NLRP2在腦缺血損傷中的作用及機制
發(fā)布時間：2018-07-29 09:49
【摘要】：研究目的腦中風,是近年來世界上導致長期致殘和死亡的主要原因,具有發(fā)病率高,死亡率高,致殘率高和復發(fā)率高的特點。其中,相比于出血性腦中風,缺血性腦中風更常見,所占比例超過80%,是由于大腦動脈阻塞從而阻斷血液流向大腦導致組織壞死。缺血性腦中風也就是腦缺血的病理生理過程是復雜的、廣泛的,包括氧化應激,興奮性氨基酸毒性作用,能量代謝障礙,細胞離子動態(tài)平衡的改變,細胞內(nèi)鈣水平增加,活性氧介導的毒性,炎癥細胞因子介導的細胞毒性作用等。最近的研究結果表明先天免疫系統(tǒng)的炎癥反應與腦缺血發(fā)病過程和進展密切相關。先天免疫系統(tǒng)是限制宿主不受損傷的第一道防線,細胞內(nèi)和細胞外含有模式識別受體(PRRs),它們可以檢測到急性損傷,從而啟動炎癥反應。核苷酸結合寡聚化結構域(NOD)——樣受體(NLRs)是細胞內(nèi)模式識別受體家族的一個成員,在炎癥反應中發(fā)揮關鍵作用。在人類22類NLRs中14類包含熱蛋白結構域并形成NLRP亞型(NLRPs)。在NLRPs家族中,一些成員如NLRP1,NLRP3,NLRP6等已經(jīng)被深入研究,它們可以通過結合蛋白ASC招募pro-caspase-1,激活炎性小體,使無活性的pro-caspase-1激活為成熟的caspase-1,從而導致IL-1β和IL-18的產(chǎn)生和釋放并引發(fā)炎癥反應。最近一項研究表明體外培養(yǎng)的人類星形膠質(zhì)細胞表達NLRP2炎性小體,而NLRP2在體內(nèi)的分布和作用卻很少研究,特別是在中樞神經(jīng)系統(tǒng)。那么NLRP2是否在體內(nèi)中樞神經(jīng)系統(tǒng)中表達以及是否與腦缺血等神經(jīng)系統(tǒng)疾病相關仍需進一步探討。在本次實驗研究中,通過建立在體大腦中動脈栓塞(MCAO)模型和培養(yǎng)細胞的糖氧剝奪(OGD)模型從體內(nèi)和體外兩方面來探究NLRP2在腦缺血中的作用及可能的機制。研究方法1 NLRP2在小鼠腦缺血后的表達變化1.1 C57BL/6野生型小鼠腦缺血模型的構建及神經(jīng)學評分1.1.1選取小鼠并構建缺血模型選取雄性野生型C57BL/6實驗小鼠,體重23g左右,建立大腦中動脈栓塞模型。頸部正中切口,切開皮膚,在顯微鏡下鈍性分離出左側頸總動脈,頸內(nèi)動脈及頸外動脈,將尼龍線栓小心插入頸總動脈,當腦血流儀檢測到血流突然下降時停止繼續(xù)插入,并開始計時,12、24、48小時后處死小鼠取材。1.1.2通過神經(jīng)學評分評價模型是否建立成功。1.2腦缺血后NLRP2在小鼠腦中的表達變化及分布1.2.1使用蛋白質(zhì)免疫印跡法(western blot,WB)、Real-time PCR和免疫組織化學法(immunohistochemistry,IHC)檢測在腦缺血后NLRP2的表達變化情況。1.2.2采用組織免疫熒光(immunofluorescence,IF)方法對NLRP2與小鼠大腦的不同細胞的標記物蛋白進行熒光雙染檢測NLRP2在腦中的分布。1.3體外模型的構建及NLRP2的檢測體外培養(yǎng)原代星形膠質(zhì)細胞,建立糖氧剝奪(Oxygen Glucose Deprivation,OGD)模型,分別進行OGD處理0.5、1、1.5、2h,使用蛋白質(zhì)免疫印跡法(western blot,WB)、組織免疫熒光(immunofluorescence,IF)和 Real-time PCR 檢測OGD后原代星形膠質(zhì)細胞中NLRP2的表達變化。2 NLRP2在小鼠腦缺血損傷中的作用2.1注射腺相關病毒沉默小鼠腦內(nèi)NLRP2基因利用腦立體定位注射技術將攜帶NLRP2沉默基因的腺相關病毒載體注射到神經(jīng)系統(tǒng)的特定部位,以沉默這個部位的NLRP2基因。2.2注射空病毒與注射攜帶NLRP2沉默基因的小鼠大腦形態(tài)學損傷對比注射空病毒與注射攜帶NLRP2沉默基因的小鼠腦缺血后處理,對注射空病毒與注射攜帶NLRP2沉默基因的小鼠大腦進行TTC染色,并對其形態(tài)學損傷進行評分。2.3小鼠腦缺血模型中炎癥因子的檢測利用CBA、Real-time PCR方法檢測注射空病毒與注射攜帶NLRP2沉默基因的小鼠大腦勻漿中促炎因子的含量。2.4 NLRP2對OGD誘導的原代星形膠質(zhì)細胞凋亡的影響2.4.1對星形膠質(zhì)細胞進行轉染2.4.2星形膠質(zhì)細胞的凋亡檢測對轉染si-NLRP2的星形膠質(zhì)細胞進行OGD處理,Annexin V/PI染色后,使用流式細胞儀檢測OGD處理條件下,原代星形膠質(zhì)細胞凋亡的影響。3腦缺血損傷中NLRP2通路的調(diào)控作用WB檢測小鼠腦缺血后NLRP2對小鼠大腦caspase-1、ASC、IL-1β等相關蛋白水平的影響。WB檢測NLRP2對注射空病毒與注射攜帶NLRP2沉默基因的病毒小鼠大腦caspase-1、ASC、IL-1β等相關蛋白水平。WB檢測轉染空si-RNA和轉染si-NLRP2的星形膠質(zhì)細胞OGD處理后,NLRP2對caspase-1、p-p65、IL-1β等蛋白表達的影響。研究結果1小鼠腦缺血后NLRP2及其下游分子在腦內(nèi)的表達及作用1.1 C57BL/6小鼠腦缺血模型的構建及神經(jīng)學評分由于死亡或者是神經(jīng)學評分小于2分,每組大約有l(wèi)5%小鼠被剔除。1.2腦缺血后NLRP2在小鼠腦中的表達變化及分布1.2.1使用蛋白質(zhì)免疫印跡法(western blot,WB)、Real-time PCR和免疫組織化學法(immunohistochemistry,IHC)檢測腦缺血后NLRP2的表達變化,通過WB、Real-time PCR分析,我們發(fā)現(xiàn)NLRP2在腦缺血后表達明顯升高,并且在24小時達到高峰,免疫組化結果也證明這一點。1.2.2通過免疫熒光染色得出,NLRP2主要表達于原代星形膠質(zhì)細胞,在神經(jīng)元中也有少量表達,在小膠質(zhì)細胞中幾乎沒有表達。1.3體外模型的構建及NLRP2的檢測體外培養(yǎng)原代星形膠質(zhì)細胞,建立糖氧剝奪OGD模型,通過WB、Real-time PCR結果分析,我們發(fā)現(xiàn)OGD后NLRP2表達明顯升高,并且在OGD1.5小時達到高峰,免疫熒光結果顯示細胞OGD1.5小時后,NLRP2表達升高。2 NLRP2在小鼠腦缺血損傷中的作用2.1注射腺相關病毒沉默小鼠腦內(nèi)NLRP2基因注射病毒4周后,OCT包埋小鼠大腦組織,冰凍切片于顯微鏡下觀察病毒侵染范圍,范圍較大,覆蓋了大腦中動脈所支配的區(qū)域。2.2注射空病毒與注射攜帶NLRP2沉默基因的病毒小鼠大腦形態(tài)學損傷對比TTC染色結果顯示,注射NLRP2沉默基因腦缺血后,小鼠神經(jīng)學評分明顯降低,腦梗死面積也明顯減少,。2.3小鼠腦缺血模型中炎癥因子的檢測對注射空病毒和注射NLRP2沉默基因的病毒的小鼠進行腦缺血處理,CBA、Real-time PCR結果顯示注射病毒所引起的NLRP2表達下降可以減輕腦缺血所引起的IL-1β、IL-18、IL-6、TNF-α MCP-1等炎性因子表達升高。2.4 NLRP2對OGD誘導的原代星形膠質(zhì)細胞凋亡的影響流式結果顯示,對轉染si-NLRP2的星形膠質(zhì)細胞進行OGD處理,凋亡的星形膠質(zhì)細胞明顯減少。