本文選題：miR-376b-5p + 血管新生��； 參考：《中南大學(xué)》2014年博士論文

【摘要】：背景和目的：腦梗死是一類發(fā)病率高、致殘率高以及復(fù)發(fā)率高的急性缺血性腦血管病,占全部腦卒中的60%-80%。缺血可以導(dǎo)致大量神經(jīng)細(xì)胞出現(xiàn)不可逆的變性壞死,導(dǎo)致嚴(yán)重的神經(jīng)功能缺損癥狀。促進(jìn)缺血區(qū)新生血管的形成增加新的側(cè)枝循環(huán),可通過改善局部腦血流供應(yīng),挽救缺血半暗帶內(nèi)瀕臨死亡的神經(jīng)細(xì)胞,促進(jìn)神經(jīng)功能的恢復(fù),這對腦梗死有重要的治療作用。已有研究證明,腦缺血可誘導(dǎo)血管新生,而HIF-1-VEGF-NOTCH通路是缺血缺氧后調(diào)節(jié)血管新生的重要的信號通路。微小RNA(microRNA,miRNA)是一類內(nèi)源性單鏈小分子RNA,參與了生物體的生長發(fā)育、器官形成、細(xì)胞增殖、分化、凋亡等多種生物學(xué)過程,與多種疾病密切相關(guān)。研究發(fā)現(xiàn),miR-376b-5p在腦缺血后缺血區(qū)腦組織內(nèi)表達(dá)降低,我們通過生物學(xué)軟件分析發(fā)現(xiàn)HIF-1a與miR-376b-5p的序列存在配對關(guān)系,故我們推測miR-376b-5p可能參與了血管新生的調(diào)控過程,并且可能通過HIF-1-VEGF-Notch信號通路來調(diào)控血管新生。 本研究通過體內(nèi)外實驗觀察腦缺血缺氧后miR-376b-5p、 HIF-1-VEGF-Notch信號分子的表達(dá)變化和血管新生情況,并通過體外實驗觀察過表達(dá)和抑制miR-376b-5p對內(nèi)皮細(xì)胞血管新生能力及HIF-1-VEGF-Notch信號通路分子表達(dá)的影響,探討miR-376b-5p對血管新生的調(diào)控作用及可能作用的信號途徑,以期為腦梗死的治療提供一種新策略。 方法： 1.體內(nèi)實驗 1.1構(gòu)建大鼠永久性局灶性大腦中動脈腦缺血(Permanent middle cerebral artery occlusion,pMCAO)模型,于pMCAO后1天,3天,7天收集腦組織標(biāo)本。 1.2通過神經(jīng)缺損評分和TTC染色方法鑒定建模是否成功。 1.3通過免疫組化方法檢測缺血腦皮質(zhì)區(qū)第八因子相關(guān)抗原因子(vWF)的表達(dá)情況從而判斷血管新生情況。 1.4采用實時熒光定量PCR的方法檢測缺血腦皮質(zhì)區(qū)miR-376b-5p、 HIF-1a mRNA、 VEGFA mRNA、 Notchl mRNA的表達(dá)變化。 1.5運(yùn)用Western blot的方法觀察缺血腦皮質(zhì)區(qū)HIF-1α、 VEGFA、 Notchl的蛋白表達(dá)變化。 2.體外實驗 2.1建立了人臍靜脈內(nèi)皮細(xì)胞(HUVEC)缺氧模型。 2.2運(yùn)用MTT,Transwell和Matrigel基質(zhì)膠實驗比較常氧組和低氧組細(xì)胞的細(xì)胞增殖,細(xì)胞遷移以及小管形成情況,從而可以判斷缺氧對血管新生的影響。運(yùn)用實時熒光定量PCR和Western blot的方法觀察常氧組和低氧組中HIF-1α、 VEGFA.Notchl在mRNA和蛋白水平的表達(dá)變化；運(yùn)用實時熒光定量PCR的方法檢測缺氧對細(xì)胞中miR-376b-5p表達(dá)的影響。 2.3進(jìn)一步通過在缺氧HUVEC細(xì)胞中轉(zhuǎn)染miR-376b-5p模擬物或抑制物使miR-376b-5p過表達(dá)或者抑制表達(dá),觀察轉(zhuǎn)染后內(nèi)皮細(xì)胞血管新生的能力以及細(xì)胞中HIF-1α、 VEGFA、Notchl在mRNA和蛋白水平的表達(dá)變化,從而明確miR-376b-5p對血管新生的調(diào)控作用。 2.4為了進(jìn)一步研究miR-376b-5p對血管新生的調(diào)控機(jī)制,我們構(gòu)建了HIF-1a shRNA表達(dá)載體,將其轉(zhuǎn)染入缺氧HUVEC細(xì)胞中以敲除HIF-1α的作用,通過比較HIF-1α被抑制前后,miR-376b-5p模擬物或抑制物對內(nèi)皮細(xì)胞血管新生能力的影響以及細(xì)胞中HIF-1α、 VEGFA、Notchl分子在mRNA和蛋白水平的表達(dá)變化,從而探討miR-376b-5p是否通過HIF-1-VEGF-Notch信號通路影響血管新生。 結(jié)果： 1.