本文關鍵詞： 缺血性腦卒中 lncRNAs 血管新生 MEG3 生物信息學分析 VEGF　出處：《南昌大學》2014年碩士論文　論文類型：學位論文

【摘要】：研究背景和目的： 缺血性腦卒中是一種嚴重危害人類健康和生命安全的腦血管疾病，具有發(fā)病率高、致殘率高和死亡率高等特點，其治療的關鍵是及時、充分恢復腦缺血區(qū)的血供。臨床觀察發(fā)現(xiàn)腦卒中后可誘發(fā)缺血區(qū)反應性血管新生，而且腦組織微血管密度與卒中患者的預后存有密切關系。因此，進一步闡明腦卒中后血管新生的分子調(diào)控機制，尋找到有效的分子調(diào)控靶點，利用藥物或基因干預促進治療性血管新生有望根本改變目前腦卒中的治療現(xiàn)狀。 長鏈非編碼RNA（long non-coding RNAs）是一類由RNA聚合酶II轉(zhuǎn)錄的轉(zhuǎn)錄本長度超過200nt的長鏈非編碼RNA，近年來，研究發(fā)現(xiàn)lncRNAs的表達具有組織特異性和發(fā)育特異性，某些lncRNAs還與參與血管新生過程的多種基因和信號分子關系密切，但是腦卒中后lncRNAs的表達變化如何，lncRNAs是否與其他非編碼RNAs如microRNAs等一樣參與腦卒中后的血管新生調(diào)控，目前尚不清楚。本研究通過Solexa高通量測序檢測大鼠腦缺血后腦組織lncRNAs的表達譜變化，生物信息學分析腦卒中后差異表達的lncRNAs，篩選出血管新生相關lncRNAs，并初步探討腦缺血后血管新生相關lncRNAs的功能及其可能的調(diào)控機制。 研究內(nèi)容和方法： 1、體內(nèi)實驗篩選腦卒中后血管新生相關lncRNAs （1）采用zea-longa法建立建立雄性SD大鼠MCAO模型，神經(jīng)功能評分、TTC染色及MRI評估模型。 （2）采用Solexa基因測序建立腦卒中后缺血腦組織lncRNAs的表達譜，real-time PCR隨機驗證lncRNAs的表達水平。 （3）生物信息學分析腦卒中后差異表達的lncRNAs，篩選血管新生相關lncRNAs。 （4）采用real-time PCR動態(tài)觀察腦卒中后所篩選出血管新生lncRNAsMEG3的表達變化。 2、體外實驗分析所篩選的lncRNA MEG3對血管新生的調(diào)控功能 （1）原代人臍靜脈血管內(nèi)皮細胞（HUVEC）的分離培養(yǎng)與鑒定。 （2） HUVEC缺氧模型的建立：采用real-time PCR檢測HUVEC缺氧24hVEGFa的表達變化評估HUVEC缺氧模型。 （3）缺氧對MEG3和VEGFa表達的影響：將正常培養(yǎng)的HUVEC置于缺氧與常氧環(huán)境中培養(yǎng)12h、24h和48h，real-time PCR檢測HUVEC缺氧后各個時間點MEG3和VEGFa的表達變化。 （4）構建pCDH-MEG3質(zhì)粒并包裝Lentiviral-meg3慢病毒。 （5）慢病毒感染過表達HUVEC的MEG3，real-time PCR驗證MEG3的上調(diào)水平。 （6）MEG3過表達的HUVEC血管生成能力的檢測：CCK-8法檢測MEG3過表達HUVEC的增殖能力，RTCA檢測MEG3過表達HUVEC的遷移能力，matrigel成管實驗檢測MEG3過表達HUVEC的成管能力。 結(jié)果： 1、成功建立SD大鼠MCAO模型：MCAO大鼠均出現(xiàn)對側(cè)肢體偏癱、行走時原地轉(zhuǎn)圈和提尾向?qū)?cè)側(cè)身等神經(jīng)功能缺失表現(xiàn)，，神經(jīng)功能評分2分；TTC染色可見右側(cè)大腦中動脈供血區(qū)呈蒼白色而周圍正常腦組織呈現(xiàn)紅色；MRI結(jié)果顯示腦梗死T1像及DWI像呈高信號；缺血模型梗死灶一致性較好，可用于后續(xù)基因測序。 2、腦卒中后缺血腦組織lncRNAs的表達譜發(fā)生顯著改變：腦缺血后Solexa基因測序共檢測出9287條lncRNAs，與健側(cè)相比缺血側(cè)上調(diào)2倍以上及下調(diào)0.5倍且P＜0.05的lncRNAs共448條，其中上調(diào)的lncRNAs有414條，下調(diào)的lncRNAs34條；real-time PCR隨機驗證lncR-14、lncR-30、lncR-4、lncR-40、lncR-64、lncR-65、lncR-76及l(fā)ncR-44的表達分別上調(diào)2.364±1.074倍、3.836±3.754倍、1.643±1.095倍、5.544±4.774倍、2.504±1.749倍、2.206±1.416倍、1.572±1.095倍、2.389±1.305倍(P＜0.05)，lncR-41的表達下調(diào)0.5803±0.4459倍(P＜0.05)，與Solexa高通量測序的結(jié)果趨勢一致，成功建立了腦缺血lncRNAs的表達譜。 3、腦缺血后448條差異表達的lncRNAs中，生物信息學篩選出47條與血管新生相關lncRNAs，其中功能尚未注釋的lncR41與血管新生相關，生物信息學分析lncR41與小鼠MEG3序列高度同源，提示lncR41為大鼠MEG3。 4、腦缺血后MEG3的表達下調(diào)：real-time PCR結(jié)果顯示腦缺血24h后MEG3的表達較健側(cè)下調(diào)0.7840.124倍（P＞0.05），腦缺血48h后MEG3的表達較健側(cè)下調(diào)0.5020.133倍（P＜0.05），腦缺血72h后MEG3的表達較健側(cè)下調(diào)0.7230.106倍（P＜0.05）。 5、缺氧后MEG3和VEGFa表達發(fā)生變化：real-time PCR結(jié)果顯示缺氧后MEG3的表達呈先下降后上升的動態(tài)變化，即缺氧12h后MEG3的表達下調(diào)0.3790±0.0187倍（P＜0.05），24h后MEG3的表達下調(diào)0.6618±0.0767（P＞0.05），48h后MEG3的表達上調(diào)3.75590.1915倍（P＜0.05）；缺氧后12h VEGFa的表達上調(diào)1.7498±0.1916倍（P＜0.05），24h后VEGFa的表達上調(diào)2.3618±0.2725倍（P＜0.05），48h后VEGFa的表達上調(diào)2.7400±0.1761倍（P＜0.05）。 6、成功構建出pCDH-MEG3質(zhì)粒并包裝出Lentiviral-meg3慢病毒，病毒滴度＞6x106/ml。 7、慢病毒感染過表達MEG3抑制HUVEC的增殖、遷移和成管等血管形成能力，抑制HUVEC VEGFa和nontch1的表達。 結(jié)論： 1、腦卒中后缺血腦組織lncRNAs的表達譜發(fā)生顯著變化，lncRNAs可能參與了腦卒中后血管新生調(diào)控。 2、meg3抑制血管內(nèi)皮細胞的增殖、遷移和成管等功能進而影響腦卒中后血管新生，其機制可能為影響了VEGF信號通路。
[Abstract]:Research background and purpose:
