不同程度腦外傷后Bcl-2、Bax、Caspase-1、Caspase-3,NF-κB表達(dá)變化與細(xì)胞凋亡及其相互關(guān)系的實(shí)
發(fā)布時間：2019-06-05 23:42
【摘要】： 前言 腦外傷(traumatic brain injury,TBI)是暴力因素所致各器官損傷中引起死亡的主要原因之一，在暴力死中占首要位置。腦外傷后并發(fā)一系列的病理、生理、生化方面的改變，統(tǒng)稱為繼發(fā)性改變，如蛛網(wǎng)膜下腔出血、腦血管痙攣、顱內(nèi)血腫、腦水腫、腦循環(huán)障礙、腦脊液的分泌吸收障礙、腦代謝障礙、內(nèi)分泌障礙及腦缺血損害等，造成腦的二次損傷和多次損傷。腦外傷后腦組織發(fā)生的這些改變由許多基因、細(xì)胞因子參與調(diào)節(jié)并影響著損傷程度，除腦水腫外，腦損傷程度主要取決于細(xì)胞因子，這些因子包括腫瘤壞死因子-α(tumor necrosis factor-α,TNF-α)，B細(xì)胞淋巴瘤／白血病-2(B-cell lymphoma／Leukemia-2,Bcl-2)基因中的Bcl-2、Bax，CED基因家族中的半胱天冬酶(cystein-dependent aspartate-specific protease,caspase)等，它們都與腦損傷后的神經(jīng)細(xì)胞死亡有關(guān)，控制著引起細(xì)胞死亡的不同層面。 程序性細(xì)胞死亡(programmed cell death,PCD)參與了神經(jīng)細(xì)胞損傷的發(fā)病機(jī)制。細(xì)胞進(jìn)行PCD通常經(jīng)過一個嚴(yán)格的形態(tài)學(xué)變化被稱之為凋亡(apoptosis)。控制細(xì)胞凋亡的基因包括Bcl-2等位基因家族和促進(jìn)細(xì)胞凋亡的蛋白激酶caspase家族。Bcl-2家族即促進(jìn)(如Bax)又阻止(如Bcl-2和bcl-xl)細(xì)胞凋亡，兩組成員的相互作用決定細(xì)胞是否進(jìn)行PCD。細(xì)胞核因子κB(nuclear factor-κB,NF-κB)是一類普遍存在的細(xì)胞轉(zhuǎn)錄因子，功能性NF-κB復(fù)合物存在于神經(jīng)系統(tǒng)的各種類型的細(xì)胞中，包括神經(jīng)元、星形膠質(zhì)細(xì)胞、小膠質(zhì)細(xì)胞、少突膠質(zhì)細(xì)胞中，在神經(jīng)細(xì)胞變性和凋亡過程中起十分重要的作用。 TBI的形成機(jī)制、損傷形成時間、損傷程度分析以及預(yù)后判斷一直受到法醫(yī)及臨床工作者的重視，，其中損傷程度的判定成為實(shí)際案例中判定案件性質(zhì)及量刑的主要依據(jù)，成為法醫(yī)病理學(xué)工作者亟待解決的一個重要的科研課題。大量實(shí)驗(yàn)證明TBI后的嚴(yán)重?fù)p害是由于繼發(fā)損傷造成的，因此，繼發(fā)性損害程度影響著TBI的預(yù)后。然而原發(fā)性損傷程度和繼發(fā)性損傷程度之間的關(guān)系，細(xì)胞凋亡與損傷程度的關(guān)系，對調(diào)節(jié)繼發(fā)性損傷的基因和細(xì)胞因子的檢測能否用來判定腦損傷程度和判斷預(yù)后等研究尚未見報道。 