【作者】 夏源；

【导师】 邹飞；

【作者基本信息】 南方医科大学 ， 劳动卫生与环境医学， 2007， 硕士

【摘要】 研究背景细胞在应对外界激刺激时,通过活化自身的信号通路,激活相关因子,促进基因转录,从而适应外界条件的转变,而p53是细胞在面对遗传毒性、非遗传毒性刺激时,细胞所激活的关键分子,其活化可激活不同类型靶基因,诱导细胞出现细胞周期停滞、凋亡、坏死等变化,同时参与DNA损伤修复。细胞内对p53的调控主要是通过转录后修饰,当细胞受到DNA损伤,缺氧和高温等刺激时,MDM2催化的泛素蛋白酶体依赖的降解通路受到抑制,从而导致p53降解以及其从核内的转出减少,核内p53聚集,进而诱导或抑制其下游调控细胞周期停滞、细胞凋亡的靶基因转录,最终产生对应于相应生理应激的细胞反应。目前的研究显示p53的活化受细胞种类,刺激形式和细胞周围环境等多方面因素的影响,即对应不同的刺激信号p53激活不同组合的下游靶基因。缺氧是一种重要的生理刺激,在正常发育和肿瘤形成以及转移中都担任着重要的角色。严重缺氧时可以引起胞内p53的积累,从而引起细胞凋亡。与其他引起DNA断裂的胞外刺激不同,缺氧活化的p53几乎没有转录激活功能,而是以转录抑制功能为主。中枢神经系统中的海马CA1神经元对缺氧最为敏感,同时不同发育时期,大脑对缺氧的耐受能力有所差异,因此本实验选取海马神经元为研究对象,对不同发育阶段p53在缺氧时的作用以及缺氧时p53对其下游基因的调控方式进行初步探讨,以期为大脑缺血缺氧损伤的进一步的研究提供一些线索。目的研究不同发育时期,大鼠海马神经元缺氧耐受性差异的分子机制,以及缺氧时大鼠海马神经元中的p53对其下游基因的转录调控模式,以期为大脑缺血缺氧损伤的进一步研究提供线索。方法将取材于出生后1d和5d的大鼠海马神经元分为常氧组,缺氧组和给药组。神经元培养7天后,进行缺氧处理（0%O₂,5%CO₂,and 95%N₂）12h或24h。药物则在缺氧处理前2h加入培养基中。采用倒置显微镜和NF免疫组织化学法观察细胞生长和形态,比较不同发育阶段大鼠海马神经元耐受缺氧的能力;Trizol法提取细胞中的总RNA,RT-PCR检测相关基因mRNA水平的表达,以了解缺氧应激下ATP依赖的钾离子通道(K_ATP)与p53信号通路之间的关系。免疫荧光检测膜蛋白UNC5B在各组的表达变化,探讨缺氧应激下p53对UNC5B的调控方式;MTT比色法和AO/EB双染法检测细胞存活率,分析不同处理组之间细胞存活率的差异。结果1.①充氧组中出生后5d大鼠海马神经元生长状态要优于对照组中培养的神经元。②出生后1d以及5d大鼠海马神经元缺氧12h时细胞凋亡率分别为81.04±2.25%（n=6）和89.4±2.197%（n=7）（p＜0.01）;缺氧24h时二者凋亡率分别为74.99±2.751%（n=7）和85.02±2.242%（n=7）（p＜0.01）。③5d海马神经元缺氧耐受能力优于1d大鼠海马神经元。2.①给予细胞K_ATP通道的激活剂和阻断剂后,通过RT-PCR检测海马神经元中p53下游靶基因mRNA表达水平变化,以了解K_ATP通道与p53信号通路的关系。缺氧+二氮嗪组中Bax的mRNA表达水平与单纯缺氧组相比明显下降（n=5）（p＜0.01）,在缺氧+甲苯磺丁脲组中Bax的mRNA表达水平相对与单纯缺氧组也有提高（n=5）（p＜0.05）;Fas的mRNA表达水平缺氧+二氮嗪组（n=5）（p＜0.01）和缺氧+甲苯磺丁脲组（n=5）（p＜0.05）与单纯缺氧组比都有差异;缺氧组中α-tubulin的mRNA表达水平与常氧时比明显下降（n=5）（p＜0.01）,缺氧+二氮嗪组和缺氧+甲苯磺丁脲组中其mRNA表达水平与单纯缺氧组相比没有明显差异。②MTT比色法检测细胞存活率。单纯缺氧组,缺氧+二氮嗪组和缺氧+甲苯磺丁脲组的细胞存活率分别是75.7±2.8%（n=7）,55.7±2.5%（n=6）和81.1±2.4%（n=6）,各加药组与单纯缺氧组比较具有显著性差异（p＜0.01）。常氧时,加药组与对照组细胞（n=6）存活率无显著性差异（p＞0.05）。③采用NF免疫组织化学法观察细胞生长和形态。单纯缺氧组,缺氧+二氮嗪组和缺氧+甲苯磺丁脲组的多极神经元的百分率分别是20.6±2.0%（n=6）,26.1±1.7%（n=6）和8.7±1.7%（n=6）,各加药组与单纯缺氧组比较都具有显著性差异（p＜0.01）,常氧时,加药组与对照组细胞（n=6）存活率无显著性差异（p＞0.05）。3.①为了研究p53对UNC5B的调控方式,我们采用RT-PCR分别检测了UNC5B在常氧和缺氧时加入p53抑制剂PFT-α（100nM）和p53激活剂nutlin-3（2μM）时的转录水平。结果显示,与常氧比较缺氧时UNC5B转录水平下降,（p＜0.01,n=5）。常氧时nutlin-3（p＜0.01,n=5）激活UNC5B的转录,而PFT-α对其无显著影响。缺氧时nutlin-3（p＜0.01,n=5）抑制UNC5B的转录,而PFT-α（p＜0.01,n=5）则激活UNC5B的转录。②采用MTT比色法检测细胞存活率。缺氧时细胞存活率（75.0±2.8%）要明显低于常氧时（p＜0.01,n=7）。常氧时PFT-α和nutlin-3都不影响细胞的存活（p＞0.05,n=5）。而缺氧时却不同,PFT-α可以保护细胞免于凋亡（85.2±5.6%）,存活率与单纯缺氧组相比有显著性差异（p＜0.01,n=6）。nutlin-3促进细胞凋亡（57.1±4.1%）,存活率与单纯缺氧组比有显著性差异（p＜0.01,n=7）。③免疫荧光检测各处理组海马神经元上UNC5B的表达水平,实验结果显示与常氧（129.959±21.633,n=122）比较缺氧（104.078±19.058,n=98）时UNC5B蛋白表达水平下降（p=0.000＜0.01）。常氧时nutlin-3（150.014±26.303,n=123）促进UNC5B的蛋白表达（p=0.000＜0.01）,而PFT-α（124.072±28.016,n=106）对其无显著影响（p=0.709＞0.05）。缺氧时nutlin-3（95.244±12.354,n=108）与单纯缺氧组相比显著抑制UNC5B蛋白表达（p=0.002＜0.01）,而PFT-α（104.541±19.080,n=104）同单纯缺氧组比无显著性变化（p=1.000）。结论1.5d大鼠海马神经元分离培养过程中充氧有利于神经元的原代培养,出生后5d大鼠海马神经元缺氧耐受力强于出生后1d大鼠海马神经元。2.实验结果显示:缺氧时K_ATP通道的开放可保护细胞免受缺氧损伤,并且K_ATP主要通过影响p53转录激活调控的基因表达来实现对神经元的缺氧保护功能。3.本实验分析了缺氧应激下海马神经元中p53对其下游靶基因的调控方式,并发现缺氧时p53转录激活基因Bax,并转录抑制UNC5B。更多还原

