腦缺血缺氧中缺氧誘導(dǎo)因子HIF-1α對小膠質(zhì)細(xì)胞自噬調(diào)節(jié)的機(jī)制研究
發(fā)布時間：2018-09-06 17:59
【摘要】：缺血性腦卒中是一種由于腦血流中斷并導(dǎo)致神經(jīng)功能障礙的疾病。缺血性腦卒中的發(fā)病率在急性腦血管中占有很高的比例，并且具有很高的傷殘率，已嚴(yán)重危害了人類的健康。根據(jù)相關(guān)研究調(diào)查，我國老年患者每年新發(fā)缺血性腦卒中的人數(shù)達(dá)到120-150萬左右，其中死亡人數(shù)達(dá)到80-100萬左右。在幸存患者中，半數(shù)以上伴有不同程度的神經(jīng)功能殘疾。隨著我國的老齡人口的逐漸增加，缺血性腦卒中的發(fā)病人數(shù)將進(jìn)一步增加。因此，缺血性腦卒中成為人類健康的頭號敵人，深入研究缺血性腦卒中的發(fā)病機(jī)制及治療策略具有重要的科學(xué)意義和臨床價值。 當(dāng)前對于缺血性腦卒中的發(fā)病機(jī)制及治療策略提出了全新的概念-血管神經(jīng)網(wǎng)絡(luò)學(xué)說。該學(xué)說研究認(rèn)為：血管內(nèi)皮細(xì)胞、神經(jīng)元、膠質(zhì)細(xì)胞以及周圍細(xì)胞外基質(zhì)等成分有機(jī)組成了血管神經(jīng)單元復(fù)合體。復(fù)合體內(nèi)的各種細(xì)胞成分及復(fù)合體之間互相影響和調(diào)控，形成了網(wǎng)絡(luò)結(jié)構(gòu)。該學(xué)說認(rèn)為缺血性腦卒中不是單純的血管事件，也不是單存的神經(jīng)事件，而是血管與神經(jīng)之間，實(shí)質(zhì)細(xì)胞與基質(zhì)之間的相互影響和對話，互相調(diào)節(jié)和平衡的作用過程。因此，在缺血性腦卒中后，所有的神經(jīng)細(xì)胞和細(xì)胞成分都應(yīng)該受到重視和保護(hù)，而不是單純保護(hù)某種細(xì)胞。在以前的缺血性腦卒中的大量相關(guān)研究都關(guān)注神經(jīng)元細(xì)胞和血管內(nèi)皮細(xì)胞，而缺乏對膠質(zhì)細(xì)胞的深入研究。目前逐漸認(rèn)為：小膠質(zhì)細(xì)胞介導(dǎo)的炎癥反應(yīng)在缺血性腦卒中后發(fā)揮重要的損傷作用。 小膠質(zhì)細(xì)胞與正常細(xì)胞一樣，在缺氧環(huán)境下均能感受低氧信號的刺激，并能活化相應(yīng)的信號通路，啟動相應(yīng)基因的表達(dá)，適應(yīng)低氧微環(huán)境對細(xì)胞的有害作用，并維持細(xì)胞內(nèi)環(huán)境的穩(wěn)定。當(dāng)細(xì)胞受到低氧信號刺激后，有多種基因誘導(dǎo)表達(dá)。其中低氧誘導(dǎo)因子(HIF-1)發(fā)揮重要的作用。研究表明HIF-1可以在缺氧環(huán)境下迅速啟動下游相關(guān)基因的活化，調(diào)節(jié)細(xì)胞內(nèi)的能量代謝水平和對氧的利用，在機(jī)體器官或局部組織發(fā)揮對低氧適應(yīng)能力中具有重要作用。在營養(yǎng)缺乏、損傷等外界有害環(huán)境下，特別是缺氧應(yīng)激下，細(xì)胞能通過啟動HIF-1的分子開關(guān)作用，使下游基因的表達(dá)做到準(zhǔn)確的精細(xì)調(diào)控，并使細(xì)胞維持基本的能量代謝。 自噬(Autophagy)是生物在進(jìn)化過程中保留的一種特殊的現(xiàn)象。本質(zhì)是細(xì)胞在生存、發(fā)育、分化及代謝過程中的一種溶酶體降解途徑。其主要的作用是保護(hù)機(jī)體內(nèi)環(huán)境的穩(wěn)定，并避免受到外界各種因素的影響。新近研究表明，自噬除了維持細(xì)胞自身環(huán)境的平衡以外，還作為一種重要的分子調(diào)控機(jī)制，參與了眾多的基因的調(diào)節(jié)和多種疾病的發(fā)生，如代謝病、心臟病、遺傳病、腫瘤、炎癥及神經(jīng)疾病等。