咪达唑仑对神经干细胞增殖的影响及其机制的初步研究

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英文题名：The Effect of Midazolam on the Proliferation of Neural Stem Cells and the Preliminary Exploration of Underlying Mechanism 作者：赵三军 论文级别：博士 学科专业名称：麻醉学 中文关键词：神经干细胞 ; 增殖 ; GABA_A受体 ; cAMP反应元件结合蛋白 ; 海马 ; 神经再生 ; 记忆 ; cAMP反应元件结合蛋白 ; 微小RNA ; 转录 英文关键词：neural stem cells ; proliferation ; GABA_A receptor ; cAMP 英文关键词：response element binding proteinhippocampus ; neurogenesis ; memoryCREB ; microRNA ; transcription 学位年度：2012 导师：米卫东 学科代码：100217 学位授予单位：中国人民解放军军医进修学院

摘要

第一部分咪达唑仑对神经干细胞增殖的影响及其分子机制（离体实验）
     目的研究咪达唑仑对神经干细胞增殖的影响及其介导这种影响的机制。方法原代培养从新生12小时内的Sprague-Dawley大鼠海马取得的神经干细胞，使用免疫细胞化学的方法标记神经干细胞的标志性蛋白nestin，从而鉴定培养的神经干细胞。实验分组：溶剂对照组（veh组）--DMSO/生理盐水+生理盐水；药物作用组（mid组）--DMSO/生理盐水+咪达唑仑；拮抗组包括两组--1）荷包牡丹碱+咪达唑仑组（bic组）2）呋塞米+咪达唑仑组（fur组）。使用GABA_A受体的特异性抑制剂荷包牡丹碱（bic）和钠钾氯的共转运体的抑制剂呋塞米（fur）的目的是探讨咪达唑仑影响神经干细胞增殖的潜在机制。分别使用测定细胞增殖的经典试剂盒cell counting kit8(CCK8)测定咪唑安定对神经干细胞增殖的影响；使用胸腺嘧啶的类似物BrdU掺入S期的神经干细胞，使用免疫细胞化学的方法标记并计数处于S期的细胞占总的细胞比例；使用流式细胞周期描述的方法检测咪达唑仑对神经干细胞周期分布的影响，进而印证CCK8和BrdU掺入的实验结果。使用钙离子敏感荧光染料fluo3-AM记录细胞内钙离子浓度变化，实验分组：阳性标记组--给予终浓度为30mM的氯化钾溶液；药物作用组--给予终浓度为30-90μM的咪达唑仑，拮抗剂组--预先给予终浓度为10μM的荷包牡丹碱，后给予30-90μM的咪达唑仑。最后使用western blot的方法检测神经干细胞内一个与神经干细胞增殖和分化密切相关的转录因子磷酸化cAMP反应元件结合蛋白（pCREB）在给予咪达唑仑处理后表达量的变化，实验分两组：溶剂对照组（veh组）--加入生理盐水；药物作用组（mid组）--加入终浓度为30μM的咪达唑仑作用5天。结果免疫细胞化学的方法证实原代培养的细胞绝大多数(>70-80%）为表达nestin蛋白的细胞。CCK8实验中终浓度90μM的咪达唑仑显著抑制神经干细胞的增殖，这种抑制的效应可以部分被荷包牡丹碱(veh,0.5230±0.0040, mid,0.4467±0.0045, bic+mid,0.4664±0.0048, n=8for each group)或者呋塞米(veh,0.6065±0.0041, mid,0.4811±0.0044, furo+mid,0.5035±0.0042, n=8for eachgroup)的预先给药所拮抗。在BrdU掺入实验，90μM咪达唑仑显著减少处于S期的神经干细胞的百分比，这种抑制的效应完全被荷包牡丹碱或者呋塞米的预先给药所拮抗。在细胞周期描述实验中，90μM的咪达唑仑显著减少处于S期的神经干细胞的百分比，这种抑制的效应完全或者部分被荷包牡丹碱(veh,12.46±0.02, mid,8.98±0.12, bic+mid,11.28±0.20, n=3for each group)或者呋塞米(veh,11.36±0.21, mid,7.48±0.04, furo+mid,11.86±0.55, n=3for eachgroup)的预先给药所拮抗。30-90μM的咪达唑仑能导致神经干细胞内的钙离子浓度显著增高，这种由咪达唑仑导致的细胞内钙离子升高可以完全的被荷包牡丹碱所拮抗。持续的30μM咪达唑仑的作用能促进在促分化培养基中生长的神经干细胞的磷酸化CREB的表达。结论90μM咪达唑仑抑制神经干细胞的的增殖，这种抑制作用至少部分是通过激动GABA_A受体实现的。咪达唑仑能诱导神经干细胞产生内向性钙流，这种内向性的钙流也是GABA_A受体介导的，并且它通过促进下游的磷酸化CREB表达升高以及后续的效应对神经干细胞增殖产生影响。
     第二部分咪达唑仑对小鼠海马神经干细胞增殖以及空间记忆功能的影响（在体实验）
     目的探讨在体情况下咪达唑仑对小鼠海马齿状回神经干细胞增殖的影响以及对空间记忆功能的影响。方法18只3-4周ICR小鼠（体重13-17克）随机分为两组：midazolam组--连续给予3次咪达唑仑（20mg/kg），时间间隔2小时；生理盐水组--连续给予3次咪达唑仑（20ml/kg）时间间隔2小时。每组中的3只小鼠在给药（溶剂）后24小时给予BrdU (100mg/kg)标记处于S期的细胞然后深麻醉下经心灌流固定，使用免疫组化（冰冻片，漂浮片法）的方法显示BrdU标记细胞。余下每组6只小鼠在2周后使用Morris水迷宫模型检验给予咪达唑仑后对ICR小鼠空间记忆的影响，分别测试小鼠定位航行和空间探索两阶段的逃避潜伏期、空间探索穿越次数和象限停留时间。结果连续给予咪达唑仑能显著减少ICR小鼠海马齿状回的神经干细胞增殖（veh:2491.0±245.1；mid:1740.0±177.3，n=3），然而在以Morris水迷宫为模型的空间记忆实验中，两组小鼠的逃避潜伏期、空间探索穿越次数（8.40±1.29,n=5;7.33±0.80,n=6）和象限停留时间（s）(17.20±1.86,n=5;16.00±1.44,n=6)并无统计学差异。结论咪达唑仑60mg/kg（分3次间隔2小时给予）可抑制在体小鼠海马神经干细胞增殖，但是并不影响两周后由Morris水迷宫模拟的空间记忆功能。
     第三部分磷酸化CREB对神经再生相关微小RNA表达的影响
     目的初步探讨磷酸化CREB调节下游微小RNA（microRNA）表达的相关性，
     并且探讨microRNA调节神经干细胞增殖相关基因表达的机制。方法：将细胞分为两组：NSC组--神经干细胞常规在神经干细胞完全培养基中培养；diff组--神经干细胞在促分化培养基中培养，使用western blot的方法检测神经干
     细胞在促分化和促增殖两种不同培养环境下的磷酸化CREB的表达，同时使用荧光定量PCR的方法测定两种不同培养环境下神经再生相关microRNA的
