本文選題：腦缺血 + 硫化氫��； 參考：《河北醫(yī)科大學》2014年碩士論文

【摘要】：目的：硫化氫(hydrogen sulfide, H2S)是有著“一種臭雞蛋氣味的氣體”,長期接觸這種有害氣體,將導致神經(jīng)系統(tǒng)、呼吸系統(tǒng)以及消化系統(tǒng)等許多組織器官的損害。但是有大量資料證明,H2S不只有毒性作用,而且還廣泛參與機體多種生理及病理的過程,被認為是繼一氧化氮(NO)和一氧化碳(CO)之后的第三類氣體信號分子。正常動物體內(nèi)可以合成H2S,參與內(nèi)源性H2S合成的兩種重要的酶是胱硫醚β-合酶(cystathionine beta-synthase,CBS)和胱硫醚Y-裂解酶(cystathionine γ lyase, CSE)。其中CBS主要分布于中樞神經(jīng)系統(tǒng),而CSE則主要分布于神經(jīng)系統(tǒng)以外的組織。近幾年的研究表明,H2S可以通過抗氧化、抑制凋亡等發(fā)揮抗腦、心肌、肝及腎等多器官組織缺血再灌注損傷作用。炎癥在腦缺血再灌注損傷發(fā)生發(fā)展中發(fā)揮重要作用。本實驗采用血管性癡呆(Vascular Dementia, VD)大鼠模型,通過腹腔給藥的方式,研究不同劑量硫化氫供體NaHHS及H2S合成酶抑制劑羥氨(hydroxylamine, HA)和炔丙基甘氨酸(propargylglycine, PAG),對建立大鼠腦缺血再灌注模型后的海馬CA1區(qū)錐體神經(jīng)元損傷和腦組織炎癥變化的影響,并探討其炎癥反應機制。采用病理切片和HE染色的方法觀察各組大鼠海馬CA1區(qū)細胞形態(tài)的變化；免疫組化和Westen blot方法檢測大鼠腦缺血再灌注中腦組織H2S生成酶CBS表達變化；ELISA方法檢測H2S對腦缺血再灌注中炎癥介質(zhì)的影響。為了證明H2S通過抗炎發(fā)揮抗腦缺血再灌注損傷作用。因此,本實驗旨在從神經(jīng)元、炎性因子、內(nèi)源性H2S等方面,證明H2S的抗腦缺血再灌注損傷作用,并為其提供理論依據(jù)。方法：選用純系健康雄性Wistar大鼠,體重310g左右,將動物隨機分為9組,正常組(Normal group),假手術組(sham),腦缺血再灌注組(ischemia-reperfusion injury:IR),硫化氫治療組(IR+NaHS)根據(jù)注射劑量進一步分為：2mg/kg,5mg/kg,10mg/kg,20mg/kg4個亞組,硫化氫羥氨抑制組(IR+NaHS+HA),硫化氫炔丙基甘氨酸抑制組(IR+NaHS+PAG)。每組5只動物,采用四血管阻斷法(4-VO)制作VD大鼠模型,假手術組麻醉其手術過程與VD模型組相同,但不電凝雙側椎動脈,不阻斷雙側頸總動脈。實驗結束后取大鼠腦組織,制作大腦切片；HE染色,采用光鏡觀察各組大鼠海馬CA1區(qū)的形態(tài)學變化；采用免疫組化的方法檢測CBS表達情況；取雙側海馬組織并勻漿,酶聯(lián)免疫吸附法(ELISA)測定IL-1β, TNF-α;采用Western blot方法檢測各組大鼠海馬組織CBS表達情況。結果：1.形態(tài)學：Normal組和Sham組大鼠海馬CA1區(qū)錐體細胞排列整齊、致密,細胞形態(tài)完整,胞核飽滿,核仁清晰,無明顯的細胞損傷。IR組大鼠出現(xiàn)了明顯的細胞損傷,海馬CA1區(qū)錐體細胞稀疏,排列紊亂；核仁消失；硫化氫治療組中,2,5 mg/kg劑量組海馬CA1區(qū)組織形態(tài)與IR組基本一致；10 mg/kg組與IR組相比,神經(jīng)元數(shù)量有所增加,可見不完整的錐體細胞層；20 mg/kg組神經(jīng)元存活狀態(tài)明顯改善,組織學特征與Normal組和Sham組類似,神經(jīng)元排列比較整齊,細胞形態(tài)也比較完整。IR+NaHS+HA組和IR+NaHS+PAG組與硫化氫治療組20mg/kg相比出現(xiàn)了明顯的細胞損傷。2蛋白CBS的表達：免疫組化結果顯示,Normal組、Sham組大鼠海馬CA1區(qū)可見到一定量CBS陽性免疫顆粒,呈棕黃色,染色較淺,均勻分布。與Sham組相比,IR組大鼠海馬CA1的CBS陽性免疫顆粒顯著減少,甚至在錐體細胞層及其周圍出現(xiàn)大片CBS表達缺失區(qū)域。與IR組相比,低劑量硫化氫(2、5mg/kg)海馬CA1區(qū)CBS的免疫陽性染色強度均無明顯變化,隨著硫化氫的劑量加大免疫染色陽性隨之增強。硫化氫大劑量(20mg/kg)組大鼠可見大量棕褐色CBS陽性免疫顆粒分布于海馬CA1區(qū),尤其在海馬錐體細胞層可見大量、深染的CBS陽性顆粒包繞錐體神經(jīng)元細胞。IR+NaHS+HA組和 IR+NaHS+PAG組與硫化氫治療組20mg/kg相比,CBS陽性免疫顆粒明顯減少。3.westen blot結果：在Normal組和Sham組動物海馬CA1區(qū)組織內(nèi)有CBS蛋白的基礎生成。與sham組相比,全腦缺血組的CBS的蛋白表達全腦缺血后顯著降低。大劑量硫化氫可明顯上調(diào)全腦缺血大鼠CBS蛋白的表達；而低劑量硫化氫對全腦缺血大鼠CBS蛋白表達下調(diào)無明顯影響。同時注射HA和PAG后硫化氫誘導的CBS的蛋白表達上調(diào)作用消失。4. ELISA結果：在Normal組和Sham組動物海馬CA1區(qū)組織內(nèi)有IL-1p和TNF-a蛋白的基礎生成。與Normal組和Sham組相比,腦缺血組動物的IL-1β和TNF-a蛋白濃度于腦缺血后顯著上調(diào)。治療性給予硫化氫后,明顯抑制了腦缺血引起的IL-1β和TNF-a蛋白生成的上調(diào),表現(xiàn)為其上升的高峰顯著降低。并且,大劑量組(20mg/kg)的抑制作用最為明顯。IR+NaHS+HA組和IR+NaHS+PAG組與硫化氫治療組20mg/kg相比IL-1β和TNF-a蛋白顯著上調(diào)。結論：腦缺血前給予一定劑量的硫化氫可有效地減輕全腦缺血再灌注大鼠海馬CA1區(qū)細胞損傷,且大劑量組的保護作用更明顯。而H2S合成酶的抑制劑HA和PAG可以抑制上述作用；HA和PAG可以抑制硫化氫上調(diào)CBS表達和下調(diào)腦缺血過程中炎癥因子IL-1β和TNF-a的表達,可能是硫化氫抗缺血性腦損傷的機制之一,并進一步表明硫化氫的抗腦缺血作用。
[Abstract]:Objective: hydrogen sulfide (H2S) is a "Stinky egg odor". Long-term exposure to this harmful gas will cause damage to many tissues, such as the nervous system, the respiratory system and the digestive system. But there is a lot of information that H2S is not only toxic, but also widely participates in many physiological and diseases of the body. The process is considered to be the third class of gas signal molecules following nitric oxide (NO) and carbon monoxide (CO). The normal animals can synthesize H2S. The two important enzymes involved in endogenous H2S synthesis are cystetyl beta synthase (cystathionine beta-synthase, CBS) and cystthioether Y- lyase (cystathionine gamma lyase, CSE). The central nervous system is distributed in the central nervous system, while CSE is mainly distributed outside the nervous system. Recent