《microRNA论文：低温环境下microRNA对小鼠脑外伤后海马区神经干细胞增殖分化的调控作用.doc》由会员分享，可在线阅读，更多相关《microRNA论文：低温环境下microRNA对小鼠脑外伤后海马区神经干细胞增殖分化的调控作用.doc（13页珍藏版）》请在三一文库上搜索。
1、 microRNA论文：低温环境下microRNA对小鼠脑外伤后海马区神经干细胞增殖分化的调控作用【中文摘要】和近年来,世界各地极端气候事件频繁发生,如2008年初发生于我国南方地区的低温雨雪冰冻天气。低温雨雪冰冻天气可导致交通事故、建筑物倒塌等意外事件增加,颅脑外伤等创伤性疾病发生率急剧上升。受伤后,由于道路湿滑、交通运输困难等原因,病人往往容易暴露于低温环境中。研究表明,低温环境不仅可造成机体局部的损伤,如冻结、冻伤等,而且还可对机体神经系统、循环系统、消化系统、呼吸系统、泌尿生殖系统等造成损害。创伤性颅脑损伤(traumatic brain injury, TBI)是当今威胁人类生命和健
2、康的主要创伤性疾病之一,其死残率一直居高不下。但是在低温环境影响下,脑外伤后的病理生理过程会发生何种变化目前尚不完全清楚。为了更好地掌握低温环境下颅脑外伤的发病规律及其防治措施,有必要积极开展低温环境下颅脑外伤病理生理学改变的研究。脑外伤可导致大量神经元变性坏死,进而造成神经功能缺损。颅脑损伤后的神经功能重建一直是临床治疗中至今尚未解决的难题。研究证实,脑外伤可诱导成体脑海马齿状回(dentate gyrus, DG)亚颗粒区(subgranular zone, SGZ)和室下区(subventricular zone, SVZ)内源性神经干细胞(neural stem cells, NSCs
3、)增殖分化,一定程度上修复受损的神经功能。因此,尽可能地提高脑外伤后内源性NSCs增殖分化能力,对于伤后神经功能的重建无疑具有重要意义。但是,有关脑外伤后NSCs增殖分化调控机制目前尚不完全清楚。微小RNA (microRNA, miRNA)为一类单链小分子非编码RNA,可通过转录后水平调控靶基因蛋白的表达,有着重要的生理功能,研究已证实其参与了多种生物学过程。然而,哪些特异miRNA可能参与调控及其如何调控脑外伤后NSCs增殖分化目前仍所知甚少；此外,低温环境是否会影响脑外伤后miRNA的表达进而影响NSCs增殖分化过程也不明确。本研究通过建立闭合性脑外伤小鼠模型,4低温暴露4h,采用miR
4、NA芯片技术确定脑外伤后海马区niRNA的表达谱,筛选出差异表达且与NSCs增殖分化相关的特异miRNA,并进行real-time PCR验证。随后挑选1-2个miRNA,进一步观察低温环境下脑外伤后不同时间点海马区特异1miRNA和其相关靶基因的表达变化以及内源性NSCs的增殖情况,以探讨miRNA对脑外伤后NSCs增殖调控作用及低温环境对该调控作用的影响。研究内容和方法：1.闭合性脑外伤小鼠模型的建立和实验分组采用改良的自由落体法构建闭合性脑外伤小鼠模型,于脑损伤后24h处死动物,对脑组织进行HE染色,从病理学上鉴定模型是否成功。实验分为常温脑外伤组和低温脑外伤组,两组又分别分为假手术组、
5、脑外伤4h、1d和3d组。低温脑外伤组动物脑损伤后放置于4低温环境中饲养4h,随后置于25室温喂养。常温脑外伤组动物脑损伤后置于25室温喂养。假手术组不做脑损伤处理。于脑损伤后4h、1d和3d分批处死动物,收集伤侧海马组织待检。2.脑外伤后海马区miRNA表达谱的变化取常温假手术组(对照组)、常温脑外伤4h组和低温脑外伤4h组海马标本,采用miRNA芯片技术检测低温环境下脑外伤后海马区miRNA的表达谱,筛选脑外伤后差异表达且与NSCs增殖分化相关的特异miRNA,并进行real-time PCR验证。3.脑外伤后海马区miR-34a、Notch1信号分子的表达变化和内源性NSCs的增殖情况3
6、.1采用real-time PCR检测脑外伤各时间点miR-34a的表达水平,明确脑外伤后miR-34a的动态表达变化。3.2采用real-time PCR检测脑外伤后各时间Notch1 mRNA的表达水平,明确Notch1基因的表达变化；采用免疫荧光染色检测脑外伤后3d组海马DG区Notch1阳性细胞的表达,明确脑外伤后Notch1蛋白的表达变化。3.3采用免疫荧光染色检测脑外伤后3d组海马DG区Nestin阳性细胞的表达,明确脑外伤后内源性NSCs的增殖情况。结果：1.成功建立闭合性脑外伤小鼠模型：病理学观察可见脑外伤动物脑组织挫裂伤明显,细胞高度水肿,神经元坏死,数目减少,伴星形胶质细胞
7、增生,表明本研究成功建立了闭合性脑外伤小鼠模型。2.脑外伤后海马区miRNA表达谱的变化：2.1芯片结果：(1)与对照组比较,常温脑外伤组有24个miRNA上调2倍以上,7个miRNA下调2倍以上；低温脑外伤组有20个IniRNA上调2倍以上,3个miRNA下调2倍以上,结果表明脑外伤可导致海马区多个miRNA表达发生变化。(2)与对照组比较,常温脑外伤组和低温脑外伤组miR-200b表达分别上调(2.450.21)倍和(1.110.15)倍；而miR-34a则分别下调(2.370.28)倍和(1.650.16)倍,表明在脑外伤后海马区1miR-200b和miR-34a的表达均发生明显变化。2
8、.2 real-time PCR验证miR-200b和miR-34a的表达：(1)与对照组比较,常温脑外伤组和低温脑外伤组miR-200b表达分别上调(2.950.17)倍和(1.220.11)倍；而miR-34a则分别下调(2.080.09)倍和(1.470.03)倍,与芯片结果趋势相一致。(2)与对照组比较,常温脑外伤组miR-200b表达显著上调(P0.05)；常温脑外伤组和低温脑外伤组miR-34a表达均显著下调(P0.05)。结果提示,脑外伤可下调miR-34a的表达,而低温可抑制伤后miR-34a的表达下调。3.2 Notch1信号分子的表达变化：(1) Notch1 mRNA的表
9、达变化：real-time PCR结果显示,常温脑外伤4h、1d和3d组Notch1 mRNA表达均明显高于常温假手术组(P0.05),3d组表达则明显高于低温假手术组(P0.05)。结果提示,脑外伤可上调Notch1 mRNA的表达,而低温可抑制伤后Notch1 mRNA的表达上调。(2) Notch1蛋白的表达变化：免疫荧光染色结果显示,常温脑外伤3d组伤侧海马DG区Notch1阳性细胞数明显高于常温假手术组(18.23.56比0.40.55,P0.05)。结果提示,脑外伤可诱导海马DG区Notch1蛋白表达上调,而低温可抑制伤后Notch1蛋白的表达。3.3内源性NSCs的增殖情况：免疫
10、荧光染色结果显示,常温脑外伤3d组伤侧海马DG区Nestin阳性细胞数明显高于常温假手术组(21.85.07比0.80.45,P0.05)。结果提示,脑外伤可诱导海马区DG区NSCs增殖,而低温可抑制脑外伤后NSCs的增殖。结论：1.脑外伤可导致海马区miR-200b和miR-34a等多个miRNA表达发生变化,而低温环境可影响脑外伤后海马区miRNA的表达。2.脑外伤后海马区IniR-34a表达显著下调,靶基因Notch1显著上调,且NSCs显著增殖,提示miR-34a/Notch1信号通路可能参与了脑外伤后海马区NSCs增殖过程的调控。3.低温环境可抑制脑外伤后海马区miR-34a/Not