【Abstract】 BackgroudThe transcription factor p53 responds to diverse stresses to regulate many target genes that induce cell-cycle arrest,apoptosis,senescence,DNA repair or alter metabolism.Under nomal conditions,the stability of p53 protein is tightly regulated.MDM2—a ubiquitin ligases mediate ubiquitin-dependet proteasomal degradation of p53.when cell exposed different types of cellular stress,p53 is stabilized and activated,for example,DNA damage,oncogenic signalling and hypoxia.The molecular mechanisms that govern the choice between cell-cycle arrest and apoptosis in response to p53 activation are only partially understood.Also,it remains unclear why different cell types display differential response to functional p53.The biological outcome of p53 activation presumably depends on several factors, including cellular context,cell type and type of stress agent.It is plausible that p53 activates different specific and possibly overlapping set of target genes in response to different stress signals.Previous studies have indicated that several hundred genes are potentially regulated by p53,but not necessarily in the same cell in response to the same stress signal.Hypoxia represents one of the most physiologically relevant stresses,having significant roles in both normal development and malignant progression.Exposure to severe hypoxia leads to the accumulation of p53 which can in turn lead to rapid apoptosis.In contrast to the response to DNA-damaging agents,hypoxia-induced p53 has little or no transcriptional transactivation capabilities and instead seems to function primarily as a transrepressor in order to induce apoptosis.This indicates that activation of p53 in response to hypoxia leads to a gene expression pattern that is different from that induced by p53 in response toγ-irradiation,ultraviolet（UV）light and other DNA-damaging agents.In the central nervous system（CNS）,hippocampal CA1 neurons are known to be extremely vulnerable to low oxygen concentrations or anoxia.Understanding the mechanisms governing tolerance to oxygen depletion is vital for developing strategies to protect the brain from hypoxic-ischemic insult.Therefore,in this experiment,we investigated the expression of p53-dependent gene in hippocampal neurons under hypoxia treatment,in order to provide important clue for the elucidation of cerebral hypoxia mechanism.ObjectivesInvestigate the molecular mechanism of hypoxia tolerance difference during the different development stage;Studing p53-dependent gene expression pattern in response to hypoxia