3腦缺血損傷中NLRP2通路的調(diào)控作用WB結果顯示,小鼠腦缺血后,NLRP2、caspase-1、ASC、IL-1β等相關蛋白表達升高;注射攜帶NLRP2沉默基因的病毒小鼠,與注射空病毒的小鼠相比,NLRP2表達降低,而且注射攜帶NLRP2沉默基因的病毒小鼠所引起的NLRP2下降可以減輕腦缺血所引起的caspase-1、ASC、IL-1β等相關蛋白升高;培養(yǎng)原代星形膠質(zhì)細胞,WB結果顯示,轉入siRNA-NLRP2星形膠質(zhì)細胞NLRP2表達明顯降低。轉染siRNA-NLRP2的細胞所引起的NLRP2表達下降可以減輕OGD所引起的caspase-1,p-p65,IL-Iβ等相關蛋白表達升高。研究結論本實驗首次證明了 NLRP2在腦缺血損傷中的表達變化,發(fā)現(xiàn)腦缺血損傷后NLRP2表達升高,進一步導致caspase-1、ASC、IL-1β等相關蛋白升高,而NLRP2的缺失可以使腦梗死面積減少,腦缺血損傷得到改善,對于腦缺血具有一定的防治作用。
[Abstract]:In recent years, cerebral apoplexy is the main cause of long-term disability and death in the world, characterized by high morbidity, high mortality, high disability and high recurrence rate. Among them, ischemic stroke is more common than hemorrhagic stroke, with a proportion of more than 80%, which is due to obstruction of the brain artery to block the flow of blood to the brain. The pathophysiological process of ischemic cerebral apoplexy is complex and extensive, including oxidative stress, toxic effects of excitatory amino acids, energy metabolism disorders, changes in dynamic balance of cell ions, increased intracellular calcium levels, active oxygen mediated toxicity, and cytotoxic effects mediated by inflammatory cytokines. Recent studies have shown that the inflammatory response of the innate immune system is closely related to the process and progress of cerebral ischemia. The innate immune system is the first line of defense to restrict the undamaged host, and the intracellular and extracellular domain of the pattern recognition receptor (PRRs) can detect acute damage and initiate the inflammatory reaction. The oligomeric domain (NOD) - like receptor (NLRs) is a member of the intracellular pattern recognition receptor family and plays a key role in the inflammatory response. In the 22 category of human NLRs, 14 types include the thermal protein domain and form a NLRP subtype (NLRPs). In the NLRPs family, some members, such as NLRP1, NLRP3, and NLRP6, have been deeply studied, and they are available The recruitment of pro-caspase-1 through binding protein ASC activates the inflammatory corpuscles and activates the inactive pro-caspase-1 to mature caspase-1, resulting in the production and release of IL-1 beta and IL-18 and triggering the inflammatory reaction. And the role is rarely studied, especially in the central nervous system. Then whether NLRP2 is expressed in the central nervous system in the body and whether it is associated with cerebral ischemia, such as cerebral ischemia, is still needed to be further explored. In this experimental study, the model of the middle cerebral artery embolism (MCAO) and the glucose deprivation (OGD) model of the cultured cells were established. To explore the role and possible mechanism of NLRP2 in cerebral ischemia from two aspects in vivo and in vitro. Method 1 NLRP2 expression changes after cerebral ischemia in mice and the construction of cerebral ischemia model in 1.1 C57BL/6 wild type mice and neurology score 1.1.1 selected mice and construct ischemic model to select male wild type C57BL/6 experimental mice, weight 23g left Right, establish a middle cerebral artery embolism model. Neck median incision, incision of