11、ch1信号通路的表达,进而影响伤后NSCs增殖,提示低温环境有可能影响脑外伤的预后恢复。【英文摘要】Background and :Recently years, more and more extremely weather and climate events frequently happen all over the world. For example, the extremely cold and snowy weather event happened in southern China in the beginning of the year 2008. The extreme
12、ly cold and snowy weather event not only can result traffic accidents and building collapse, increasing the incidence rate of trauma, but also often can get trauma patients exposed to low temperature a period of time following injury because of slippery road and traffic jams. Traumatic brain injury
13、(TBI) is a leading cause of death and disability wordwide thar threatens our life and health seriously. However, it is still unclear that what changes will occur about the pathophisiology of TBI following low temperature exposure. Hence, it is necessary to carry out pathophisiological studies of TBI
14、 following low temperature exposure actively in order to mastering its pathogenesis regularity and preventive and therapeutic strategis better.A large number of neural cells necrosis leads to permanent loss of neurological function after TBI. So far, there are no effective treatments that could faci
15、litate the repair of the loss of neurological functional. More and more studies have demonstrated that TBI can induce endogenous neural stem cells (NSCs) proliferation and differentiation in the subgranular zone (SGZ) of hippocampal dentate gyrus (DG) and subventricular zone (SVZ) and reapair the lo
16、ss of neurological function. Therefore, improving the proliferation and differentiation ability of NSCs after injury will be a new therapeutic stratege of TBI. MicroRNAs (miRNAs) are a class of small noncoding RNAs that act as important post-transcriptional repressors and have been implicated in man
17、y biological processes. However, which and how miRNAs are involved in trauma-induced proliferation and differentiation of NSCs are still largely unknown; whether low temperature exposure can affect miRNAs expression after TBI is still unclear yet.In our study, mice were suffered closed head injury (
18、CHI), and then were exposed at 4for 4 hours. We detected hippocampal miRNA profiles after TBI by miRNA microarray, screened abnormally expressed miRNAs that are involved in NSCs proliferation and differentiation according to references and bioinformatics, and then identified their expression by real
19、-time PCR. Subsequently, we investigated expression levels of 1-2 of these miRNAs and their targets and endogenous proliferation of NSCs in the hippocampus after TBI following low temperature exposure to explore the regulation role of miRNAs on neural stem cells proliferation after traumatic brain i
20、njury following low temperature exposure.Methods:1. Closed head injury mice model and experiment groupsMice were suffered closed head injury using a modified weight-drop device. At 24 hours after injury, brain tissues were isolated and stained by HE staining to assess whether CHI model was successfu
