本文關(guān)鍵詞：硫化氫對新生大鼠HIBD神經(jīng)細(xì)胞保護(hù)作用的研究　出處：《南方醫(yī)科大學(xué)》2013年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 硫化氫 缺氧缺血性腦損傷 神經(jīng)元細(xì)胞凋亡 caspase-3

【摘要】：研究背景 新生兒缺氧缺血性腦損傷(Hypoxic-ischemic brain damage, HIBD)是導(dǎo)致新生兒死亡和神經(jīng)系統(tǒng)永久性損傷的重要原因之一。將遺留腦性癱瘓、學(xué)習(xí)障礙、智力低下、癲癇、精神運(yùn)動(dòng)發(fā)育遲滯等神經(jīng)系統(tǒng)后遺癥。研究發(fā)現(xiàn)0.2～0.4%的足月兒在分娩中窒息,其中15-20%因繼發(fā)新生兒缺氧缺血性腦損傷死亡,幸存者近25%存在神經(jīng)系統(tǒng)障礙。隨著新生兒重癥監(jiān)護(hù)室的建立及醫(yī)療技術(shù)水平的提高,中、重度新生兒窒息患兒的存活率顯著提高,但存活兒中神經(jīng)系統(tǒng)后遺癥的發(fā)病率仍較高。因此深入探討HIBD發(fā)病機(jī)制,尋找有效的治療措施提局HIBD的治愈率和降低神經(jīng)系統(tǒng)后遺癥有重要意義。 HIBD發(fā)病機(jī)制復(fù)雜,其核心是因?yàn)槿毖跛?主要病變部位為皮質(zhì)、海馬、基底節(jié)、丘腦等部位,由多種機(jī)制共同導(dǎo)致的缺氧缺血性生化連鎖反應(yīng)包括：①缺氧早期腦內(nèi)血液動(dòng)力學(xué)改變。②腦細(xì)胞能量代謝的變化。③興奮性氨基酸的神經(jīng)毒性作用。④氧自由基的作用。⑤Ca2+內(nèi)流與再灌注損傷。⑥NO、炎癥因子及細(xì)胞因子的作用。⑦神經(jīng)細(xì)胞的凋亡。實(shí)驗(yàn)證明神經(jīng)元細(xì)胞的凋亡是缺氧缺血性腦損傷發(fā)病機(jī)制中重要作用之一。 硫化氫(H2S)是繼一氧化氮(NO)和一氧化碳(CO)后第三種小分子量氣體信號(hào)分子,對H2S最早的認(rèn)識(shí)是作為有毒氣體,但在90年代后期發(fā)現(xiàn)其生理濃度是一種新型氣體信號(hào)分子,研究發(fā)現(xiàn),內(nèi)源性H2S廣泛存在于哺乳動(dòng)物的組織及器官中,在生理濃度時(shí)通過多種信號(hào)轉(zhuǎn)導(dǎo)形式及調(diào)節(jié)方式發(fā)揮廣泛病理生理作用。1996年Abe等首次證實(shí)內(nèi)源性H2S是一種神經(jīng)活性分子,參與神經(jīng)調(diào)節(jié)和信號(hào)轉(zhuǎn)導(dǎo)的過程。內(nèi)源性H2S的生成受體內(nèi)胱硫醚-p-合成酶(CBS)和胱硫醚-γ-裂解酶(CSE)兩種關(guān)鍵酶的調(diào)節(jié)。CBS主要分布于神經(jīng)系統(tǒng),如海馬、皮層、小腦、腦干等,而CSE則以神經(jīng)系統(tǒng)外組織為主。在體內(nèi)1/3以H2S氣體形式存在,2/3以硫氫化鈉(NaHS)形式存在,并在體內(nèi)水解為Na+和HS-,而HS-在與H+結(jié)合成H2S。研究證明大鼠腦內(nèi)內(nèi)源性H2S的濃度可達(dá)50-160μmol/L,而鼠腦內(nèi)游離的H2S生理濃度約14±3.0μmol/L,其多種生理功能：①選擇性增強(qiáng)NMDA受體介導(dǎo)的興奮性電流而加強(qiáng)神經(jīng)反應(yīng)效應(yīng)。②調(diào)節(jié)突觸的活動(dòng),影響海馬長時(shí)程增強(qiáng)(LTP)。③通過下丘腦-垂體-腎上腺軸調(diào)節(jié)神經(jīng)內(nèi)分泌功能。