skin, separation of the left common carotid artery, internal carotid artery and external carotid artery under the microscope. The nylon thread plug is carefully inserted into the common carotid artery. When the cerebral blood flow meter detects the sudden drop of blood flow, it stops continuously and starts the time, after 12,24,48 hours, the mice are killed and taken to take mice fetch. Material.1.1.2 was used to evaluate the expression of NLRP2 in the brain of.1.2 after cerebral ischemia and its distribution and distribution of 1.2.1 using protein immunoblotting (Western blot, WB), Real-time PCR and immunohistochemistry (immunohistochemistry, IHC) for the detection of the changes in the expression of NLRP2 in the brain after cerebral ischemia. Immunofluorescence (IF) method for the detection of NLRP2 and the marker proteins of different cells in the brain of the mice, the distribution of NLRP2 in the brain, the construction of the.1.3 in vitro model and the detection of the original astrocytes by NLRP2 in vitro, and the establishment of oxygen deprivation (Oxygen Glucose Deprivation, OGD) model, respectively, for OGD. Treatment of 0.5,1,1.5,2h, Western blot (WB), tissue immunofluorescence (immunofluorescence, IF) and Real-time PCR detection of the expression of NLRP2 in the primary astrocytes after OGD;.2 NLRP2 in the brain ischemia injury in mice 2.1 injection of adeno-associated virus in mouse brain to use brain stereotaxis Injection of adeno-related virus vectors carrying NLRP2 silencing gene into specific parts of the nervous system by injection technique, the NLRP2 gene.2.2 injected into this site is injected into the brain of mice and the mice with NLRP2 silencing genes are injected into the brain. The brain of mice injected with NLRP2 silencing gene was stained with TTC, and the morphological damage was evaluated in.2.3 mice model of cerebral ischemia. CBA, Real-time PCR method was used to detect the level of pro-inflammatory factors in the brain homogenate of mice with NLRP2 silencing basis and.2. The effect of.4 NLRP2 on the apoptosis of primary astrocytes induced by OGD 2.4.1 the apoptosis of astrocytes transfected with 2.4.2 astrocytes, OGD treatment of astrocytes transfected with si-NLRP2, and the effect of Annexin V/PI staining on the apoptosis of primary astrocytes using flow cytometry to detect the apoptosis of astrocytes The regulation of NLRP2 pathway in.3 cerebral ischemia injury WB detection of NLRP2 effect on the level of Caspase-1, ASC, IL-1 beta in the brain of mice after cerebral ischemia The effect of NLRP2 on the expression of Caspase-1, p-p65, IL-1 beta in si-NLRP2 astrocytes after OGD treatment. Results 1 the expression of NLRP2 and its downstream molecules in the brain after cerebral ischemia in mice and the construction of the 1.1 C57BL/6 mouse model of cerebral ischemia and the neurology score were less than 2 in each group due to death or neurology score. The expression of NLRP2 in the brain of mice after.1.2 cerebral ischemia was removed and the distribution of NLRP2 in the brain of mice was removed and the distribution of 1.2.1 using protein immunoblotting (Western blot, WB), Real-time PCR and immunohistochemistry (immunohistochemistry, IHC) were