in hippocampal neurons,in order to provide important clue for the elucidation of cerebral hypoxia mechanism.MethodsTo establish a primary culture technique for rat hippocampal tissue,We divided the neurons into control group,medication administration group and hypoxia group. Inverted microscope and immunohistochemistry of NF was performed to assess the growth and morphology of the cells.Investigate protein expression of UNC5B by immunofluorescence.The neurons were exposed to hypoxia condition（0%O₂, 5%CO₂,and 95%N₂）for 12h or 24h after 1 week seeding.MTT cell assays and AO/EB staining measured cell viability.Medicine was added to the culture medium 2 h before hypoxia condition were introduced and included for the full hypoxia period. Total RNA was isolated using TRIZOL according to the manufacturer’s instructions. RT-PCR using RT-PCR kit.Results1.①The growth of hippocampal neurons from 5 days old rat in oxygenation group was more vigorous than in control group.②The cell viability of neurons from 1 day old rat and 5 days old rat with 12h hypoxia exposure were 89.4±2.197% （n=6）and 81.04±2.25%（n=7）（p＜0.01）respectively,with 24h hypoxia exposure were 85.02±2.242%（n=7）and 74.99±2.751%（n=7）（p＜0.01）respectively.③The growth of hippocampal neurons from 5 days old rat in oxygenation group was more vigorous than in control group.During hypoxia,the hippocampal neurons disassociated from 5 days old rat were more tolerant than from 1 day old rat.2.①Multipolar neuron ratio of primary cultured hippocampal neurons in hypoxia group was 20.6±2.0%（n=6）,in diazoxide and tolbutamide with hypoxia groups were 26.1±1.7%（n=6）and 8.7±1.7%（n=6）.Those two kinds of medicine had effect on multipolar neuron ratio of cell under hypoxia condition（p＜0.01）.In normoxia,diazoxide and tolbutamide had no significant effect on multipolar neuron ratio of cell compared with those of in control group（p＞0.05,n=6）.②Using MTT analysis,we observed a much lower survival rate（75.7±2.8%）in neurons exposed to severe hypoxia（PO2=0 mmHg）compared with those exposed to normoxia（p＜0.01, n=7）.At the same time,tolbutamide（100μM）reduced the survival rate of neurons （55.7±2.5%）,however,diazoxide（100μM）increased the survival rate of neurons （81.1±2.4%）（p＜0.01,n=6）.In normoxia,neither diazoxide nor tolbutamide had a significant effect on cell viability（p＞0.05,n=6）.③The experiments showed a significant increase in Bax（p＜0.01）and Fas（p＜0.01）mRNA levels in neurons cultured at severe hypoxia condition（PO2=0 mmHg）compared with those cultured at normoxia（PO2=144 mmHg）.In hypoxia groups,tolbutamide increased Bax（p＜0.05）and Fas（p＜0.05）mRNA expression,while diazoxide reduced it to levels observed in normoxic condition（n=5 for each）,a-tubulin had a different expression pattern.It’s mRNA levels decreased in neurons cultured at severe hypoxia condition compared with those cultured at normoxia.In hypoxia groups,tolbutamide and diazoxide didn’t change a-tubulin mRNA expression.3.