acupuncture can reduce the hippocampus HDAC2 protein table. Da, promote histone H3K9 acetylation, increase BDNF mRNA expression, alleviate the depressive behavior of model rats,.4. chronic stress induced depression like behavior in rats is related to the inhibition of PKA signaling pathway in the hippocampus. Acupuncture can play antidepressant in hippocampus BDNF, acH3K9, HDAC2, and PKA signaling pathway by activating the PKA signaling pathway in the hippocampus. It plays an important role in the pathophysiology of depression. This study confirms that acupuncture can relieve the depressive behavior of rats by regulating these indexes and provide a scientific basis for clarifying the mechanism of antidepressant action of acupuncture.
【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R245
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本文編號(hào)：1899294