used to detect the changes of NLRP2 expression after cerebral ischemia. We found that after cerebral ischemia, we found that the NLRP2 was after cerebral ischemia. The expression rose obviously and reached the peak in 24 hours. The results of immunohistochemical staining showed that.1.2.2 was mainly expressed in the primary astrocytes by immunofluorescence staining, and that NLRP2 was also expressed in the neurons. In the microglia, there was little expression of the expression of.1.3 in vitro model and the detection of NLRP2 in vitro. On behalf of astrocytes, the OGD model of oxygen deprivation was established. Through the analysis of WB and Real-time PCR, we found that the expression of NLRP2 increased significantly after OGD and reached the peak in OGD1.5 hours. The results of immunofluorescence showed that after the cell OGD1.5 hours, the expression of NLRP2 expression increased.2 NLRP2 in the cerebral ischemia injury of mice 2.1 injection of adeno-associated virus silencing. 4 weeks after the NLRP2 gene injection of the virus in the brain of mice, OCT was embedded in the brain tissue of mice. The scope of the virus infection was observed under the microscope under the microscope. The scope of the virus infection was larger, covering the.2.2 injection of the middle cerebral artery and the.2.2 virus with the NLRP2 silencing gene. After NLRP2 silencing gene cerebral ischemia, the neurology score of mice decreased obviously and the area of cerebral infarction decreased obviously. The detection of inflammatory factors in the.2.3 mice model of cerebral ischemia had cerebral ischemia treatment on mice injected with the virus and the virus injected with the NLRP2 silencing gene. The results of CBA and Real-time PCR showed the decline of NLRP2 expression caused by the injection virus. The effects of IL-1 beta, IL-18, IL-6, TNF- alpha MCP-1 and other inflammatory factors on the apoptosis of primary astrocytes induced by OGD can be alleviated by the flow cytometry results showed that the astrocytes transfected with si-NLRP2 were treated with OGD, and apoptotic astrocytes significantly reduced NLRP2 pathways in.3 cerebral ischemia. The WB results showed that the expression of NLRP2, caspase-1, ASC, IL-1 beta and other related proteins increased after cerebral ischemia in mice, and the mice injected with NLRP2 silencing genes decreased the NLRP2 expression compared with the mice injected with the empty virus, and the decrease of NLRP2 caused by the mice injected with the NLRP2 silent gene could reduce the cerebral ischemia. The associated proteins such as caspase-1, ASC, IL-1 beta and other related proteins were raised, and primary astrocytes were cultured. WB results showed that the expression of NLRP2 in siRNA-NLRP2 astrocytes decreased significantly. The decrease of NLRP2 expression caused by transfection of siRNA-NLRP2 cells could reduce caspase-1, p-p65, IL-I beta and other related proteins caused by OGD. This experiment is the first time to demonstrate the expression of NLRP2 in cerebral ischemia injury. It is found that the expression of NLRP2 increases after cerebral ischemia injury, which leads to the increase of Caspase-1, ASC, IL-1 beta and other related proteins, and the loss of NLRP2 can reduce the area of cerebral infarction and improve the cerebral ischemia injury, which has some preventive effect on cerebral ischemia.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R743