studies have shown that H2S can play an important role in the ischemic reperfusion injury of multi organ tissues, such as brain, myocardium, liver and kidney, through antioxidation and inhibition of apoptosis. The inflammation plays an important role in the development of cerebral ischemia-reperfusion injury. The rat model of Vascular Dementia (VD) was used to study the NaHHS and H2S synthetase inhibitors (hydroxylamine, HA) and propargyl glycine (propargylglycine, PAG) of different doses of hydrogen sulfide donor NaHHS and H2S synthase inhibitors by intraperitoneal administration. The damage of pyramidal neurons in the hippocampus CA1 region after the rat model of cerebral ischemia reperfusion was established. The effects of inflammatory changes in brain tissue and the mechanism of inflammatory reaction were investigated. The morphological changes of hippocampal CA1 area were observed by pathological section and HE staining. Immunohistochemical and Westen blot methods were used to detect the expression of H2S producing enzyme CBS in brain tissue of rat cerebral ischemia reperfusion, and ELISA method was used to detect the cerebral ischemia reperfusion. The effect of medium inflammatory mediators. In order to prove that H2S plays the role of anti cerebral ischemia and reperfusion injury through anti-inflammatory. Therefore, this experiment aims to prove the anti cerebral ischemia reperfusion injury effect of H2S from the aspects of neurons, inflammatory factors and endogenous H2S, and provide a theoretical basis for it. Methods: a healthy male Wistar rat was selected, and the weight of the rat was about 310G, The animals were randomly divided into 9 groups, the normal group (Normal group), the sham operation group (sham), the cerebral ischemia reperfusion group (ischemia-reperfusion injury:IR), and the hydrogen sulfide treatment group (IR+NaHS) were further divided into 2mg/kg, 5mg/kg, 10mg/kg, 20mg/kg4 subgroups, hydrogen sulfide hydroxyl ammonia inhibition group (IR+NaHS+HA), and hydrogen sulfide propargyl glycine inhibition group. (IR+NaHS+PAG) 5 animals in each group were used to make the VD rat model by the four vascular blocking method (4-VO). The sham operation group was anesthetized with the same procedure as the VD model group, but did not electrocoagulation the bilateral vertebral arteries and did not block the bilateral common carotid artery. After the experiment, the rat brain tissue was taken to make the large brain slices; HE staining was used to observe the CA1 area of the hippocampus in each group by light microscope. The expression of CBS expression was detected by immunohistochemical method; IL-1 beta and TNF- a were measured by bilateral hippocampal tissue and homogenate and enzyme linked immunosorbent assay (ELISA). The expression of CBS in hippocampus of each group was detected by Western blot. Results: 1. morphology: Normal and Sham group rat hippocampal pyramidal cell lines. Neat, dense, complete cell morphology, full nucleolus, clear nucleolus, no obvious cell damage in the.IR group, there were obvious cell damage, the pyramidal cells in the hippocampal CA1 region were sparse, and the nucleolus disappeared; in the hydrogen sulfide treatment group, the CA1 area of the hippocampus in the 2,5 mg/kg dose group was basically the same as that in the IR group; 10 mg/kg group and IR group phase. The number of neurons increased, and the incomplete pyramidal cell layer was seen. The survival state of the 20 mg/kg group was obviously improved. The histological features were similar to that of the Normal group and