本文關鍵詞： miR-296 血管新生 腦缺血 VEGF Notch　出處：《中南大學》2014年博士論文　論文類型：學位論文

【摘要】：背景與目的：側枝循環(huán)的建立是改善腦梗死后腦組織血流供應的有效途徑,治療性血管生成為缺血性腦卒中血運重建提供了一種新的治療策略。微小RNA (microRNA, miRNA)是近年發(fā)現(xiàn)的一類內源性非編碼小分子RNA,在轉錄后水平調控細胞增殖分化、凋亡、分裂及器官的發(fā)育,與人類生命活動的多種疾病密切相關,已成為醫(yī)學領域的研究熱點。在腦腫瘤實體及體外實驗中已證實miR-296通過直接抑制其靶基因HGS表達,呈現(xiàn)出促進血管新生的作用。本研究體內實驗擬通過構建大鼠大腦中動脈閉塞(middle cerebral artery occlusion,MCAO)腦缺血梗死模型,檢測缺血梗死后腦組織中miR-296及其靶基因HGS的表達變化,同時動態(tài)觀察腦缺血梗死后血管新生的變化,初步明確miR-296是否參與腦缺血梗死后血管新生過程；進一步在體外環(huán)境下培育人臍靜脈內皮細胞(human umbilical vein endothelial cell, HUVEC),利用重組腺病毒技術過表達miR-296于HUVEC內,觀察血管內皮細胞中HGS及VEGF-Notch通路相關分子VEGF、VEGFR2、DLL4和Notchl的mRNA及蛋白表達水平的變化,明確miR-296對VEGF-Notch信號通路的調控作用,深入探討闡明miR-296參與調控腦缺血梗死后血管新生的分子機制,為缺血性腦卒中治療提供新的靶點。 研究內容與方法： 1.體內實驗 1.1構建大鼠MCAO模型,行TTC染色以鑒定腦缺血梗死區(qū)域。于缺血梗死后第1天,第3天,第7天留取腦組織標本行相關檢測。 1.2采用qRT-PCR方法檢測腦缺血梗死后不同時間點大鼠缺血腦皮質區(qū)miR-296的表達水平,明確腦缺血梗死后miR-296動態(tài)表達變化。 1.3采用Western blot法檢測腦缺血梗死后不同時間點大鼠缺血腦皮質區(qū)HGS的蛋白表達,明確腦缺血梗死后miR-296的靶基因HGS的蛋白表達變化。 1.4采用免疫組化染色法檢測腦缺血梗死后不同時間點缺血腦皮質區(qū)CD105標記的新生微血管以判斷血管新生的情況。 2.體外實驗 2.1采用貼壁法培養(yǎng)人臍靜脈內皮細胞HUVEC-12。構建腺病毒載體,將包裝AdV-miR-296-GFP及AdV-GFP的重組腺病毒分別轉染至HUVEC-12;熒光顯微鏡下觀察GFP綠色熒光的陽性率。實驗分為miR-296過表達組(AdV-miR-296-GFP)和對照組(AdV-GFP)。 2.2行內皮細胞小管形成實驗,比較miR-296過表達組與對照組內皮細胞血管形成數,明確miR-296對血管內皮細胞血管形成能力的影響。 2.3采用qRT-PCR法比較miR-296過表達組與對照組中miR-296的表達水平,以明確AdV-miR-296-GFP上調miR-296表達的水平。 2.4采用RT-PCR、Western Blot法分別比較miR-296過表達組與對照組中HGS及VEGF-Notch信號通路相關分子VEGF、VEGFR2、DLL4、 Notch1的mRNA和蛋白表達水平,明確miR-296對VEGF-Notch信號通路的調控途徑。 結果： 1.體內實驗 1.1成功構建大鼠MCAO模型。TTC染色可見左側大腦中動脈供血區(qū)的梗死灶呈白色,正常腦組織呈紅色。 1.2qRT-PCR結果顯示,假手術組腦組織中可見miR-296的表達,腦缺血梗死后各時間點實驗組大鼠缺血腦皮質區(qū)miR-296表達均高于假手術組(各P0.05),miR-296在腦缺血梗死后第1天表達即開始上調(2.25±0.36),第三天表達量繼續(xù)上調(5.35±0.35),7天時最明顯(7.91±0.21),表明正常非缺血梗死腦組織中存在miR-296的表達,且腦缺血損傷可刺激miR-296表達水平的逐漸上調。 1.3Western blot結果顯示檢測腦缺血梗死后Id,3d,7d實驗組大鼠缺血腦皮質區(qū)HGS的蛋白表達逐漸下降,驗證了腦缺血梗死后過表達的miR-296可抑制其靶基因HGS的蛋白表達。 1.4免疫組化染色結果顯示,腦缺血梗死后第一天即出現(xiàn)CD105陽性細胞(1.8±0.84),第3天時陽性細胞數增多(6.8±2.17),第7天可見更多的新生血管(10±2.17),與miR-296表達水平呈正相關(r=0.95,P0.05),提示miR-296促進腦缺血梗死后血管新生過程。 2.體外實驗 2.1成功構建腺病毒載體,將包裝AdV-miR-296-GFP及AdV-GFP的重組腺病毒轉染至HUVEC-12細胞,熒光顯微鏡下可見絕大部分細胞均表達GFP綠色熒光,GFP陽性細胞率達90%以上。 2.2qRTHPCR結果顯示,與對照組比較,miR-296過表達組miR-296上調了320±30倍。 2.3內皮細胞小管形成實驗結果顯示,與對照組比較,miR-296過表達組HUVEC-12細胞形成血管管腔數明顯增多(28±1.5vs.8±2.5,P0.05),表明miR-296可促進血管內皮細胞形成管腔樣結構。 2.4RT-PCR、western blot結果顯示,與對照組比較,miR-296過表達組HUVEC-12細胞中HGS mRNA的表達水平下降,其蛋白水平則顯著下降。與此同時,VEGF、VEGFR2的mRNA和蛋白表達水平均顯著上調而DLL4、Notchl的mRNA和蛋白表達水平均顯著下降(各P0.05)。 結論： 1.大鼠正常(非梗死)腦組織中存在miR-296的表達。 2.miR-296促進大鼠腦缺血梗死后血管新生的過程。 3.miR-296通過抑制其靶基因HGS的表達,上調VEGF-VEGFR2信號通路的活性,促進血管新生的過程。 4.促血管生成因子miR-296抑制DLL4-Notchl信號通路的活性,其機制有待進一步研究。圖16幅,表2個,參考文獻116篇。
[Abstract]:Background and purpose: the establishment of collateral circulation is an effective way to improve cerebral blood supply, provides a new therapeutic strategy for the treatment of angiogenesis in ischemic stroke revascularization. Micro RNA (microRNA, miRNA) is a kind of newly discovered endogenous small molecule non encoding RNA, apoptosis at the post transcriptional level the regulation of cell proliferation, differentiation, proliferation and organ development, a variety of diseases and is closely related to the activities of human life, has become a hot research topic in the field of medicine. In the brain tumor and in vitro experiments have demonstrated that miR-296 through direct inhibition of HGS