本文編號：2152292
[Abstract]:In recent years, cerebral apoplexy is the main cause of long-term disability and death in the world, characterized by high morbidity, high mortality, high disability and high recurrence rate. Among them, ischemic stroke is more common than hemorrhagic stroke, with a proportion of more than 80%, which is due to obstruction of the brain artery to block the flow of blood to the brain. The pathophysiological process of ischemic cerebral apoplexy is complex and extensive, including oxidative stress, toxic effects of excitatory amino acids, energy metabolism disorders, changes in dynamic balance of cell ions, increased intracellular calcium levels, active oxygen mediated toxicity, and cytotoxic effects mediated by inflammatory cytokines. Recent studies have shown that the inflammatory response of the innate immune system is closely related to the process and progress of cerebral ischemia. The innate immune system is the first line of defense to restrict the undamaged host, and the intracellular and extracellular domain of the pattern recognition receptor (PRRs) can detect acute damage and initiate the inflammatory reaction. The oligomeric domain (NOD) - like receptor (NLRs) is a member of the intracellular pattern recognition receptor family and plays a key role in the inflammatory response. In the 22 category of human NLRs, 14 types include the thermal protein domain and form a NLRP subtype (NLRPs). In the NLRPs family, some members, such as NLRP1, NLRP3, and NLRP6, have been deeply studied, and they are available The recruitment of pro-caspase-1 through binding protein ASC activates the inflammatory corpuscles and activates the inactive pro-caspase-1 to mature caspase-1, resulting in the production and release of IL-1 beta and IL-18 and triggering the inflammatory reaction. And the role is rarely studied, especially in the central nervous system. Then whether NLRP2 is expressed in the central nervous system in the body and whether it is associated with cerebral ischemia, such as cerebral ischemia, is still needed to be further explored. In this experimental study, the model of the middle cerebral artery embolism (MCAO) and the glucose deprivation (OGD) model of the cultured cells were established. To explore the role and possible mechanism of NLRP2 in cerebral ischemia from two aspects in vivo and in vitro. Method 1 NLRP2 expression changes after cerebral ischemia in mice and the construction of cerebral ischemia model in 1.1 C57BL/6 wild type mice and neurology score 1.1.1 selected mice and construct ischemic model to select male wild type C57BL/6 experimental mice, weight 23g left Right, establish a middle cerebral artery embolism model. Neck median incision, incision of skin, separation of the left common carotid artery, internal carotid artery and external carotid artery under the microscope. The nylon thread plug is carefully inserted into the common carotid artery. When the cerebral blood flow meter detects the sudden drop of blood flow, it