體內(nèi)實驗 1.1成功構(gòu)建了大鼠永久性局灶性大腦中動脈腦缺血(pMCAO)模型,TTC染色顯示：右側(cè)大腦中動脈供血區(qū)的梗死區(qū)域呈白色,正常腦組織呈紅色。 1.2免疫組化結(jié)果顯示：pMCAO1天,3天,7天組缺血腦組織第八因子相關(guān)抗原因子(vWF)表達(dá)水平與假手術(shù)組相比均上調(diào),反映了pMCAO1天,3天,7天組缺血區(qū)中微血管密度比假手術(shù)組均增加,提示腦缺血損傷可導(dǎo)致反應(yīng)性血管新生。 1.3實時熒光定量PCR結(jié)果顯示：pMCAO后1天,3天,7天組缺血腦皮質(zhì)區(qū)miR-376b-5p的表達(dá)水平與假手術(shù)組相比均降低,并且HIF-1a mRNA、 VEGFA mRNA、 NotchlmRNA的表達(dá)均增加。 1.4Western blot結(jié)果顯示：pMCAO后1天,3天,7天組缺血腦皮質(zhì)區(qū)HIF-1α、VEGFA、 Notchl的蛋白表達(dá)與假手術(shù)組相比均增加。 2.體外實驗 2.1缺氧組與常氧組相比,miR-376b-5p的表達(dá)水平降低,而HUVEC的細(xì)胞增殖,細(xì)胞遷移以及小管形成能力增強(qiáng),即血管新生能力增強(qiáng),同時HIF-1α、 VEGFA、 Notchl在mRNA水平和蛋白水平的表達(dá)也增加,提示miR-376b-5p可能對內(nèi)皮細(xì)胞的血管新生具有抑制作用。 2.2將miR-376b-5p模擬物轉(zhuǎn)染缺氧細(xì)胞后,內(nèi)皮細(xì)胞形成新生血管的能力減弱,同時HIF-1α、VEGFA、 Notchl在mRNA水平和蛋白水平的表達(dá)也降低；而miR-376b-5p抑制物轉(zhuǎn)染缺氧細(xì)胞后,內(nèi)皮細(xì)胞形成新生血管的能力增強(qiáng),同時HIF-1α、VEGFA、 Notch1在mRNA水平和蛋白水平的表達(dá)也增加,初步明確了缺氧后miR-376b-5p可能通過HIF-1-VEGF-Notch信號通路調(diào)控了血管新生。 2.3將HIF-1α shRNA表達(dá)載體轉(zhuǎn)染入缺氧HUVEC細(xì)胞敲除HIF-1α的作用后,miR-376b-5p模擬物(或抑制物)與HIF-1α shRNA共轉(zhuǎn)染組與單純miR-376b-5p模擬物(或抑制物)轉(zhuǎn)染組相比,內(nèi)皮細(xì)胞形成新生血管的能力發(fā)生改變,同時HIF-1α、 VEGFA、 Notchl在mRNA水平和蛋白水平的表達(dá)也產(chǎn)生變化,進(jìn)一步明確了miR-376b-5p是通過調(diào)控HIF-1-VEGF-Notch信號通路來發(fā)揮對血管新生的負(fù)調(diào)控作用。 結(jié)論： 1. miR-376b-5p對腦缺血缺氧后的血管新生具有抑制作用。 2. miR-376b-5p可能是通過調(diào)控HIF-1-VEGF-Notch信號通路來參與抑制血管新生的過程。 3.通過干預(yù)miR-376b-5p的表達(dá),促進(jìn)新生血管形成,為腦梗死的治療提供了一種新思路。圖32幅,表6個,參考文獻(xiàn)101篇。
[Abstract]:BACKGROUND & OBJECTIVE : Cerebral infarction is a kind of acute ischemic cerebrovascular disease with high morbidity , high disability rate and high recurrence rate , which accounts for 60 % -80 % of all cerebral apoplexy . It has been proved that the expression of HIF - 1 - VEGF - NOTCH is an important signal pathway to regulate angiogenesis after cerebral ischemia . It has been found that miR - 376b - 5p is a kind of endogenous single - chain small molecule RNA . It is found that miR - 376b - 5p can participate in the regulation of angiogenesis , and it is possible to regulate angiogenesis through HIF - 1 - VEGF - Notch signaling pathway .

