【摘要】：第一部分慢性社會挫敗應激對小鼠前額皮層miRNAs及P-catenin蛋白表達水平的影響 目的：建立小鼠慢性社會挫敗應激模型,檢測前額皮層區(qū)小RNA (microRNA, miRNA)的差異化表達及蛋白表達變化。 方法：采用慢性社會挫敗應激和慢性不可預知性應激建立抑郁癥模型；糖水偏好測試,懸尾測試和社會接觸測試等檢測抑郁樣行為；Elisa檢測模型小鼠血清皮質(zhì)酮水平；實時熒光定量PCR定量分析前額皮層區(qū)miRNAs的差異表達；Western Blotting檢測前額皮層區(qū)p-連環(huán)素(β-catenin)的表達。 結(jié)果：(1)慢性社會挫敗應激模型鼠前額皮層miR-214-3p表達相對于對照組上調(diào)4.249±0.816,miR-690相對于在對照組上調(diào)2.527±0.292；(2)慢性社會挫敗應激模型小鼠的糖水偏好率下降到40.577±4.81%,社會接觸時間縮短至60.85±13.17s；(3)慢性社會挫敗應激模型小鼠血清皮質(zhì)酮含量升高到17.943±0.86μg/L；(4)慢性社會挫敗應激和慢性不可預知性應激模型鼠前額皮層β-catenin表達相對于對照組降低至54.7±11.2%。 結(jié)論：慢性社會挫敗應激模型小鼠均可作為抑郁癥研究的理想模型,前額皮層區(qū)miR-214-3p表達上調(diào),β-catenin表達降低。 第二部分miR-214-3p和miR-690的作用靶點研究 目的：明確miR-214-3p和miR-690以ctnnb1mRNA作為靶點。 方法：采用實時熒光定量PCR的方法檢測miR-214-3p和miR-690的表達水平；Western Blotting檢測前額皮層區(qū)β-catenin的表達；雙熒光素酶酶報告系統(tǒng)檢測miR-690與ctnnb1mRNA的3'-UTR序列的結(jié)合位點。 結(jié)果：(1)神經(jīng)元轉(zhuǎn)染miR-690的模擬物后miR-690的表達量相對于對照增加44.2±0.44；(2)神經(jīng)元轉(zhuǎn)染miR-690的模擬物后β-catenin蛋白表達水平相對于對照組降低到60±12%；(3)雙熒光素酶報告實驗證明miR-690以ctnnbl的3'UTR區(qū)作為靶點；(4)第三腦室在體注射miR-214-3p和miR-690的模擬物后相對于對照組miR-214-3p的表達增加到2.465±0.032,miR-690的表達增加到1.21±0.05；(5)第三腦室在體注射miR-214-3p模擬物后,相對于對照組降低β-catenin蛋白表達至64.6±4.35,而注射miR-690不影響β-catenin蛋白表達；(6)在體沉默ctnnbl區(qū)的3'UTR區(qū)相對于對照組可以有效降低mRNA的表達至0.37±0.07,同時降低P-catenin蛋白的表達量至24±3.5%。 結(jié)論：miR-214-3p以ctnnb1的3'-UTR區(qū)為靶點調(diào)控β-catenin蛋白表達。 第三部分調(diào)控前額皮層區(qū)miR-214-3p表達對慢性社會挫敗應激所致小鼠抑郁樣行為的影響及機制 目的：研究調(diào)控前額皮層區(qū)miRNA-214-3p對小鼠抑郁樣行為的影響及機制。 方法：糖水偏好測試,懸尾測試和社會接觸測試檢測小鼠抑郁樣行為的改變；實時熒光定量PCR分析前額皮層區(qū)miR-214-3p的表達；Western Blotting檢測前額皮層區(qū)β-catenin的表達；腦片鉗記錄前額皮層區(qū)的微小興奮性突觸后電流(miniature excitatory postsynaptic current, mEPSC)。 結(jié)果：(1)相對于對照組,antagomir-214明顯降低miR-214-3p表達至1.046±0.05,增加β-catenin蛋白表達至91.3±4.7%；(2)antagomir-214明顯降低模型小鼠懸尾不動時間至27.954±4.06s,并增加社會接觸時間至64.283±10.7s；(3)antagomir-214能增加模型小鼠前額皮層區(qū)mEPSC幅度；(4)慢病毒包被miR-214-3p抑制物(lentivirus-miR-214, LV-miR-214)明顯降低miR-214-3p表達水平至1.013±0.046,增加β-catenin蛋白表達至227±59.8%；(5)LV-miR-214降低模型小鼠懸尾不動時間至45.7±12.08,并增加社會接觸時間至53.6±10.051s；(6)過表達ctnnb1降低模型小鼠懸尾不動時間至37.5±6.91s,并增加社會接觸時間至46.833±6.921s；(7)與對照組相比,經(jīng)鼻腔給予antagomir-214降低miR-214-3p表達至0.4±0.1,增加β-catenin蛋白表達至110±9.3%；(8)經(jīng)鼻腔給予antagomir-214降低模型小鼠懸尾不動時間至67.83±14.35s,增加社會接觸時間至50.55±7.44s；(9)antagomir-214, LV-miR-214和過表達ctnnbl均不影響模型小鼠的糖水偏好率。 結(jié)論：抑制miR-214-3p表達可以逆轉(zhuǎn)慢性社會挫敗應激所致小鼠的抑郁樣行為。
[Abstract]:Part I Effects of chronic social frustration stress on the expression of microRNAs and P-catenin in prefrontal cortex of mice
Objective: To establish a chronic social frustration stress model in mice and detect the differential expression and protein expression of small RNA (microRNA, microRNA) in prefrontal cortex.
Methods: Depression model was established by chronic social frustration stress and chronic unpredictable stress; depression-like behavior was detected by sugarwater preference test, tail suspension test and social contact test; serum corticosterone level was detected by Elisa; the expression of microRNAs in prefrontal cortex was quantitatively analyzed by real-time fluorescence quantitative PCR. Blotting was used to detect the expression of p- catenin (-catenin) in the prefrontal cortex.
Results: (1) Compared with the control group, the expression of microRNA-214-3p in the prefrontal cortex was up-regulated by 4.249 (+ 0.816) and that of microRNA-690 was up-regulated by 2.527 (+ 0.292) in the chronic social frustration stress model mice; (2) The sugar preference rate of the chronic social frustration model mice decreased to 40.577 (+ 4.81%) and the social contact time shortened to 60.85 (+ 13.17s); and (3) in the chronic society. The content of serum corticosterone in frustration stress model mice increased to 17.943
Conclusion: Chronic social frustration stress model mice can be used as an ideal model for depression study. The expression of microRNA214-3p is up-regulated and the expression of beta-catenin is down-regulated in prefrontal cortex.