stops continuously and starts the time, after 12,24,48 hours, the mice are killed and taken to take mice fetch. Material.1.1.2 was used to evaluate the expression of NLRP2 in the brain of.1.2 after cerebral ischemia and its distribution and distribution of 1.2.1 using protein immunoblotting (Western blot, WB), Real-time PCR and immunohistochemistry (immunohistochemistry, IHC) for the detection of the changes in the expression of NLRP2 in the brain after cerebral ischemia. Immunofluorescence (IF) method for the detection of NLRP2 and the marker proteins of different cells in the brain of the mice, the distribution of NLRP2 in the brain, the construction of the.1.3 in vitro model and the detection of the original astrocytes by NLRP2 in vitro, and the establishment of oxygen deprivation (Oxygen Glucose Deprivation, OGD) model, respectively, for OGD. Treatment of 0.5,1,1.5,2h, Western blot (WB), tissue immunofluorescence (immunofluorescence, IF) and Real-time PCR detection of the expression of NLRP2 in the primary astrocytes after OGD;.2 NLRP2 in the brain ischemia injury in mice 2.1 injection of adeno-associated virus in mouse brain to use brain stereotaxis Injection of adeno-related virus vectors carrying NLRP2 silencing gene into specific parts of the nervous system by injection technique, the NLRP2 gene.2.2 injected into this site is injected into the brain of mice and the mice with NLRP2 silencing genes are injected into the brain. The brain of mice injected with NLRP2 silencing gene was stained with TTC, and the morphological damage was evaluated in.2.3 mice model of cerebral ischemia. CBA, Real-time PCR method was used to detect the level of pro-inflammatory factors in the brain homogenate of mice with NLRP2 silencing basis and.2. The effect of.4 NLRP2 on the apoptosis of primary astrocytes induced by OGD 2.4.1 the apoptosis of astrocytes transfected with 2.4.2 astrocytes, OGD treatment of astrocytes transfected with si-NLRP2, and the effect of Annexin V/PI staining on the apoptosis of primary astrocytes using flow cytometry to detect the apoptosis of astrocytes The regulation of NLRP2 pathway in.3 cerebral ischemia injury WB detection of NLRP2 effect on the level of Caspase-1, ASC, IL-1 beta in the brain of mice after cerebral ischemia The effect of NLRP2 on the expression of Caspase-1, p-p65, IL-1 beta in si-NLRP2 astrocytes after OGD treatment. Results 1 the expression of NLRP2 and its downstream molecules in the brain after cerebral ischemia in mice and the construction of the 1.1 C57BL/6 mouse model of cerebral ischemia and the neurology score were less than 2 in each group due to death or neurology score. The expression of NLRP2 in the brain of mice after.1.2 cerebral ischemia was removed and the distribution of NLRP2 in the brain of mice was removed and the distribution of 1.2.1 using protein immunoblotting (Western blot, WB), Real-time PCR and immunohistochemistry (immunohistochemistry, IHC) were used to detect the changes of NLRP2 expression after cerebral ischemia. We found that after cerebral ischemia, we found that the NLRP2 was after cerebral ischemia. The expression rose obviously and reached the peak in 24 hours. The results of immunohistochemical