④通過血管內(nèi)皮細(xì)胞及平滑肌細(xì)胞調(diào)節(jié)大腦血供進(jìn)而影響神經(jīng)系統(tǒng)功能。⑤通過上調(diào)Y-氨基丁酸(GABA) B受體進(jìn)而調(diào)節(jié)神經(jīng)遞質(zhì)釋放而調(diào)節(jié)興奮性。⑥調(diào)節(jié)胞內(nèi)外Ca2+平衡。⑦通過小膠質(zhì)細(xì)胞及星形膠質(zhì)細(xì)胞膜上CL-/HCO3-,Na+/H+離子通道調(diào)節(jié)細(xì)胞內(nèi)PH值。⑧通過增強(qiáng)γ-谷氨酰胺半胱氨酸合成酶(γ-GCS)活力以提高胞內(nèi)γ-谷氨酰胺半胱氨酸(γ-GC)含量,并同時(shí)加強(qiáng)半胱氨酸轉(zhuǎn)運(yùn)效率進(jìn)而增加胞內(nèi)谷胱甘肽(GSH)水平,經(jīng)抗氧化應(yīng)激而保護(hù)神經(jīng)元細(xì)胞,⑨抗凋亡作用。而NO亦是氣體信號(hào)分子,起著信號(hào)傳導(dǎo)和神經(jīng)遞質(zhì)的作用,適量的NO可調(diào)節(jié)血管張力、免疫功能、神經(jīng)傳導(dǎo)及攻擊腫瘤細(xì)胞、殺死病原微生物等；過量的NO則具有神經(jīng)毒性導(dǎo)致神經(jīng)元細(xì)胞的凋亡。H2S與NO之間有相互作用,H2S可抑制NOS活性,減少NO生成,達(dá)到保護(hù)神經(jīng)細(xì)胞作用。 生后7d新生SD大鼠大腦組織與新生兒腦發(fā)育相似,并且SD大鼠頸總動(dòng)脈形成的Willis環(huán)和腦的血液供應(yīng)與人類相似,而且在缺氧缺血條件后其血流動(dòng)力學(xué)變化、能量代謝及繼發(fā)性能量衰竭等病理生理學(xué)改變與新生兒缺氧缺血性腦損傷極其相似。加之大鼠模型制備相對簡單并且死亡率相對低,因此目前廣泛利用7d新生SD大鼠參照Rice方法制備新生鼠HIBD動(dòng)物模型。任彩麗等發(fā)現(xiàn)在新生大鼠HIBD后腦皮質(zhì)中H2S濃度成先升高后降低的動(dòng)態(tài)變化。大量研究也證明H2S可減輕腦缺血-再灌注損傷而對神經(jīng)細(xì)胞有保護(hù)作用。因此在此基礎(chǔ)上,探索H2S對HIBD后神經(jīng)元細(xì)胞凋亡的影響,并通過干預(yù)措施了解H2S是否會(huì)影響細(xì)胞凋亡,探索一種新的干預(yù)方法。 細(xì)胞凋亡是體內(nèi)外因素觸發(fā)細(xì)胞內(nèi)預(yù)存的死亡程序而導(dǎo)致的細(xì)胞死亡的過程,是與壞死不同的另一種細(xì)胞死亡形式。細(xì)胞凋亡對確保機(jī)體正常發(fā)育、生長,及維持機(jī)體內(nèi)環(huán)境穩(wěn)定起著至關(guān)重要的作用,而凋亡失調(diào)也是導(dǎo)致許多疾病發(fā)病的機(jī)制之一并威脅著人類的健康。 Caspase家族始于線蟲(C.elegans)細(xì)胞程序性死亡的研究,是存在于胞質(zhì)結(jié)構(gòu)上相關(guān)的半胱氨酸蛋白酶,其共同點(diǎn)是特異斷裂天冬氨酸殘基后肽鍵。Caspase家族中caspase-1,4,11等主要參與前體的活化；Caspase-2,8,9,10參與細(xì)胞凋亡起始；caspase-3,6,7參與細(xì)胞凋亡的執(zhí)行。Caspase-3是1996年命名,是細(xì)胞凋亡過程中關(guān)鍵的終末剪切酶,也是CTL細(xì)胞殺傷機(jī)制中重要部分之一。Caspase-3是多種細(xì)胞凋亡途徑的下游效應(yīng)部分的交匯點(diǎn)介導(dǎo)死亡受體作用,是凋亡蛋白級聯(lián)反應(yīng)的必經(jīng)之路。 