①To confirm the relationship between UNC5B and p53,we measured UNC5B mRNA levels with RT-PCR in neurons that have been treated with p53 inhibitor PFT-a（100nM[5]）or p53 activator nutlin-3（2μM）in normoxia or anoxia for 24h.These experiments showed a significant decrease in UNC5B（p＜0.01,n=5）mRNA levels in neurons cultured at severe hypoxia condition（PO2=0 mmHg）compared with those cultured at normoxia（PO2=144 mmHg）.In the normoxia group,nutlin-3 increased UNC5B（p＜0.01,n=5）mRNA expression,while PFT-a had no effect on it.In hypoxia groups,PFT-a increased UNC5B（p＜0.01,n=5） mRNA expression,while nutlin-3 reduced it（p＜0.01,n=5）.②MTT cell assays measured cell viability.Using MTT analysis,we observed a much lower survival rate （75.0±2.8%）in neurons exposed to severe hypoxia（PO₂=0mmHg）compared with those exposed to normoxia（p＜0.01,n=7）.In normoxia,neither PFT-a nor nutlin-3 had a significant effect on cell viability（p＞0.05,n=5）.In anoxia,however,PFT-a increased the survival rate of neurons（85.2±5.6%）（p＜0.01,n=6）.At the same time, nutlin-3 reduced the survival rate of neurons（57.1±4.1%）（p＜0.01,n= 7）.③Immunofluorescence of UNC5B was performed to assess its protein expression in all groups.UNC5B was significantly reduced in neurons exposed to severe hypoxia（PO2 =0mmHg）（104.078±19.058,n =98）compared with those exposed to normoxia（129.959±21.633,n= 122）（p=0.000＜0.01,n=7）.In normoxia,nutlin-3 （150.014±26.303,n=123）increased the expression of UNC5B（p=0.000＜0.01）, while PFT-a（124.072±28.016,n=106）had no effect on it（p=0.709＞0.05）.In anoxia,however,nutlin-3（95.244±12.354,n=108）decreased the expression of UNC5B on neurons（p=0.002＜0.01）.At the same time, PFT-a（104.541±19.080,n=104）had no effect on it as it acted in normoxia（p=1.000） （F=100.675）.Conclusions1.The oxygenation during dissection of hippocampal tissue was beneficial to the growth of neurons,and the hypoxia tolerance of this kind of neuron was better than the one in 1 day old group.2.The result suggested that the activation of K_ATPchannels can protially reduce cell death during hypoxia.And we also proved that K_ATPinfluenced transactivation of p53 tumor suppressor.3.We present the first analysis of p53-dependent gene expression patterns in hypoxia-treated Hippocampal neurons.We have found that hypoxia induces gene Bax that were previously not known to be regulated in a p53-dependent manner.Moreover, we found that the membrane receptor UNC5B which is transactivated by p53 under other stress is downgulated by p53 in response to hypoxia.更多还原