硫化氢对新生大鼠HIBD神经细胞保护作用的研究

发布时间：2018-01-01 05:35

  本文关键词：硫化氢对新生大鼠HIBD神经细胞保护作用的研究　出处：《南方医科大学》2013年硕士论文　论文类型：学位论文

  更多相关文章： 硫化氢 缺氧缺血性脑损伤 神经元细胞凋亡 caspase-3

【摘要】：研究背景 新生儿缺氧缺血性脑损伤(Hypoxic-ischemic brain damage, HIBD)是导致新生儿死亡和神经系统永久性损伤的重要原因之一。将遗留脑性瘫痪、学习障碍、智力低下、癫痫、精神运动发育迟滞等神经系统后遗症。研究发现0.2～0.4%的足月儿在分娩中窒息,其中15-20%因继发新生儿缺氧缺血性脑损伤死亡,幸存者近25%存在神经系统障碍。随着新生儿重症监护室的建立及医疗技术水平的提高,中、重度新生儿窒息患儿的存活率显著提高,但存活儿中神经系统后遗症的发病率仍较高。因此深入探讨HIBD发病机制,寻找有效的治疗措施提局HIBD的治愈率和降低神经系统后遗症有重要意义。 HIBD发病机制复杂,其核心是因为缺氧所致,主要病变部位为皮质、海马、基底节、丘脑等部位,由多种机制共同导致的缺氧缺血性生化连锁反应包括：①缺氧早期脑内血液动力学改变。②脑细胞能量代谢的变化。③兴奋性氨基酸的神经毒性作用。④氧自由基的作用。⑤Ca2+内流与再灌注损伤。⑥NO、炎症因子及细胞因子的作用。⑦神经细胞的凋亡。实验证明神经元细胞的凋亡是缺氧缺血性脑损伤发病机制中重要作用之一。 硫化氢(H2S)是继一氧化氮(NO)和一氧化碳(CO)后第三种小分子量气体信号分子,对H2S最早的认识是作为有毒气体,但在90年代后期发现其生理浓度是一种新型气体信号分子,研究发现,内源性H2S广泛存在于哺乳动物的组织及器官中,在生理浓度时通过多种信号转导形式及调节方式发挥广泛病理生理作用。1996年Abe等首次证实内源性H2S是一种神经活性分子,参与神经调节和信号转导的过程。内源性H2S的生成受体内胱硫醚-p-合成酶(CBS)和胱硫醚-γ-裂解酶(CSE)两种关键酶的调节。CBS主要分布于神经系统,如海马、皮层、小脑、脑干等,而CSE则以神经系统外组织为主。在体内1/3以H2S气体形式存在,2/3以硫氢化钠(NaHS)形式存在,并在体内水解为Na+和HS-,而HS-在与H+结合成H2S。研究证明大鼠脑内内源性H2S的浓度可达50-160μmol/L,而鼠脑内游离的H2S生理浓度约14±3.0μmol/L,其多种生理功能：①选择性增强NMDA受体介导的兴奋性电流而加强神经反应效应。②调节突触的活动,影响海马长时程增强(LTP)。③通过下丘脑-垂体-肾上腺轴调节神经内分泌功能。④通过血管内皮细胞及平滑肌细胞调节大脑血供进而影响神经系统功能。⑤通过上调Y-氨基丁酸(GABA) B受体进而调节神经递质释放而调节兴奋性。⑥调节胞内外Ca2+平衡。⑦通过小胶质细胞及星形胶质细胞膜上CL-/HCO3-,Na+/H+离子通道调节细胞内PH值。⑧通过增强γ-谷氨酰胺半胱氨酸合成酶(γ-GCS)活力以提高胞内γ-谷氨酰胺半胱氨酸(γ-GC)含量,并同时加强半胱氨酸转运效率进而增加胞内谷胱甘肽(GSH)水平,经抗氧化应激而保护神经元细胞,⑨抗凋亡作用。而NO亦是气体信号分子,起着信号传导和神经递质的作用,适量的NO可调节血管张力、免疫功能、神经传导及攻击肿瘤细胞、杀死病原微生物等；过量的NO则具有神经毒性导致神经元细胞的凋亡。H2S与NO之间有相互作用,H2S可抑制NOS活性,减少NO生成,达到保护神经细胞作用。 生后7d新生SD大鼠大脑组织与新生儿脑发育相似,并且SD大鼠颈总动脉形成的Willis环和脑的血液供应与人类相似,而且在缺氧缺血条件后其血流动力学变化、能量代谢及继发性能量衰竭等病理生理学改变与新生儿缺氧缺血性脑损伤极其相似。加之大鼠模型制备相对简单并且死亡率相对低,因此目前广泛利用7d新生SD大鼠参照Rice方法制备新生鼠HIBD动物模型。任彩丽等发现在新生大鼠HIBD后脑皮质中H2S浓度成先升高后降低的动态变化。大量研究也证明H2S可减轻脑缺血-再灌注损伤而对神经细胞有保护作用。因此在此基础上,探索H2S对HIBD后神经元细胞凋亡的影响,并通过干预措施了解H2S是否会影响细胞凋亡,探索一种新的干预方法。 细胞凋亡是体内外因素触发细胞内预存的死亡程序而导致的细胞死亡的过程,是与坏死不同的另一种细胞死亡形式。细胞凋亡对确保机体正常发育、生长,及维持机体内环境稳定起着至关重要的作用,而凋亡失调也是导致许多疾病发病的机制之一并威胁着人类的健康。 Caspase家族始于线虫(C.elegans)细胞程序性死亡的研究,是存在于胞质结构上相关的半胱氨酸蛋白酶,其共同点是特异断裂天冬氨酸残基后肽键。Caspase家族中caspase-1,4,11等主要参与前体的活化；Caspase-2,8,9,10参与细胞凋亡起始；caspase-3,6,7参与细胞凋亡的执行。Caspase-3是1996年命名,是细胞凋亡过程中关键的终末剪切酶,也是CTL细胞杀伤机制中重要部分之一。Caspase-3是多种细胞凋亡途径的下游效应部分的交汇点介导死亡受体作用,是凋亡蛋白级联反应的必经之路。 本研究构建新生大鼠HIBD模型,观察大鼠内源性H2S和NO浓度变化及两者的相关性,并通过给予外源性H2S供体硫氢化钠(NaHS)和CBS抑制剂羟胺(HA)改变内源性H2S及NO浓度,观察HIBD后脑组织病理性改变,利用TUNEL法检测神经元细胞凋亡及免疫组织化学方法检测caspase-3表达的情况,探讨H2S对新生儿缺氧缺血性脑损伤神经元细胞的保护作用。 