     表达。使用生物信息学的方法预测miR-124原始转录本上游的可能的CREB结合位点（启动子区域），使用染色质免疫共沉淀（chromatinimmunoprecipitation，ChIP）技术检测转录因子磷酸化CREB和microRNA（miR-124）原始转录本(pri-miR-124-2, pri-mir-124-3)上游的启动子区域的结合，从而揭示磷酸化CREB调节microRNA（miR-124）表达的机制。结果神经干细胞分化时，磷酸化CREB的表达升高，同时miR-9,miR-124,let-7b（⊿Ct分别为diff：-2.053±0.217， NSC:2.707±0.213； diff：9.090±0.111，NSC:12.690±0.284; diff：-3.257±0.359，NSC:-0.310±0.040；n=3）的表达也升高。生物信息学的结果显示，miR-124转录本pri-mir-124-2和pri-mir-124-3上游都存在结合CREB的基序，并且存在特征性的CpG岛区域；染色质共沉淀的结果显示磷酸化CREB和CREB与miR-124的原始转录本pri-miR-124（pri-miR-124-2, pri-miR-124-3）的上游预测的启动子区有结合。结论神经干细胞分化初期磷酸化CREB的表达和一系列神经再生相关microRNA的表达具有相关性。CREB是结构性的结合于神经再生的相关microRNA（miR-124）的原始转录本pri-mir-124-2和pri-mir-124-3上游的启动子区域，磷酸化CREB能促进原始转录本的转录。
     结论：1、离体情况下，咪达唑仑在90μM浓度时，可抑制神经干细胞的增殖，这种抑制效应部分是由GABA_A受体所介导；
     2、咪达唑仑能诱导神经干细胞产生GABA_A受体介导的内向性钙流，并通过下游的磷酸化CREB表达升高以及后续的效应对神经干细胞增殖产生影响；
     3、咪达唑仑60mg/kg（分3次间隔2小时给予）可抑制在体小鼠海马神经干细胞增殖，但是并不影响两周后其空间记忆功能；
     4、磷酸化的CREB能通过microRNA-124原始转录本表达的增加，促进与神经再生相关的microRNA-124的表达。
Part1The Effect of Midazolam on the Proliferation of NeuralStem Cells and the Preliminary Exploration of UnderlyingMechanism (in-vitro experiments)
     Objective To investigate the effect of midazolam on the proliferation of neuralstem cells (NSCs) isolated from the hippocampus of rats and the underlyingmechanism. Methods We firstly isolated neural stem cells from Sprague-Dawleyrats within12hours of birth and then cultured them in Dulbecco's Modified EagleMedium: Nutrient Mixture F-12(DMEM/F-12) media supplemented withmitogen and B27supplement. Immunocytochemistry was used to identify theexpression of nestin, a marker of neural stem cells. Two classic experiments—cellcounting kit8(CCK8) and5-bromo-2'-deoxyuridine（BrdU）incorporation testwere used to test the effect of midazolam on the proliferation of NSCs. Cell cycledistribution analysis was also used to analyse the percentage of cells in the Sphase which had a positive correlation with proliferation activity. We also appliedGABA_Areceptor antagonist bicuculline and Na-K-2Cl cotransport blockerfurosemide in our experimrnts to investigate the mechanism of the effect inducedby midazolam. Calcium sensitive fluorescent dye Fluo3-AM was used to tracethe calcium influx induced by midazolam. Western blot was also used to identifythe expression pattern of phosphorylated cAMP response element binding protein(pCREB), a crucial transcription factor for neurogenesis in response to midazolam.Results The majority (>70-80%）of cells raised from this method expressed nestin. In the CCK8and BrdU incorporation test, the proliferation activity was reduced inresponse to90μM midazolam (ultimate concentration) and these effects werepartly or entirely rescued by the pretreatment with GABA_Areceptor antagonistbicuculline and Na-K-2Cl cotransport blocker furosemide. The intracellularcalcium concentration was elevated in response to the administration ofmidazolam (30-90μM) and this effect was completely blocked by the pretreatmentwith GABA_Areceptor antagonist bicuculline. The expression of pCREB of NSCscultured in pro-differentiation culture media was enhanced by continuousinfluence by30μM midazolam. Conclusion Midazolam decreased theproliferation of NSCs isolated from hippocampus of rats and this effect was atleast partly induced by GABA_Areceptor. The enhanced expression of pCREB inNSCs might be triggered by the calcium influx induced by midazolam.
     Part2The Effects of Midazolam on the Proliferation of NSCsLocated in the Hippocampus of Mice and the HippocampalDependent Spatial Memory (in-vivo experiments)