In this study , the expression of miR - 376b - 5p , HIF - 1 - VEGF - Notch signaling molecule and angiogenesis were observed in vivo and in vivo , and the effects of miR - 376b - 5p on angiogenesis in endothelial cells and the expression of HIF - 1 - VEGF - Notch signaling pathway were observed through in vitro experiments , and the effects of miR - 376b - 5p on angiogenesis and possible signaling pathways were investigated in order to provide a new strategy for the treatment of cerebral infarction .

Method :

1 . In vivo experiment

1.1 Rat permanent middle cerebral artery occlusion ( TIA ) model was constructed , and the brain tissue samples were collected on 1 day , 3 days and 7 days after the operation .

1.2 Identification of the success of the modeling by the neural defect score and TTC staining method .

1.3 The expression of eighth factor related antigen factor ( VWF ) in ischemic cerebral cortex was detected by immunohistochemistry .

1.4 The expression of miR - 376b - 5p , HIF - 1a mRNA , VEGFA mRNA and Notchl mRNA was detected by real - time fluorescence quantitative PCR .

1.5 The expression of HIF - 1偽 , VEGFA and Notchl was observed by Western blot .

2 . In vitro experiments

2.1 The hypoxia model of human umbilical vein endothelial cells ( HUVEC ) was established .

2.2 The expression of HIF - 1偽 , VEGFA and Notchl in normal oxygen and hypoxic groups was determined by real - time fluorescence quantitative PCR and Western blot .
The effect of hypoxia on the expression of miR - 376b - 5p in cells was detected by real - time fluorescence quantitative PCR .

2.3 Further , miR - 376b - 5p overexpression or inhibition of miR - 376b - 5p was observed or inhibited by transfection of miR - 376b - 5p in an anoxic HUVEC cell , and expression of HIF - 1偽 , VEGFA , Notchl in the cells was observed to change in mRNA and protein levels , thereby defining the regulation of miR - 376b - 5p on angiogenesis .

2.4 In order to further study the regulation mechanism of miR - 376b - 5p on angiogenesis , we constructed the expression vector of HIF - 1a shRNA , transfected into anoxic HUVEC cells to knock out HIF - 1偽 . After the inhibition of HIF - 1偽 , the expression of HIF - 1偽 , VEGFA and Notchl on endothelial cells and the expression of HIF - 1偽 , VEGFA and Notchl on endothelial cells were investigated .

Results :

1 . In vivo experiment

1.1 The rat permanent focal cerebral artery ischemia model was successfully constructed . TTC staining showed that the infarct area of the right middle cerebral artery supply area was white , and the normal brain tissue was red .

1.2 Immunohistochemical results showed that the expression level of eighth factor related antigen factor ( VWF ) in ischemic brain tissue was up - regulated in 3 days and 7 days after pMCAO1 , which reflected the increase of microvessel density ( MVD ) in the ischemic area of 7 days after the treatment of pMCAO1 , 3 days and 7 days , which suggested that cerebral ischemia injury could lead to reactive angiogenesis .

1.3 The expression level of miR - 376b - 5p in ischemic brain cortex was lower than that in sham operation group , and the expression of HIF - 1a mRNA , VEGFA mRNA and Notchloral mRNA increased 1 day , 3 days and 7 days after the operation .