staining showed that.1.2.2 was mainly expressed in the primary astrocytes by immunofluorescence staining, and that NLRP2 was also expressed in the neurons. In the microglia, there was little expression of the expression of.1.3 in vitro model and the detection of NLRP2 in vitro. On behalf of astrocytes, the OGD model of oxygen deprivation was established. Through the analysis of WB and Real-time PCR, we found that the expression of NLRP2 increased significantly after OGD and reached the peak in OGD1.5 hours. The results of immunofluorescence showed that after the cell OGD1.5 hours, the expression of NLRP2 expression increased.2 NLRP2 in the cerebral ischemia injury of mice 2.1 injection of adeno-associated virus silencing. 4 weeks after the NLRP2 gene injection of the virus in the brain of mice, OCT was embedded in the brain tissue of mice. The scope of the virus infection was observed under the microscope under the microscope. The scope of the virus infection was larger, covering the.2.2 injection of the middle cerebral artery and the.2.2 virus with the NLRP2 silencing gene. After NLRP2 silencing gene cerebral ischemia, the neurology score of mice decreased obviously and the area of cerebral infarction decreased obviously. The detection of inflammatory factors in the.2.3 mice model of cerebral ischemia had cerebral ischemia treatment on mice injected with the virus and the virus injected with the NLRP2 silencing gene. The results of CBA and Real-time PCR showed the decline of NLRP2 expression caused by the injection virus. The effects of IL-1 beta, IL-18, IL-6, TNF- alpha MCP-1 and other inflammatory factors on the apoptosis of primary astrocytes induced by OGD can be alleviated by the flow cytometry results showed that the astrocytes transfected with si-NLRP2 were treated with OGD, and apoptotic astrocytes significantly reduced NLRP2 pathways in.3 cerebral ischemia. The WB results showed that the expression of NLRP2, caspase-1, ASC, IL-1 beta and other related proteins increased after cerebral ischemia in mice, and the mice injected with NLRP2 silencing genes decreased the NLRP2 expression compared with the mice injected with the empty virus, and the decrease of NLRP2 caused by the mice injected with the NLRP2 silent gene could reduce the cerebral ischemia. The associated proteins such as caspase-1, ASC, IL-1 beta and other related proteins were raised, and primary astrocytes were cultured. WB results showed that the expression of NLRP2 in siRNA-NLRP2 astrocytes decreased significantly. The decrease of NLRP2 expression caused by transfection of siRNA-NLRP2 cells could reduce caspase-1, p-p65, IL-I beta and other related proteins caused by OGD. This experiment is the first time to demonstrate the expression of NLRP2 in cerebral ischemia injury. It is found that the expression of NLRP2 increases after cerebral ischemia injury, which leads to the increase of Caspase-1, ASC, IL-1 beta and other related proteins, and the loss of NLRP2 can reduce the area of cerebral infarction and improve the cerebral ischemia injury, which has some preventive effect on cerebral ischemia.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R743
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