     Objective To investigate the effects of midazolam on the proliferation of NSCslocated in the hippocampus of mice and the hippocampal-dependent spatialmemory in vivo. Methods3-4weeks old mice were randomly devided into twogroups—midazolam group (mid group) and normal saline group (veh group). Weused BrdU to label the cells in S phase in vivo and immumohistochemistry(floating frozen-section) was also used to display these cells. Morris water mazewas used to test the effect of midazolam on the the hippocampal dependent spatialmemory of mice. Results3times administration of midazolam (20mg/kg, with2hinterval) decreased the cells incorporatin BrdU in mice dentate gyrus. However,we didn’t detect any statistical significance between the two groups in the performance of Morris water maze with respect to escape latency, residence timeand crossing frequency. Conclusion Midazolam limitedly influence theneurogenesis of mice hippocampus or has different effects on the different phaseof NSCs. Further researches are required before we get a systemic evaluation onthe effect of midazolam on the neurogenesis of hippocampus.
     Part3The Effect of Phosphorylated CREB on the Expressionof Neurogenesis Related MicroRNAs
     Objective To investigate the relationship of expression of phosphorylated CREBand neurogenesis related microRNA and further explore the mechanism throughwhich pCREB regulate the expression of miR-124. Methods Firstly, western blotwas used to identify the different expression of pCREB in the NSCs cultured intwo different media. Special real-time quantitative PCR to microRNA was used toquanify the expression of3neurogenesis related microRNAs (miR-124, miR-9,let-7b) in the NSCs cultured in two different media. Then, bioinformatics wasused to predict the potential binding sites in the upstream loci of primarymicroRNA-124. Then, chromatin immunoprecipitation (ChIP) was used toidentify these potential binding sites in the promoters. Results When cultured inpro-differentiation media, the NSCs significantly expressed higher pCREB andneurogenesis related microRNAs (miR-124, miR-9, let-7b). Bioinformaticsindicated that there were potential binding sites in upstream loci of primarymicroRNA-124(pri-mir-124-2and pri-mir-124-3). Chromatin immuno-precipitation identified the same results with those predicted.Conclusion In theearly phase of NSCs differentiation, the enhanced expression of pCREB promotesthe differentiation of NSCs and may be the inducement of many neurogenesisrelated microRNAs. CREB binds constitutively to the upstream loci ofpri-mir-124-2and pri-mir-124-3and the phosphorylation of CREB may promotethe transcription of these two primary microRNAs.
     Conclusion
     190μM Midazolam decreased the proliferation of NSCs isolated fromhippocampus of rats and this effect was at least partly induced by GABAAreceptor.
     2The enhanced expression of pCREB in NSCs might be triggered by thecalcium influx induced by midazolam.
     320mg/kg (repeatedly administered for3times with2hours interval)midazolam limitedly influence the neurogenesis of mice hippocampus or hasdifferent effects on the different phase of NSCs. Further researches are requiredbefore we get a systemic evaluation on the effect of midazolam on theneurogenesis of hippocampus.
     4CREB constitutively binds to the upstream loci of pri-mir-124-2andpri-mir-124-3and the phosphorylation of CREB may promote the transcription ofthese two primary microRNAs.

引文

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