gene expression, showing a role in promoting angiogenesis in vivo. This study intends to by constructing a rat model of middle cerebral artery occlusion (middle cerebral artery occlusion, MCAO) model of ischemic cerebral infarction, miR-296 and target detection based brain infarction due to the expression of HGS At the same time, changes in angiogenesis of ischemic infarction after dynamic observation of brain changes, preliminary to determine whether miR-296 is involved in ischemic brain infarction angiogenesis; further cultivating human umbilical vein endothelial cells in vitro environment (human umbilical vein endothelial cell, HUVEC), using the technology of recombinant adenovirus overexpression of miR-296 in HUVEC, the observation of vascular endothelial cells in the HGS and VEGF-Notch pathway related molecules VEGF, VEGFR2, mRNA and protein expression of DLL4 and Notchl, miR-296 on the clear regulation of the VEGF-Notch signaling pathway, explore the molecular mechanism of angiogenesis to clarify miR-296 involved in the regulation of cerebral ischemia after myocardial infarction and provide a new target for the treatment of ischemic stroke.
Research contents and methods:
1 in vivo experiment
1.1 the rat model of MCAO was constructed, and TTC staining was used to identify the cerebral ischemic infarct area. The brain tissue specimens were collected at first days, third days and seventh days after the ischemic infarction.
1.2 qRT-PCR method was used to detect the expression level of miR-296 in the ischemic cortex of rats at different time points after cerebral ischemia, and to clarify the dynamic expression of miR-296 after cerebral ischemia.
1.3 Western blot method was used to detect the protein expression of HGS in the ischemic cortex of rats at different time points after cerebral ischemia, and to clarify the protein expression of target gene HGS after cerebral ischemia and infarction in miR-296.
1.4 immuno histochemical staining was used to detect the neovascularization of CD105 markers in ischemic cerebral cortex at different time points after cerebral ischemia in order to determine angiogenesis.
2. in vitro experiment
2.1 the cultured human umbilical vein endothelial cells HUVEC-12. to construct adenovirus vector, the recombinant adenovirus packaging AdV-miR-296-GFP and AdV-GFP were transfected into HUVEC-12; observe the positive rate of GFP green fluorescence under fluorescence microscope. The experiment was divided into miR-296 overexpression group (AdV-miR-296-GFP) and control group (AdV-GFP).
2.2 endothelial cell canaliculus formation experiments were performed to compare the number of endothelial cells in the miR-296 overexpressing group and the control group, and to clarify the effect of miR-296 on the angiogenesis of vascular endothelial cells.
2.3 qRT-PCR was used to compare the expression level of miR-296 in the miR-296 overexpression group and the control group, so as to determine the level of AdV-miR-296-GFP to increase the expression of miR-296.
2.4, we used RT-PCR and Western Blot to compare HGS and VEGF-Notch signal pathway related molecules VEGF, VEGFR2, DLL4, Notch1 mRNA and protein expression levels between miR-296 overexpressing group and control group, and to clarify the regulation pathway of DLL4 on the signal pathway.
Result錛，

本文編號：1448342