本研究構(gòu)建新生大鼠HIBD模型,觀察大鼠內(nèi)源性H2S和NO濃度變化及兩者的相關(guān)性,并通過給予外源性H2S供體硫氫化鈉(NaHS)和CBS抑制劑羥胺(HA)改變內(nèi)源性H2S及NO濃度,觀察HIBD后腦組織病理性改變,利用TUNEL法檢測神經(jīng)元細(xì)胞凋亡及免疫組織化學(xué)方法檢測caspase-3表達(dá)的情況,探討H2S對新生兒缺氧缺血性腦損傷神經(jīng)元細(xì)胞的保護(hù)作用。 具體研究內(nèi)容包括以下兩個(gè)部分： 第一部分新生大鼠HIBD后腦組織H2S與NO濃度的變化 目的 構(gòu)建新生大鼠缺氧缺血性腦損傷(Hypoxic-ischemic brain damage, HIBD)模型后觀察血清硫化氫(H2S)和腦組織H2S、NO濃度變化規(guī)律,并分析腦組織H2S與NO之間的相互作用。 方法 124只7日齡新生SD大鼠隨機(jī)分為對照組(n=4)、假手術(shù)組(n=30)、HIBD組(n=30)、HIBD+硫氫化鈉(NaHS)組(n=30)和HIBD+羥胺(HA)組(n=30),HIBD組參照Rice方法制備新生鼠HIBD模型,假手術(shù)組僅予分離左頸總動(dòng)脈不予缺氧處理。HIBD+NaHS組予HIBD后30min給予NaHS(14μmol/kg), HIBD+HA組予HIBD后30min給予HA (12.5mg/kg),用生理鹽水稀釋后腹腔注射。分別于HIBD及干預(yù)后6h、12h、24h、48h及72h采血用生化反應(yīng)方法檢測新生大鼠血清H2S濃度,分離左側(cè)大腦皮質(zhì)分別檢測腦組織H2S濃度及用硝酸還原酶法檢測NO濃度,假手術(shù)組及對照組檢測措施與HIBD組相同。分析HIBD后H2S和NO的動(dòng)態(tài)變化規(guī)律,并進(jìn)行H2S和NO相關(guān)性分析。結(jié)果 HIBD后6h新生大鼠血清和腦組織H2S顯著升高,12h達(dá)到高峰,后逐漸降低；腦組織NO于6h后逐漸上升。給予H2S供體NaHS后血清和腦組織內(nèi)源性H2S在各時(shí)間點(diǎn)上較HIBD組明顯升高,相反NO則下降；而給予酶抑制劑后內(nèi)源性H2S明顯降低,而NO則升高。血清H2S同一時(shí)間點(diǎn)中72h假手術(shù)組與HA組差異無統(tǒng)計(jì)學(xué)意義(P0.05)。HIBD組、NaHS組及HA組中6h與24h,HIBD組48h與72h,NaHS組24h與48h均無統(tǒng)計(jì)學(xué)意義(P0.05)；腦組織H2S同時(shí)間點(diǎn)四組均有統(tǒng)計(jì)學(xué)差異(P0.05)。HIBD組6h與12h、12h與其他四個(gè)時(shí)間點(diǎn)有統(tǒng)計(jì)學(xué)差異(P0.05), NaHS組6h與24h,48h與72h,HA組6h與48h、72h,12h與24h均無統(tǒng)計(jì)學(xué)差異(P0.05)；腦組織NO6h時(shí)假手術(shù)組與NaHS組無統(tǒng)計(jì)學(xué)差異(P0.05)。NaHS組中6h與12h無統(tǒng)計(jì)學(xué)差異(P0.05)。HIBD組、NaHS組腦組織H2S與NO負(fù)相關(guān)(r值分別為-0.537、-0.838,P均0.05)。 結(jié)論 H2S可能參與了HIBD新生大鼠的病理生理過程。給予NaHS和HA后內(nèi)源性H2S和NO的動(dòng)態(tài)變化,提示外源性干預(yù)能改變HIBD新生大鼠內(nèi)源性H2S和NO濃度,為臨床提供一新的治療方法。 第二部分硫化氫對新生大鼠HIBD后神經(jīng)細(xì)胞凋亡的影響 目的 探討H2S對新生大鼠HIBD的腦神經(jīng)細(xì)胞凋亡的影響。 