1 . Western blot showed that the expression of HIF - 1偽 , VEGFA and Notchl in the ischemic brain cortex increased in 1 day , 3 day and 7 days after the treatment .

2 . In vitro experiments

2.1 The expression level of miR - 376b - 5p decreased compared with the normal oxygen group , while the cell proliferation , cell migration and small tube formation ability of HUVEC were enhanced , that is , the ability of angiogenesis was enhanced , while the expression of HIF - 1偽 , VEGFA and Notchl increased at mRNA level and protein level , suggesting that miR - 376b - 5p could inhibit angiogenesis in endothelial cells .

2.2 After transfection of hypoxia cells with miR - 376b - 5p , the ability of endothelial cells to form new blood vessels decreased , while HIF - 1偽 , VEGFA and Notchl decreased at mRNA level and protein level ;
At the same time , the expression of HIF - 1偽 , VEGFA , Notch1at mRNA level and protein level increased , and the expression of HIF - 1偽 , VEGFA and Notch1at mRNA level and protein level was also increased . After hypoxia , the expression of miR - 376b - 5p could regulate angiogenesis through HIF - 1 - VEGF - Notch signaling pathway .

2.3 After the HIF - 1偽 shRNA expression vector was transfected into the hypoxia HUVEC cells to knock out the HIF - 1偽 , the ability of the miR - 376b - 5p mimetic ( or inhibitor ) to co - transfected with the HIF - 1偽 shRNA was changed , and the expression of HIF - 1偽 , VEGFA and Notchl also changed at the mRNA level and protein level , and further clarified that miR - 376b - 5p plays a negative regulatory role in angiogenesis by regulating the HIF - 1 - VEGF - Notch signaling pathway .

Conclusion :

1.miR - 376b - 5p has an inhibitory effect on angiogenesis after cerebral ischemia .

2 . miR - 376b - 5p may be involved in inhibiting angiogenesis by modulating the HIF - 1 - VEGF - Notch signaling pathway .

3 . The expression of miR - 376b - 5p is intervened to promote the formation of neovascularization , which provides a new idea for the treatment of cerebral infarction .
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R743.33

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 萬賽英,黎杏群,梁清華,智屹惠,羅云,張花先;腦溢安對缺氧大鼠腦微血管內(nèi)皮細(xì)胞VEGF蛋白表達(dá)的影響[J];中南大學(xué)學(xué)報(醫(yī)學(xué)版);2005年02期

2 張愛梅,李憲章,杜留春,王紀(jì)佐;局灶性腦缺血大鼠血管內(nèi)皮生長因子的表達(dá)及意義[J];中國臨床神經(jīng)科學(xué);2002年04期

3 張曼,沈樂,周愛儒;缺氧條件下血管活性因子對血管內(nèi)皮細(xì)胞中VEGF表達(dá)的影響[J];中國生物化學(xué)與分子生物學(xué)報;2001年01期

4 梅愛農(nóng);王玨;湛彥強(qiáng);張?zhí)K明;姜亞平;;通心絡(luò)膠囊對腦梗死大鼠腦皮質(zhì)血管新生和皮層神經(jīng)元凋亡的影響[J];實用醫(yī)學(xué)雜志;2006年03期

5 洪艷;鄧長生;章軍建;朱江;李芹;;Neuroprotective Effect of Granulocyte Colony-stimulating Factor in a Focal Cerebral Ischemic Rat Model with Hyperlipidemia[J];Journal of Huazhong University of Science and Technology(Medical Sciences);2012年06期

6 徐冰;評估血管內(nèi)皮細(xì)胞生長因子及其受體在大鼠大腦中動脈短暫閉塞再灌注后對缺血腦組織的保護(hù)作用[J];中國臨床康復(fù);2002年01期

7 李芳;張先慧;陳臨溪;;血管新生機(jī)制的研究進(jìn)展[J];中國心血管病研究;2007年11期

8 陳治;杜鈺;;血管內(nèi)皮細(xì)胞特異性受體在腫瘤血管靶向治療中的應(yīng)用[J];世界華人消化雜志;2001年06期

9 王仁剛,朱興族;永久性大腦局灶缺血后angiopoietin-2和血管內(nèi)皮生長因子在小鼠大腦皮層的表達(dá)(英文)[J];Acta Pharmacologica Sinica;2002年05期

10 文全慶;賈延R，

本文編號：2007804