The second part is about the target of miR-214-3p and miR-690.
Objective: to make clear that miR-214-3p and miR-690 take ctnnb1mRNA as the target.
Methods: Real-time fluorescence quantitative PCR was used to detect the expression of microRNAs-214-3p and microRNAs-690, Western Blotting was used to detect the expression of beta-catenin in prefrontal cortex, and double luciferase reporter system was used to detect the 3'-UTR binding sites of microRNAs-690 and CTNNB1 mRNA.
Results: (1) The expression of microRNA-690 in neurons transfected with mimics of microRNA-690 increased by 44.2 (+ 0.44) compared with the control; (2) The expression of beta-catenin protein in neurons transfected with mimics of microRNA-690 decreased to 60 (+ 12%); (3) Diluciferase reporter assay showed that microRNA-690 targeted the 3'UTR region of ctnnbl; (4) The third brain. Compared with the control group, the expression of Mi-214-3p and Mi-690 mimics increased to 2.465 (+ 0.032) and the expression of Mi-690 increased to 1.21 (+ 0.05) after intraventricular injection of Mi-214-3p mimics, the expression of beta-catenin protein decreased to 64.6 (+ 4.35) after intraventricular injection of Mi-214-3p mimics, while the expression of Mi-690 did not affect the expression of beta-catenin protein. (6) Compared with the control group, the 3'UTR region of ctnnbl silenced in vivo could effectively reduce the expression of mRNA to 0.37 [0.07] and the expression of P-catenin protein to 24 [3.5].
Conclusion: miR-214-3p regulates the expression of beta -catenin protein in CTNNB1 3'-UTR region.
The third part is about the effect and mechanism of regulating the expression of microRNA-214-3p in prefrontal cortex on depression-like behavior induced by chronic social frustration stress in mice
Objective: To study the effect and mechanism of prefrontal cortex miRNA-214-3p on depressive behavior in mice.
Methods: Sugar preference test, tail suspension test and social contact test were used to detect depression-like behavior changes in mice; real-time fluorescence quantitative PCR was used to analyze the expression of microRNA-214-3p in prefrontal cortex; Western Blotting was used to detect the expression of beta-catenin in prefrontal cortex; and miniature excitatory postsynaptic currents (miniature ex) in prefrontal cortex were recorded by brain patch clamp. Citatory postsynaptic current, mEPSC).
Results: (1) Compared with the control group, antagomir-214 significantly decreased the expression of microRNAs-214-3p to 1.046 [0.05] and increased the expression of beta-catenin protein to 91.3 [4.7%]; (2) antagomir-214 significantly decreased the tail-suspension immobility time to 27.954 [4.06s] and increased the social contact time to 64.283 [10.7s]; (3) antagomir-214 significantly increased the forehead skin of the model mice. Layered mEPSC amplitude; (4) Lentivirus-coated microRNA-214-3p inhibitor (LV-microRNA-214) significantly decreased the expression of microRNA-214-3p to 1.013 (+0.046) and increased the expression of beta-catenin protein to 227 (+59.8%); (5) LV-microRNA-214 decreased the tail immobility time to 45.7 (+12.08) and increased the social contact time to 53.6 (+10.051s); (6) Over The expression of CTNNB1 decreased the immobility time of mice tail suspension to 37.5 65 (9) Ant agomir-214, LV-miR-214 and over-expression of ctnnbl did not affect the sugar preference rate of the model mice.
Conclusion: inhibition of miR-214-3p expression can reverse the depressive behavior of mice with chronic social stress.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R749.4

【共引文獻】

相關期刊論文 前10條

1 王姬;王曉斌;;MicroRNA與抑郁癥[J];國際精神病學雜志;2013年01期

2 Stella Chai;Stephanie Ma;;Clinical implications of microRNAs in liver cancer stem cells[J];癌癥(英文版);2013年08期

3 肖玲;舒暢;唐記華;王曉萍;王高華;;不同時程的慢性不可預計溫和應激對大鼠海馬神經(jīng)可塑性相關蛋白水平的影響[J];神經(jīng)損傷與功能重建;2013年06期

4 白玫;朱熊兆;張晟;張逸;張麗;薛亮;周晟;;海馬microRNA let-7a在應激致大鼠抑郁樣行為反應中的作用[J];國際精神病學雜志;2013年04期

5 閔U，

本文編號：2213969