21、lly established. Animals were randomly divided into normal temperature exposure TBI group (NTBI) and low temperature exposure TBI group (LTBI). Each group was divided into sham group,4 hours,1 day and 3 days group post-TBI. NTBI groups were kept at25/. LTBI groups were kept at4for 4 hours, and then
22、were kept at25. Sham group was not suffered CHI as control. Animals were killed at 4 hours,1 day and 3 days after injury. Ipsilateral hippocampus tissues were extracted for examining.2. Expression changes and validation of hippocampal miRNA profiles after TBImiRNA microarry was used to determine hip
23、pocampal miRNA profiles at 4 hours after TBI. Abnomally expressive miRNAs that regulate NSCs proliferation and differentiation after TBI were screened according to references and bioinformatics. And then, their expression levels were validated using real-time PCR.3. Expression changes of miR-34a, No
24、tch1 and proliferation of endogenous NSCs in hippocampus after TBI3.1 real-time PCR was used to examine miR-34a expression changes in the hippocampus at different time points after TBI3.2 real-time PCR was used to detect Notch1 mRNA expression changes in the hippocampus at different time points afte
25、r TBI; Notchl immunofluorescence was used to identify Notch1 protein expression changes in the DG at 3 days post TBI. 3.2 Immunofluorescence was used to identify NSCs proliferation in DG at 3 days after TBI by Nestin staning.Results:1. Assessment of CHI mice model:Pathology observation showed that s
26、eriously cerebral contusion, highly cellular edema, a large number of neurons necrosis and astrocyte proliferation after TBI, demonstrating CHI mice model was successfully established.2. Expression changes of hippocampal miRNA profiles after TBI2.1 miRNAs microarray results:(1) Compared to sham grou
27、p,24 miRNAs were more than 2-fold up-regulated,7 miRNAs were more than 2-fold down-regulated in NTBI group; 20 miRNAs were more than 2-fold up-regulated,3 miRNAs were more than 2-fold down-regulated in LTBI group, reaveling that TBI can induce lots of miRNAs expression changed in the hippocampus.(2)
28、 Compared to control group, the expression levels of miR-200b in NTBI group and LTBI group were up-regulated (2.450.21) fold and (1.10.15) fold, respectively; the expression levels of miR-34a were down-regulated (2.370.28) and (1.650.16) fold, respectively, suggesting TBI can change miR-200b and miR
29、-34a expression in the hippocampus.2.2 Validation of miR-200b and miR-34a expression levels by real-time PCR:(1) Compared to control group, the expression levels of miR-200b were were up-regulated (2.950.17) fold and (1.220.11) fold, respectively; the expression levels of miR-34a were down-regulated
30、 (2.080.09) and (1.470.03) fold, respectively. Real-time PCR essentially showed, the same pattern of expression changes of the two miRNAs as observed with the microarray analysis.(2) The expression levels of miR-200b in NTBI group were apparently up-regulated (P0.05) compared to sham group; the expr