具体研究内容包括以下两个部分： 第一部分新生大鼠HIBD后脑组织H2S与NO浓度的变化 目的 构建新生大鼠缺氧缺血性脑损伤(Hypoxic-ischemic brain damage, HIBD)模型后观察血清硫化氢(H2S)和脑组织H2S、NO浓度变化规律,并分析脑组织H2S与NO之间的相互作用。 方法 124只7日龄新生SD大鼠随机分为对照组(n=4)、假手术组(n=30)、HIBD组(n=30)、HIBD+硫氢化钠(NaHS)组(n=30)和HIBD+羟胺(HA)组(n=30),HIBD组参照Rice方法制备新生鼠HIBD模型,假手术组仅予分离左颈总动脉不予缺氧处理。HIBD+NaHS组予HIBD后30min给予NaHS(14μmol/kg), HIBD+HA组予HIBD后30min给予HA (12.5mg/kg),用生理盐水稀释后腹腔注射。分别于HIBD及干预后6h、12h、24h、48h及72h采血用生化反应方法检测新生大鼠血清H2S浓度,分离左侧大脑皮质分别检测脑组织H2S浓度及用硝酸还原酶法检测NO浓度,假手术组及对照组检测措施与HIBD组相同。分析HIBD后H2S和NO的动态变化规律,并进行H2S和NO相关性分析。结果 HIBD后6h新生大鼠血清和脑组织H2S显著升高,12h达到高峰,后逐渐降低；脑组织NO于6h后逐渐上升。给予H2S供体NaHS后血清和脑组织内源性H2S在各时间点上较HIBD组明显升高,相反NO则下降；而给予酶抑制剂后内源性H2S明显降低,而NO则升高。血清H2S同一时间点中72h假手术组与HA组差异无统计学意义(P0.05)。HIBD组、NaHS组及HA组中6h与24h,HIBD组48h与72h,NaHS组24h与48h均无统计学意义(P0.05)；脑组织H2S同时间点四组均有统计学差异(P0.05)。HIBD组6h与12h、12h与其他四个时间点有统计学差异(P0.05), NaHS组6h与24h,48h与72h,HA组6h与48h、72h,12h与24h均无统计学差异(P0.05)；脑组织NO6h时假手术组与NaHS组无统计学差异(P0.05)。NaHS组中6h与12h无统计学差异(P0.05)。HIBD组、NaHS组脑组织H2S与NO负相关(r值分别为-0.537、-0.838,P均0.05)。 结论 H2S可能参与了HIBD新生大鼠的病理生理过程。给予NaHS和HA后内源性H2S和NO的动态变化,提示外源性干预能改变HIBD新生大鼠内源性H2S和NO浓度,为临床提供一新的治疗方法。 第二部分硫化氢对新生大鼠HIBD后神经细胞凋亡的影响 目的 探讨H2S对新生大鼠HIBD的脑神经细胞凋亡的影响。 方法 124只7日龄新生SD大鼠随机分为对照组(n=4)、假手术组(n=30)、HIBD组(n=30)、HIBD+NaHS组(n=30)和HIBD+HA组(n=30),分别于HIBD及干预后6h、12h、24h、48h及72h取大脑皮质、海马观察HE染色脑组织病理改变,检测神经细胞凋亡指数及caspase-3表达的平均光密度。 结果 HE染色提示假手术组细胞排列整齐,形体基本正常,核仁清楚,无神经细胞缺失；HIBD组左侧脑组织细胞变性,排列紊乱,神经元细胞明显减少,可见变性坏死的神经元的形成；NaHS组变性程度较HIBD组减轻,变性坏死的神经元减少；HA组与HIBD组无明显区别。假手术组大脑海马和皮层可见少量凋亡阳性细胞。HIBD组损伤侧凋亡细胞6h开始逐渐升高,至48h达到高峰后逐渐降低。NaHS组亦6h开始逐渐升高,48h至高峰后降低,但各时间点凋亡细胞较HIBD组明显减少。HA组凋亡细胞亦呈现逐渐升高至48h后降低趋势,较HIBD组则显著增加。HIBD组大鼠左侧脑海马和皮层区内可见较多免疫反应阳性细胞caspase-3表达,6h开始增多,12-24h逐渐升高,至48h后达到高峰分布密集后逐渐降低。HIBD+NaHS组左侧脑海马区阳性细胞亦6h开始逐渐升高,但12h稍有下降,24h时再次上升至48h达到最高峰,72h阳性细胞降低,较HIBD组减少,染色亦变浅。HA组海马和皮层区阳性细胞亦呈现先上升后降低趋势,与HIBD组比较明显增多,染色加深。神经细胞凋亡指数,海马区6h、12h、24h、48h假手术组与NaHS组差异无统计学意义(P0.05),海马中NaHS组6h分别与12h、24h、72h,12h分别与24h、72h,24h与72h差异无统计学意义(P0.05)；皮层的HIBD组24h与72h,NaHS组6h与24h及24h与72h,HA组12h与24h、24h与72h及48h与72h差异无统计学意义(P0.05)。Caspase-3阳性表达,海马中同一时间点上6h假手术组与NaHS组及HIBD组与NaHS组,12h假手术组与NaHS组差异无统计学意义(P0.05)；海马中HIBD组6h与12h,12分别与24h、72h及24h与72h,NaHS组6h分别与12h、24h、72h及24h分别与48h、72h, HA组6h与12h,12h与24h,24h与72h及48h与72h差异无统计学意义(P0.05)。皮层的6h假手术组分别与HIBD组、NaHS组及HIBD组分别与NaHS组、HA组,12h、24h和72h假手术组与NaHS组,24h、72hHIBD组与HA组差异无统计学意义(P0.05)；皮层中HIBD组6h与12h,12h与24h,24h分别与48h、72h及48与72h,NaHS组6h与12h,24h与72h及48h与72h,HA组12h分别与24h、72h,24h与72h及48h与72h差异无统计学意义(P0.05)。 结论 H2S参与HIBD后神经元细胞凋亡过程,给予外源性H2S供体NaHS后可以抑制神经元细胞的凋亡,而给予CBS抑制剂HA后则促进神经元细胞的凋亡。
