调控脑卒中后血管新生lncRNAs的发现及其功能的初步探讨

发布时间：2018-01-20 04:23

  本文关键词： 缺血性脑卒中 lncRNAs 血管新生 MEG3 生物信息学分析 VEGF　出处：《南昌大学》2014年硕士论文　论文类型：学位论文

【摘要】：研究背景和目的： 缺血性脑卒中是一种严重危害人类健康和生命安全的脑血管疾病，具有发病率高、致残率高和死亡率高等特点，其治疗的关键是及时、充分恢复脑缺血区的血供。临床观察发现脑卒中后可诱发缺血区反应性血管新生，而且脑组织微血管密度与卒中患者的预后存有密切关系。因此，进一步阐明脑卒中后血管新生的分子调控机制，寻找到有效的分子调控靶点，利用药物或基因干预促进治疗性血管新生有望根本改变目前脑卒中的治疗现状。 长链非编码RNA（long non-coding RNAs）是一类由RNA聚合酶II转录的转录本长度超过200nt的长链非编码RNA，近年来，研究发现lncRNAs的表达具有组织特异性和发育特异性，某些lncRNAs还与参与血管新生过程的多种基因和信号分子关系密切，但是脑卒中后lncRNAs的表达变化如何，lncRNAs是否与其他非编码RNAs如microRNAs等一样参与脑卒中后的血管新生调控，目前尚不清楚。本研究通过Solexa高通量测序检测大鼠脑缺血后脑组织lncRNAs的表达谱变化，生物信息学分析脑卒中后差异表达的lncRNAs，筛选出血管新生相关lncRNAs，并初步探讨脑缺血后血管新生相关lncRNAs的功能及其可能的调控机制。 研究内容和方法： 1、体内实验筛选脑卒中后血管新生相关lncRNAs （1）采用zea-longa法建立建立雄性SD大鼠MCAO模型，神经功能评分、TTC染色及MRI评估模型。 （2）采用Solexa基因测序建立脑卒中后缺血脑组织lncRNAs的表达谱，real-time PCR随机验证lncRNAs的表达水平。 （3）生物信息学分析脑卒中后差异表达的lncRNAs，筛选血管新生相关lncRNAs。 （4）采用real-time PCR动态观察脑卒中后所筛选出血管新生lncRNAsMEG3的表达变化。 2、体外实验分析所筛选的lncRNA MEG3对血管新生的调控功能 （1）原代人脐静脉血管内皮细胞（HUVEC）的分离培养与鉴定。 （2） HUVEC缺氧模型的建立：采用real-time PCR检测HUVEC缺氧24hVEGFa的表达变化评估HUVEC缺氧模型。 （3）缺氧对MEG3和VEGFa表达的影响：将正常培养的HUVEC置于缺氧与常氧环境中培养12h、24h和48h，real-time PCR检测HUVEC缺氧后各个时间点MEG3和VEGFa的表达变化。 （4）构建pCDH-MEG3质粒并包装Lentiviral-meg3慢病毒。 （5）慢病毒感染过表达HUVEC的MEG3，real-time PCR验证MEG3的上调水平。 （6）MEG3过表达的HUVEC血管生成能力的检测：CCK-8法检测MEG3过表达HUVEC的增殖能力，RTCA检测MEG3过表达HUVEC的迁移能力，matrigel成管实验检测MEG3过表达HUVEC的成管能力。 结果： 1、成功建立SD大鼠MCAO模型：MCAO大鼠均出现对侧肢体偏瘫、行走时原地转圈和提尾向对侧侧身等神经功能缺失表现，，神经功能评分2分；TTC染色可见右侧大脑中动脉供血区呈苍白色而周围正常脑组织呈现红色；MRI结果显示脑梗死T1像及DWI像呈高信号；缺血模型梗死灶一致性较好，可用于后续基因测序。 2、脑卒中后缺血脑组织lncRNAs的表达谱发生显著改变：脑缺血后Solexa基因测序共检测出9287条lncRNAs，与健侧相比缺血侧上调2倍以上及下调0.5倍且P＜0.05的lncRNAs共448条，其中上调的lncRNAs有414条，下调的lncRNAs34条；real-time PCR随机验证lncR-14、lncR-30、lncR-4、lncR-40、lncR-64、lncR-65、lncR-76及lncR-44的表达分别上调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的表达下调0.5803±0.4459倍(P＜0.05)，与Solexa高通量测序的结果趋势一致，成功建立了脑缺血lncRNAs的表达谱。 3、脑缺血后448条差异表达的lncRNAs中，生物信息学筛选出47条与血管新生相关lncRNAs，其中功能尚未注释的lncR41与血管新生相关，生物信息学分析lncR41与小鼠MEG3序列高度同源，提示lncR41为大鼠MEG3。 4、脑缺血后MEG3的表达下调：real-time PCR结果显示脑缺血24h后MEG3的表达较健侧下调0.7840.124倍（P＞0.05），脑缺血48h后MEG3的表达较健侧下调0.5020.133倍（P＜0.05），脑缺血72h后MEG3的表达较健侧下调0.7230.106倍（P＜0.05）。 5、缺氧后MEG3和VEGFa表达发生变化：real-time PCR结果显示缺氧后MEG3的表达呈先下降后上升的动态变化，即缺氧12h后MEG3的表达下调0.3790±0.0187倍（P＜0.05），24h后MEG3的表达下调0.6618±0.0767（P＞0.05），48h后MEG3的表达上调3.75590.1915倍（P＜0.05）；缺氧后12h VEGFa的表达上调1.7498±0.1916倍（P＜0.05），24h后VEGFa的表达上调2.3618±0.2725倍（P＜0.05），48h后VEGFa的表达上调2.7400±0.1761倍（P＜0.05）。 6、成功构建出pCDH-MEG3质粒并包装出Lentiviral-meg3慢病毒，病毒滴度＞6x106/ml。 7、慢病毒感染过表达MEG3抑制HUVEC的增殖、迁移和成管等血管形成能力，抑制HUVEC VEGFa和nontch1的表达。 结论： 1、脑卒中后缺血脑组织lncRNAs的表达谱发生显著变化，lncRNAs可能参与了脑卒中后血管新生调控。 2、meg3抑制血管内皮细胞的增殖、迁移和成管等功能进而影响脑卒中后血管新生，其机制可能为影响了VEGF信号通路。
[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锛

本文编号：1446833