31、ession levels of miR-34a in NTBI group and LTBI group were both apparently down-regulated compared to sham gourp (P0.05), but up-regulated significantly at 3 days (P0.05); (3) the up-regulation levels of Notchl mRNA at 4 hours,1 day and 3 days post injury were less compared to NTBI relative time poi
32、nt groups (P0.05), suggesting TBI can induce Notchl mRNA up-regulation, whereas low temperature can inhibit its up-regulation.(2) Expression changes of Notchl protein:immunofluorescence show that, (1) the number of Notch1+cells in the ipsilateral hippocampal DG of NTBI 3 days group was significantly
33、 increased compared to NTBI sham group (18.23.56 vs 0.40.55, P0.01); (2) the number of Notchl+ cells in the ipsilateral hippocampal DG of LTBI 3 days group was also significantly increased compared to LTBI sham group (102.55 vs 0.60.55, P0.01); (3) the number of Notchl+ cells in the ipsilateral hipp
34、ocampal DG of LTBI 3 days group was less compared to NTBI 3 days group (P0.01), suggesting TBI can induce Notch1 protein up-regulation, whereas low temperature can inhibit its up-regulation.3.3 Endogenous NSCs proliferation:Immunofluorescence show that, (1) the number of Nestin+ cells in the ipsilat
35、eral hippocampal DG of NTBI 3 days group was significantly enhanced compared to NTBI sham group (21.85.07 vs 0.80.45, P0.01); (2) the number of Nestin+ cells in the ipsilateral hippocampal DG of LTBI 3 days group was also significantly enhanced compared to LTBI sham group (16.01.59 vs 0.40.55, P0.01
36、); (3) the number of Nestin+ cells in the ipsilateral hippocampal DG of LTBI 3 days group was less compared to NTBI 3 days group (P0.05), suggesting TBI can induce NSCs proliferation in DG, whereas low temperature can inhibit its proliferation.Conclussions:1. Lots of miRNAs (such as miR-200b and miR
37、-34a) expression were changed in the hippocampus after TBI, whereas low temperature can affect their expression post injury.2. TBI can significantly decrease miR-34a expression, increase its target Notch1 signaling expression and induce NSCs proliferation in the hippocampus, suggesting miR-34a/Notch
38、1 pathway might be involved in trauma-induced NSCs proliferation.3. Low temperature exposure might affect NSCs proliferation by inhibiting the role of miR-34a/Notch1 pathway in the hippocampus after TBI, suggesting low temperature exposure may affect TBI prognosis.【关键词】microRNA 神经干细胞 Notch1 脑外伤 低温 基
39、因芯片【英文关键词】microRNA neural stem cells Notch1 traumatic brain injury low temperature microarray【目录】低温环境下microRNA对小鼠脑外伤后海马区神经干细胞增殖分化的调控作用摘要3-7ABSTRACT7-11第1章 引言14-17第2章 材料与方法17-312.1 实验材料17-192.2 实验方法19-31第3章 结果31-383.1 闭合性脑外伤小鼠模型的建立313.2 脑外伤后海马区MIRNA表达谱的变化31-333.2.1 总RNA质量鉴定313.2.2 脑外伤后海马区miRNA表达谱的变化3
40、1-333.2.3 real-time PCR验证芯片结果333.3 脑外伤后海马区MIR-34A、NOTCH1信号分子的表达变化和内源性NSCs增殖情况33-383.3.1 miR-34a的表达变化343.3.2 Notch1信号分子的表达变化34-363.3.3 内源性NSCs的增殖情况36-38第4章 讨论38-494.1 脑外伤动物的低温暴露处理38-394.2 NSCs与脑外伤39-404.3 MIRNA与NSCs40-434.4 脑损伤后NSCs增殖分化相关MIRNA的表达变化43-454.5 MIR34A/NOTCH1信号通路对脑外伤后海马区NSCs增殖的调控作用45-49第5章 结论49-50致谢50-51参考文献51-57附图57-60攻读学位期间的研究成果60-61综述61-72参考文献69-72