[Abstract]:Research background
Neonatal hypoxic ischemic brain damage (Hypoxic-ischemic brain, damage, HIBD) is one of the important causes of neonatal death and permanent damage to the nervous system. The left cerebral palsy, learning disabilities, mental retardation, epilepsy, psychomotor retardation and other sequelae of nervous system development. The study found that 0.2 ~ 0.4% full-term infants during birth asphyxia, including 15-20% because of secondary neonatal hypoxic ischemic brain injury, there are nearly 25% survivors of nervous system disorders. As the level of medical technology and the establishment of NICU increased in severe neonatal asphyxia, the survival rate increased significantly, but the sequelae of nervous system work in the incidence rate is still high. Therefore, in-depth study of the pathogenesis of HIBD and to find effective treatment measures to improve the cure rate of HIBD and have important significance to reduce neurological sequelae.
The pathogenesis of HIBD is complex, its core is caused by lack of oxygen, the main lesion site of cortex, hippocampus, basal ganglia and thalamus and other parts, caused by multiple mechanisms of hypoxic ischemic brain biochemical chain reaction include: early hemodynamic changes of hypoxia. The change of energy metabolism of brain cells. The neurotoxicity of excitatory amino acid. 4. The role of oxygen free radicals. The internal flow of Ca2+ and reperfusion injury, NO, inflammatory factors and cytokines. The apoptosis of neural cells. Experiments show that neuronal apoptosis is one of the important role in the pathogenesis of hypoxic ischemic brain damage.
Hydrogen sulfide (H2S) is the second nitric oxide (NO) and carbon monoxide (CO) after third kinds of small molecular gas signal molecule, H2S is the first to recognize as a toxic gas, but in late 90s found that the physiological concentration is a new gaseous signal molecule, the study found that tissues and organs of endogenous H2S exist in in mammals, play an important pathophysiological role of.1996 Abe for the first time that