不同程度脑外伤后Bcl-2、Bax、Caspase-1、Caspase-3,NF-κB表达变化与细胞凋亡及其相互关系的实
发布时间：2019-06-05 23:42
【摘要】： 前言 脑外伤(traumatic brain injury,TBI)是暴力因素所致各器官损伤中引起死亡的主要原因之一，在暴力死中占首要位置。脑外伤后并发一系列的病理、生理、生化方面的改变，统称为继发性改变，如蛛网膜下腔出血、脑血管痉挛、颅内血肿、脑水肿、脑循环障碍、脑脊液的分泌吸收障碍、脑代谢障碍、内分泌障碍及脑缺血损害等，造成脑的二次损伤和多次损伤。脑外伤后脑组织发生的这些改变由许多基因、细胞因子参与调节并影响着损伤程度，除脑水肿外，脑损伤程度主要取决于细胞因子，这些因子包括肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)，B细胞淋巴瘤／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，它们都与脑损伤后的神经细胞死亡有关，控制着引起细胞死亡的不同层面。 程序性细胞死亡(programmed cell death,PCD)参与了神经细胞损伤的发病机制。细胞进行PCD通常经过一个严格的形态学变化被称之为凋亡(apoptosis)。控制细胞凋亡的基因包括Bcl-2等位基因家族和促进细胞凋亡的蛋白激酶caspase家族。Bcl-2家族即促进(如Bax)又阻止(如Bcl-2和bcl-xl)细胞凋亡，两组成员的相互作用决定细胞是否进行PCD。细胞核因子κB(nuclear factor-κB,NF-κB)是一类普遍存在的细胞转录因子，功能性NF-κB复合物存在于神经系统的各种类型的细胞中，包括神经元、星形胶质细胞、小胶质细胞、少突胶质细胞中，在神经细胞变性和凋亡过程中起十分重要的作用。 TBI的形成机制、损伤形成时间、损伤程度分析以及预后判断一直受到法医及临床工作者的重视，，其中损伤程度的判定成为实际案例中判定案件性质及量刑的主要依据，成为法医病理学工作者亟待解决的一个重要的科研课题。大量实验证明TBI后的严重损害是由于继发损伤造成的，因此，继发性损害程度影响着TBI的预后。然而原发性损伤程度和继发性损伤程度之间的关系，细胞凋亡与损伤程度的关系，对调节继发性损伤的基因和细胞因子的检测能否用来判定脑损伤程度和判断预后等研究尚未见报道。 本研究拟建立不同程度的脑外伤动物模型，应用HE染色和免疫组织 化学染色方法，从形态学上研究不同程度脑外伤的损伤分布以及Bcl一2、 Bax、caspase一1、caspase一3和NF一KB这些凋亡相关因子表达的变化和阳 性细胞分布与损伤分布的关系，同时应用细胞凋亡的形态学检测方法(末 端脱氧核昔酸转移酶介导的生物素脱氧尿嗜吮核昔酸缺口末端标记法; Terminal deoxynueleoti勿1 transferase一mediated biotinylated dUTP niek一end 协eling，TuNEL)研究凋亡细胞的时空分布与脑外伤程度以及上述细胞因 子分布和表达变化之间的关系;应用Westem blot方法检测Bcl一2、Bax、 casPase一1、casPase一3和NF一KB激活和酶原裂解情况，从蛋白质水平研 究脑外伤程度及经过时间与上述活性细胞因子变化的关系;应用RT一PcR 方法研究Bel一2、Bax、easpase一l、easpase一3和NF一KB mRNA表达变化 与脑外伤程度和经过时间的关系。以期为判定脑损伤程度、形成时间和临 床治疗TBI病人提供一定的实验性理论依据，并从分子水平研究脑损伤程 度、继发性脑损伤、细胞凋亡、细胞凋亡相关因子之间相互关系的可能机制。 实验材料与方法 实验材料 1.实验动物 健康Wistar大鼠54只，雌雄不限，体重1 809一2809，由中国医科大学实 验动物中心提供。 2.主要试剂. 鼠抗人Bel一2(e一2)单克隆抗体(产品编号:s。一7352，Santa Cruz Bio- teChnolo盯U.s.A) 鼠抗人Bax(B一9)单克隆抗体(产品编号:sc一7450，Santa CruzBi。- technofogy U.S.A) 兔抗人caspase一lp10(M一20)多克隆抗体(产品编号:sc一514， Santa Cruz Bioteehnolo盯U.S.A) 鼠抗人caspase一3(cpp一32)单克隆抗体(产品编号:BA0588，武汉博 士德公司) 鼠抗人NF一KB颐5(F一6)单克隆抗体(产品编号:se一8008，Santa Co BIOteehn01o留U.S.A) 辣根过氧化物酶标记或碱性磷酸酶标记的羊抗鼠或羊抗兔艳G以及免 疫组织化学S一p试剂盒、DAB试剂盒均购于北京中山生物技术有限公司; 十二烷基磺酸钠(sodium dodeeyl sulfate，SDS)和丙烯酞胺(ae单amide standard，AS)购于法国Biomol公司;甲叉双丙烯酞胺购于美国Sino公司; Bel一2、Bax、eas衅e一1、easpase一3和NF一KB的mRNA引物均由北京奥 科生物工程公司合成;RNA裂解液购于华美生物工程公司;PCR试剂盒购 于日本TAKARA公司。Te丽耐deo材Inucleotidrl Transferase(TdT)、Tris、p 一琉基乙醇、四甲基乙二胺(N，N，N’，N’一tetr别rne吻l一e吻len。ai俪ne， ，rE MED)购于美国Promega公司;DIG一dUTp购于德国B .M公司;Anti- DIG一Biotin、Westem blot显色剂购于美国51目叮a公司。SABC和TUNEL染 色试剂盒购于中国BOS几R公司。标准参照蛋白、小牛血清白蛋白购于华 美公司 3.主要仪器 oly哪us显微镜(BX51，日本) 图像分析仪(Metamo印h/Dplo/BX51，美国/?
[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
本文编号：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
本文编号：2493902
本文链接：https://www.wllwen.com/shekelunwen/gongan/2493902.html