本研究擬建立不同程度的腦外傷動物模型，應(yīng)用HE染色和免疫組織 化學(xué)染色方法，從形態(tài)學(xué)上研究不同程度腦外傷的損傷分布以及Bcl一2、 Bax、caspase一1、caspase一3和NF一KB這些凋亡相關(guān)因子表達(dá)的變化和陽 性細(xì)胞分布與損傷分布的關(guān)系，同時應(yīng)用細(xì)胞凋亡的形態(tài)學(xué)檢測方法(末 端脫氧核昔酸轉(zhuǎn)移酶介導(dǎo)的生物素脫氧尿嗜吮核昔酸缺口末端標(biāo)記法; Terminal deoxynueleoti勿1 transferase一mediated biotinylated dUTP niek一end 協(xié)eling，TuNEL)研究凋亡細(xì)胞的時空分布與腦外傷程度以及上述細(xì)胞因 子分布和表達(dá)變化之間的關(guān)系;應(yīng)用Westem blot方法檢測Bcl一2、Bax、 casPase一1、casPase一3和NF一KB激活和酶原裂解情況，從蛋白質(zhì)水平研 究腦外傷程度及經(jīng)過時間與上述活性細(xì)胞因子變化的關(guān)系;應(yīng)用RT一PcR 方法研究Bel一2、Bax、easpase一l、easpase一3和NF一KB mRNA表達(dá)變化 與腦外傷程度和經(jīng)過時間的關(guān)系。以期為判定腦損傷程度、形成時間和臨 床治療TBI病人提供一定的實(shí)驗(yàn)性理論依據(jù)，并從分子水平研究腦損傷程 度、繼發(fā)性腦損傷、細(xì)胞凋亡、細(xì)胞凋亡相關(guān)因子之間相互關(guān)系的可能機(jī)制。 實(shí)驗(yàn)材料與方法 實(shí)驗(yàn)材料 1.實(shí)驗(yàn)動物 健康Wistar大鼠54只，雌雄不限，體重1 809一2809，由中國醫(yī)科大學(xué)實(shí) 驗(yàn)動物中心提供。 2.主要試劑. 鼠抗人Bel一2(e一2)單克隆抗體(產(chǎn)品編號:s。一7352，Santa Cruz Bio- teChnolo盯U.s.A) 鼠抗人Bax(B一9)單克隆抗體(產(chǎn)品編號:sc一7450，Santa CruzBi。- technofogy U.S.A) 兔抗人caspase一lp10(M一20)多克隆抗體(產(chǎn)品編號:sc一514， Santa Cruz Bioteehnolo盯U.S.A) 鼠抗人caspase一3(cpp一32)單克隆抗體(產(chǎn)品編號:BA0588，武漢博 士德公司) 鼠抗人NF一KB頤5(F一6)單克隆抗體(產(chǎn)品編號:se一8008，Santa Co BIOteehn01o留U.S.A) 辣根過氧化物酶標(biāo)記或堿性磷酸酶標(biāo)記的羊抗鼠或羊抗兔艷G以及免 疫組織化學(xué)S一p試劑盒、DAB試劑盒均購于北京中山生物技術(shù)有限公司; 十二烷基磺酸鈉(sodium dodeeyl sulfate，SDS)和丙烯酞胺(ae單amide standard，AS)購于法國Biomol公司;甲叉雙丙烯酞胺購于美國Sino公司; Bel一2、Bax、eas釁e一1、easpase一3和NF一KB的mRNA引物均由北京奧 科生物工程公司合成;RNA裂解液購于華美生物工程公司;PCR試劑盒購 于日本TAKARA公司。Te麗耐deo材Inucleotidrl Transferase(TdT)、Tris、p 一琉基乙醇、四甲基乙二胺(N，N，N’，N’一tetr別rne吻l一e吻len。ai儷ne， ，rE MED)購于美國Promega公司;DIG一dUTp購于德國B .M公司;Anti- DIG一Biotin、Westem blot顯色劑購于美國51目叮a公司。SABC和TUNEL染 色試劑盒購于中國BOS幾R公司。標(biāo)準(zhǔn)參照蛋白、小牛血清白蛋白購于華 美公司 3.主要儀器 oly哪us顯微鏡(BX51，日本) 圖像分析儀(Metamo印h/Dplo/BX51，美國/?