H2S is a kind of endogenous neural active molecules form a variety of signal transduction and regulation by physiological concentrations, participate in the process of neural regulation and signal transduction. The generation of endogenous H2S receptor in cystathionine synthase -p- (CBS) and cystathionine gamma lyase (CSE) are two key enzymes regulating.CBS mainly distributed in the nervous system, such as the hippocampus, cortex, cerebellum, brainstem, and CSE in the nervous system. In H2S gas organization form in the body 1/3, 2/3 Sodium hydrosulfide (NaHS) form, and in vivo hydrolysis of Na+ and HS-, and HS- in combination with H+ H2S. of high concentration in the rat brain of endogenous H2S 50-160 mol/L, H2S and physiological concentrations in rat brain free about 14 + 3 mol/L and its physiological functions: 1. Selective enhanced excitatory currents mediated by NMDA receptors and strengthen the neural response effect. The regulation of synaptic activity, affect hippocampal long-term potentiation (LTP). Through the hypothalamic pituitary adrenal axis and neuroendocrine function. For the influence function of the nervous system through blood brain vascular endothelial cells and vascular smooth muscle cells 5. Through upregulation of Y- amino butyric acid (GABA) and B receptors modulate neurotransmitter release and excitability regulation. The regulation of intracellular Ca2+. The balance by microglia and astrocyte cell membrane CL-/HCO3-, Na+/H+ ion channel regulation of intracellular P The value of H. Through the enhancement of gamma glutamyl cysteine synthetase (gamma -GCS) to improve the activity of intracellular gamma glutamyl cysteine (gamma -GC) content, and at the same time to strengthen the transport efficiency and increase the intracellular cysteine glutathione (GSH) level, the oxidative stress and protect neuronal cells, to anti apoptosis and NO. Is the gas signal molecule, signal transduction and plays the role of neurotransmitters, the amount of NO can regulate vascular tone, immune function, nerve conduction and attack tumor cells and kill the pathogenic microorganisms; excessive NO is neurotoxic leads to the interaction between.H2S and NO neurons apoptosis, H2S can inhibit the activity of NOS reduce the production of NO, to protect the nerve cells.