the Sham group, the neurons were arranged neatly, and the cell morphology was also more complete in the.IR+NaHS+HA group and in the IR+NaHS+PAG group than in the hydrogen sulfide treatment group 20mg/kg. The expression of cell damage.2 protein CBS: the immunohistochemical results showed that a certain amount of CBS positive immune granules in the hippocampus CA1 area of group Sham rats were found to be brown, and the staining was shallow and evenly distributed. Compared with the Sham group, the CBS positive immune particles of the CA1 in the hippocampus of the IR group were significantly reduced, even in the pyramidal cells and around the cell layer and around a large scale CBS table. Compared with the IR group, the immune positive staining intensity of the CBS in the hippocampus CA1 area of the low dose hydrogen sulfide (2,5mg/kg) was not significantly changed, and the positive staining of the immunostaining increased with the increase of the dose of hydrogen sulfide. The large dose of hydrogen sulfide (20mg/kg) group showed that a large number of brown CBS positive immune particles were distributed in the hippocampal CA1 region, especially in the hippocampal cones. In the body cell layer, a large number of CBS positive particles wrapped around the pyramidal neurons were found in the.IR+NaHS+HA group and the IR+NaHS+PAG group, compared with the hydrogen sulfide treatment group 20mg/kg, the CBS positive immune particles significantly reduced the.3.westen blot results: the base formation of CBS protein in the Normal and Sham group CA1 region tissues. Compared with the sham group, the whole brain was compared with the sham group. The protein expression of CBS in the ischemic group decreased significantly after the whole brain ischemia. The high dose of hydrogen sulfide could obviously increase the expression of CBS protein in the rats with whole brain ischemia, while the low dose of hydrogen sulfide had no obvious effect on the down regulation of CBS protein expression in the whole brain ischemia rats. At the same time, the up regulation of the protein expression of CBS induced by hydrogen sulfide induced by injection of HA and PAG disappeared in the.4. ELISA junction. Fruit: the basal formation of IL-1p and TNF-a protein in the hippocampal CA1 region of the Normal and Sham groups. Compared with the Normal and Sham groups, the concentration of IL-1 beta and TNF-a protein in the cerebral ischemia group was significantly up-regulated after cerebral ischemia. After the treatment of hydrogen sulfide, the up regulation of IL-1 beta and TNF-a protein production caused by cerebral ischemia was obviously inhibited. The maximum inhibitory effect of the high dose group (20mg/kg) was most obvious in the.IR+NaHS+HA group and the IR+NaHS+PAG group, and the IL-1 beta and TNF-a protein increased significantly compared with the hydrogen sulfide treatment group 20mg/kg. Conclusion: a certain dose of hydrogen sulfide before cerebral ischemia can effectively reduce the CA1 region of the hippocampus of the rats with whole cerebral ischemia reperfusion. H2S synthase inhibitors, HA and PAG, can inhibit the above action. HA and PAG can inhibit the up-regulated CBS expression of hydrogen sulfide and down regulation of the expression of IL-1 beta and TNF-a in the process of cerebral ischemia. It may be one of the mechanisms of hydrogen sulfide against ischemic brain damage and further indicates sulfur. The effect of hydrogen on cerebral ischemia.
【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R743.3

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