Ischemic stroke is a serious hazard to human health and life safety of the cerebrovascular disease, with high incidence rate, high disability rate and mortality rate higher characteristic, and the key of treatment is timely, full recovery of cerebral ischemia and blood supply. The clinical observation that after stroke can induce ischemic area reactive angiogenesis, and the prognosis of brain microvascular density and stroke patients have a close relationship. Therefore, to further clarify the mechanism of molecular regulation of angiogenesis after stroke, to find the molecular target, the use of drugs or gene intervention to promote therapeutic angiogenesis is expected to fundamentally change the current status of treatment of stroke.
Long chain non RNA (long non-coding RNAs) encoding is a class of transcription by RNA polymerase II transcription of the length of more than 200nt long chain non encoding RNA, in recent years, studies have found that lncRNAs expression is tissue-specific and developmental specificity, some lncRNAs with multiple genes and signaling molecules involved in the process of angiogenesis is closely related but after stroke, the expression of lncRNAs, angiogenesis regulation whether lncRNAs and other non RNAs encoding such as microRNAs as involved in the stroke, it is unclear. This study by Solexa high-throughput sequencing to detect the expression of brain tissue after cerebral ischemia in rats lncRNAs spectrum, lncRNAs bioinformatics analysis of differentially expressed in brain after stroke, screening of angiogenesis related lncRNAs, and to investigate the cerebral ischemia angiogenesis related function of lncRNAs and its possible mechanism.
Research contents and methods:
1, in vivo experimental screening of vascular neovascularization related lncRNAs after stroke
(1) the MCAO model of male SD rats, neural function score, TTC staining and MRI evaluation model were established by zea-longa method.
(2) the expression of lncRNAs in ischemic brain tissue after stroke was set up by Solexa gene sequencing, and real-time PCR was used to verify the expression level of lncRNAs randomly.
(3) bioinformatics analysis of differential expression of lncRNAs after cerebral apoplexy and screening of angiogenesis related lncRNAs.
(4) real-time PCR was used to dynamically observe the changes in the expression of angiogenesis lncRNAsMEG3 after cerebral apoplexy.
2, in vitro analysis of the effects of lncRNA MEG3 on angiogenesis
(1) isolation, culture and identification of primary human umbilical vein endothelial cells (HUVEC).
(2) the establishment of HUVEC hypoxia model: real-time PCR was used to detect the expression of HUVEC hypoxia 24hVEGFa to evaluate the HUVEC hypoxia model.
(3) the effect of hypoxia on the expression of MEG3 and VEGFa: normal cultured HUVEC was cultured in hypoxia and normoxic environment, 12h, 24h and 48h, real-time PCR were used to detect the expression changes of MEG3 and VEGFa at different time points after HUVEC hypoxia.
(4) construct pCDH-MEG3 plasmids and package Lentiviral-meg3 lentivirus.
(5) the lentivirus was infected with MEG3 of the expression of HUVEC, and real-time PCR verified the up-regulated level of MEG3.
(6) MEG3 overexpression of HUVEC angiogenesis ability detection: CCK-8 method to detect MEG3 over expression of HUVEC proliferation ability, RTCA detection MEG3 over expression HUVEC migration ability, Matrigel tube test to detect MEG3 over expression of HUVEC tube ability.
Result錛

本文編號：1446833