After 7d SD neonatal brain tissue with neonatal rat brain development, and SD in rat carotid artery to form Willis ring and cerebral blood supply and humans, and in the condition of hypoxia ischemia after the hemodynamic changes, energy metabolism and secondary energy failure pathophysiology and neonatal hypoxic ischemic brain damage very similar. And the preparation of rat model is relatively simple and the mortality rate is relatively low, so the current widespread use of 7D newborn SD rats by neonatal rat HIBD animal model by Rice method. Ren Caili found in the neonatal rat cerebral cortex HIBD H2S concentration changes increased firstly and then decreased. A lot of research has proved that H2S can relieve the cerebral ischemia reperfusion injury and has protective effect on nerve cells. Therefore, on this basis, to explore the influence of H2S on neuronal apoptosis after HIBD, and through the intervention measures about H2S Whether it will affect cell apoptosis and explore a new method of intervention.
Apoptosis is a process of in vivo factors to trigger cell death program stored in caused cell death, is a form of cell death and necrosis in different cell apoptosis. The growth of the body, and to ensure the normal development, maintain homeostasis plays a vital role, but also lead to apoptosis mechanism of many diseases the incidence of one threat to human health.
The Caspase family in nematodes (C.elegans) of the cell death program is present in the cytoplasm of structurally related cysteine protease, their common point is the activation of specific fracture aspartic acid residues after caspase-1,4,11 peptide in the.Caspase family are mainly involved in the body; Caspase-2,8,9,10 involved in apoptosis initiation; caspase-3,6,7 involved in the execution of apoptosis.Caspase-3 1996 is named, is the end of shear key enzyme in the process of cell apoptosis, CTL cell killing is one of the most important part in.Caspase-3 mechanism is a variety of apoptotic pathways downstream part of the intersection effect mediated by death receptor, apoptosis protein cascade is the only way which must be passed.
The construction of HIBD model of neonatal rats, rats to observe the correlation between the endogenous H2S and NO concentration and the two, and by the exogenous H2S donor sodium hydrosulfide (NaHS) and CBS inhibitor hydroxylamine (HA) changes of endogenous H2S and NO concentration of HIBD in brain tissue, observe pathological change detected by TUNEL neurons cell apoptosis and immunohistochemistry to detect the expression of Caspase-3, to investigate the protective effect of H2S on neurons of neonatal hypoxic-ischemic brain injury.
The specific research contents include the following two parts:
Changes in the concentration of H2S and NO in the HIBD posterior brain tissue of the first part of the neonatal rats
objective
After constructing the model of Hypoxic-ischemic brain damage (HIBD) in neonatal rats, we observed the change rule of serum hydrogen sulfide (H2S) and H2S and NO concentration in brain tissue, and analyzed the interaction between H2S and NO in brain tissue.
Method
124 7 day old SD rats were randomly divided into control group (n=4), sham operation group (n=30), HIBD group (n=30 HIBD+), sodium hydrosulfide (NaHS) group (n=30) and HIBD+ hydroxylamine (HA) group (n=30), HIBD group according to Rice preparation method of neonatal rat HIBD model the sham operation group, only to separate the left common carotid artery without.HIBD+NaHS hypoxia group HIBD 30min after administration of NaHS (14 mol/kg), HIBD+HA group, HIBD 30min HA (12.5mg/kg), with the injection of saline diluted peritoneal cavity. After the intervention of 6h and HIBD respectively, 12h, 24h, 48h and 72h the blood serum H2S concentration in neonatal rats by biochemical methods, the concentration of brain tissue H2S and NO concentrations were detected by nitrate reductase method was used to detect the separation of the left cerebral cortex of sham operation group and control group and HIBD group. The same detection measures analysis of dynamic changes of H2S and NO HIBD, and H2S and NO correlation analysis of the results.
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