[Abstract]:foreword Brain trauma (TBI) is one of the main causes of death in various organ damage caused by violent factors. The first place of violent death. A series of pathological, physiological and biochemical changes after brain trauma are collectively referred to as secondary changes, such as subarachnoid hemorrhage, cerebral vasospasm, intracranial hematoma, cerebral edema, cerebral circulation disorder, and secretion of cerebrospinal fluid. The brain is caused by the obstruction of the brain, the metabolic disorder of the brain, the endocrine disturbance, the cerebral ischemia and the like. Secondary injury and multiple injury. These changes in brain tissue after brain trauma are regulated by many genes and cytokines and affect the degree of injury. In addition to brain edema, the degree of brain injury is mainly dependent on the cytokines, which include tumor necrosis factor-necrosis factor. Bcl-2, Bax, cystein-dependent caspase-specific protein (caspase) in the family of B-cell lymphoma/ Leukebia-2 and Bcl-2, which are related to the death of the nerve cells after brain injury, which are controlled by the control Different levels of cell death. programmed cell death PCD) is involved in the pathogenesis of nerve cell injury. PCD usually has a strict morphology. The change is called apotosis. The genes that control the apoptosis of the cells include the Bcl-2 allele family and the promoter. The expression of Bcl-2 and Bcl-2, such as Bcl-2 and bcl-xl, and the apoptosis of Bcl-2 and bcl-xl cells. The interaction of group members determines whether the cells are PCD. The nuclear factor B (nullar factor-B, NF-B) is a common type of cell transcription factor, and the functional NF-B complex is present in various types of cells of the nervous system, including neurons, stars, Glial, microglia, oligodendrocytes, in God. It plays an important role in the process of cell degeneration and apoptosis. The formation mechanism of TBI, the formation time, the degree of damage and the prognosis of TBI have been paid more and more attention by the forensic and clinical workers. The judgment of the degree of injury in the case is the judgment of the nature of the case and the sentencing in the actual case. The main basis is to be an important scientific research subject to be solved by the forensic pathologists. A large number of experiments prove that the serious damage after TBI is As a result of secondary injury, the degree of secondary damage affects the prognosis of the TBI. However, the relationship between the degree of primary injury and the degree of secondary injury, the relationship between the degree of apoptosis and the degree of injury, the genes and cells that regulate secondary injury, Whether the detection of the factor can be used to determine the degree of brain injury and to judge the prognosis has not been reported. The study is to establish a different degree of head injury Animal model with HE staining and immunity To study the different degree of the chemical staining of the tissue from the morphology. Injury distribution of head injury and Bcl-2, Bax, ca The apoptosis-related factors of spase-1, caspase-3 and NF-KB The relationship between the change of expression and the distribution of the positive cell and the distribution of the damage, and the morphological detection of the cell apoptosis (the end-to-end deoxy-celecoxib) Enzyme-mediated deoxidization of the end-to-end labeling method of the deoxy-urophilic nucleicexic acid; Term inal deoxynueleoti鍕
本文編號：2493902
[Abstract]:foreword Brain trauma (TBI) is one of the main causes of death in various organ damage caused by violent factors. The first place of violent death. A series of pathological, physiological and biochemical changes after brain trauma are collectively referred to as secondary changes, such as subarachnoid hemorrhage, cerebral vasospasm, intracranial hematoma, cerebral edema, cerebral circulation disorder, and secretion of cerebrospinal fluid. The brain is caused by the obstruction of the brain, the metabolic disorder of the brain, the endocrine disturbance, the cerebral ischemia and the like. Secondary injury and multiple injury. These changes in brain tissue after brain trauma are regulated by many genes and cytokines and affect the degree of injury. In addition to brain edema, the degree of brain injury is mainly dependent on the cytokines, which include tumor necrosis factor-necrosis factor. Bcl-2, Bax, cystein-dependent caspase-specific protein (caspase) in the family of B-cell lymphoma/ Leukebia-2 and Bcl-2, which are related to the death of the nerve cells after brain injury, which are controlled by the control Different levels of cell death. programmed cell death PCD) is involved in the pathogenesis of nerve cell injury. PCD usually has a strict morphology. The change is called apotosis. The genes that control the apoptosis of the cells include the Bcl-2 allele family and the promoter. The expression of Bcl-2 and Bcl-2, such as Bcl-2 and bcl-xl, and the apoptosis of Bcl-2 and bcl-xl cells. The interaction of group members determines whether the cells are PCD. The nuclear factor B (nullar factor-B, NF-B) is a common type of cell transcription factor, and the functional NF-B complex is present in various types of cells of the nervous system, including neurons, stars, Glial, microglia, oligodendrocytes, in God. It plays an important role in the process of cell degeneration and apoptosis. The formation mechanism of TBI, the formation time, the degree of damage and the prognosis of TBI have been paid more and more attention by the forensic and clinical workers. The judgment of the degree of injury in the case is the judgment of the nature of the case and the sentencing in the actual case. The main basis is to be an important scientific research subject to be solved by the forensic pathologists. A large number of experiments prove that the serious damage after TBI is As a result of secondary injury, the degree of secondary damage affects the prognosis of the TBI. However, the relationship between the degree of primary injury and the degree of secondary injury, the relationship between the degree of apoptosis and the degree of injury, the genes and cells that regulate secondary injury, Whether the detection of the factor can be used to determine the degree of brain injury and to judge the prognosis has not been reported. The study is to establish a different degree of head injury Animal model with HE staining and immunity To study the different degree of the chemical staining of the tissue from the morphology. Injury distribution of head injury and Bcl-2, Bax, ca The apoptosis-related factors of spase-1, caspase-3 and NF-KB The relationship between the change of expression and the distribution of the positive cell and the distribution of the damage, and the morphological detection of the cell apoptosis (the end-to-end deoxy-celecoxib) Enzyme-mediated deoxidization of the end-to-end labeling method of the deoxy-urophilic nucleicexic acid; Term inal deoxynueleoti鍕
本文編號：2493902
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