方法 124只7日齡新生SD大鼠隨機(jī)分為對照組(n=4)、假手術(shù)組(n=30)、HIBD組(n=30)、HIBD+NaHS組(n=30)和HIBD+HA組(n=30),分別于HIBD及干預(yù)后6h、12h、24h、48h及72h取大腦皮質(zhì)、海馬觀察HE染色腦組織病理改變,檢測神經(jīng)細(xì)胞凋亡指數(shù)及caspase-3表達(dá)的平均光密度。 結(jié)果 HE染色提示假手術(shù)組細(xì)胞排列整齊,形體基本正常,核仁清楚,無神經(jīng)細(xì)胞缺失；HIBD組左側(cè)腦組織細(xì)胞變性,排列紊亂,神經(jīng)元細(xì)胞明顯減少,可見變性壞死的神經(jīng)元的形成；NaHS組變性程度較HIBD組減輕,變性壞死的神經(jīng)元減少；HA組與HIBD組無明顯區(qū)別。假手術(shù)組大腦海馬和皮層可見少量凋亡陽性細(xì)胞。HIBD組損傷側(cè)凋亡細(xì)胞6h開始逐漸升高,至48h達(dá)到高峰后逐漸降低。NaHS組亦6h開始逐漸升高,48h至高峰后降低,但各時(shí)間點(diǎn)凋亡細(xì)胞較HIBD組明顯減少。HA組凋亡細(xì)胞亦呈現(xiàn)逐漸升高至48h后降低趨勢,較HIBD組則顯著增加。HIBD組大鼠左側(cè)腦海馬和皮層區(qū)內(nèi)可見較多免疫反應(yīng)陽性細(xì)胞caspase-3表達(dá),6h開始增多,12-24h逐漸升高,至48h后達(dá)到高峰分布密集后逐漸降低。HIBD+NaHS組左側(cè)腦海馬區(qū)陽性細(xì)胞亦6h開始逐漸升高,但12h稍有下降,24h時(shí)再次上升至48h達(dá)到最高峰,72h陽性細(xì)胞降低,較HIBD組減少,染色亦變淺。HA組海馬和皮層區(qū)陽性細(xì)胞亦呈現(xiàn)先上升后降低趨勢,與HIBD組比較明顯增多,染色加深。神經(jīng)細(xì)胞凋亡指數(shù),海馬區(qū)6h、12h、24h、48h假手術(shù)組與NaHS組差異無統(tǒng)計(jì)學(xué)意義(P0.05),海馬中NaHS組6h分別與12h、24h、72h,12h分別與24h、72h,24h與72h差異無統(tǒng)計(jì)學(xué)意義(P0.05)；皮層的HIBD組24h與72h,NaHS組6h與24h及24h與72h,HA組12h與24h、24h與72h及48h與72h差異無統(tǒng)計(jì)學(xué)意義(P0.05)。Caspase-3陽性表達(dá),海馬中同一時(shí)間點(diǎn)上6h假手術(shù)組與NaHS組及HIBD組與NaHS組,12h假手術(shù)組與NaHS組差異無統(tǒng)計(jì)學(xué)意義(P0.05)；海馬中HIBD組6h與12h,12分別與24h、72h及24h與72h,NaHS組6h分別與12h、24h、72h及24h分別與48h、72h, HA組6h與12h,12h與24h,24h與72h及48h與72h差異無統(tǒng)計(jì)學(xué)意義(P0.05)。皮層的6h假手術(shù)組分別與HIBD組、NaHS組及HIBD組分別與NaHS組、HA組,12h、24h和72h假手術(shù)組與NaHS組,24h、72hHIBD組與HA組差異無統(tǒng)計(jì)學(xué)意義(P0.05)；皮層中HIBD組6h與12h,12h與24h,24h分別與48h、72h及48與72h,NaHS組6h與12h,24h與72h及48h與72h,HA組12h分別與24h、72h,24h與72h及48h與72h差異無統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論 H2S參與HIBD后神經(jīng)元細(xì)胞凋亡過程,給予外源性H2S供體NaHS后可以抑制神經(jīng)元細(xì)胞的凋亡,而給予CBS抑制劑HA后則促進(jìn)神經(jīng)元細(xì)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.
HIBD鍚，

本文編號(hào)：1363170