研究報道在缺氧過程中，自噬變化明顯，并影響細(xì)胞的病理及生理學(xué)效應(yīng)。 然后在腦缺血缺氧環(huán)境下，小膠質(zhì)細(xì)胞的病理生理學(xué)特點(diǎn)是怎樣的？HIF-1α的在小膠質(zhì)細(xì)胞上的表達(dá)情況如何？小膠質(zhì)細(xì)胞是否有自噬發(fā)生？其具體發(fā)生及調(diào)節(jié)機(jī)制又是怎樣的？干擾上述環(huán)節(jié)是否對腦缺血的神經(jīng)功能具有保護(hù)作用？為了證實(shí)上述的疑問，我們在本研究中設(shè)計系列實(shí)驗(yàn)，以明確在腦缺血缺氧環(huán)境下HIF-1α的表達(dá)對小膠質(zhì)細(xì)胞的自噬影響及生物學(xué)意義。 研究內(nèi)容：①在體外應(yīng)用氧糖剝奪模型，在0-48小時的不同時間點(diǎn)，應(yīng)用PI和MTT檢測小膠質(zhì)細(xì)胞的生存率；②體外應(yīng)用氧糖剝奪模型，在0-48小時的不同時間點(diǎn)，應(yīng)用RT-PCR和ELISA檢測小膠質(zhì)細(xì)胞的炎癥細(xì)胞因子IL-8和TNF-α表達(dá)量；③體外應(yīng)用氧糖剝奪模型，在0-48小時的不同時間點(diǎn)，應(yīng)用western blot檢測小膠質(zhì)細(xì)胞的HIF-1α表達(dá)量；④體外應(yīng)用氧糖剝奪模型，分別用HIF-1α的抑制劑2ME和YC-1以及HIF-1α的RNA阻斷HIF-1α的表達(dá)，應(yīng)用PI和MTT檢測小膠質(zhì)細(xì)胞的生存率；⑤體外應(yīng)用氧糖剝奪模型，分別用HIF-1α的抑制劑2ME和YC-1以及HIF-1α的RNA阻斷HIF-1α的表達(dá)，應(yīng)用ELISA檢測小膠質(zhì)細(xì)胞的炎癥細(xì)胞因子IL-8和TNF-α表達(dá)量；⑥體外應(yīng)用氧糖剝奪模型，在0-48小時的不同時間點(diǎn)，應(yīng)用western blot檢測檢測小膠質(zhì)細(xì)胞自噬相關(guān)蛋白LC3的表達(dá)變化；應(yīng)用激光共聚焦顯微鏡觀察小膠質(zhì)細(xì)胞的GFP-LC3質(zhì)粒表達(dá)變化；應(yīng)用MDC (單丹磺酰尸胺)及丫啶橙染色，流式細(xì)胞儀分析小膠質(zhì)細(xì)胞自噬相關(guān)的情況；電子顯微鏡檢測小膠質(zhì)細(xì)胞自噬囊泡的形成；⑦體外應(yīng)用氧糖剝奪模型，分別用HIF-1α的抑制劑2ME和YC-1以及HIF-1α的RNA阻斷HIF-1α的表達(dá)，通過上述手段檢測對小膠質(zhì)細(xì)胞自噬的影響；⑧體外應(yīng)用氧糖剝奪模型，分別用自噬的抑制劑3-MA和BafA1，以及促進(jìn)劑Rapa作用小膠質(zhì)細(xì)胞，檢測小膠質(zhì)細(xì)胞的存活率；運(yùn)用自噬關(guān)鍵調(diào)節(jié)蛋白Beclin1的RNAi作用小膠質(zhì)細(xì)胞，檢測小膠質(zhì)細(xì)胞的存活率；⑨建立小鼠大腦中動脈栓塞模型，檢測缺血區(qū)小膠質(zhì)細(xì)胞的自噬及小鼠神經(jīng)功能情況；⑩建立小鼠大腦中動脈栓塞模型，側(cè)腦室注射自噬的抑制劑3-MA，檢測抑制自噬后，缺血區(qū)小膠質(zhì)細(xì)胞的自噬及小鼠神經(jīng)功能情況。 我們的研究得出了以下結(jié)果：①在體外氧糖剝奪模型中，隨著時間延長，小膠質(zhì)細(xì)胞的死亡率逐漸增加，存活率逐漸下降；②在體外氧糖剝奪模型中，隨著時間延長，炎癥細(xì)胞因子IL-8和TNF-α表達(dá)量逐漸增加；③體外氧糖剝奪模型型中，阻斷HIF-1α的表達(dá)，小膠質(zhì)細(xì)胞的死亡率下降，存活率增加；⑤體外氧糖剝奪模型中，阻斷HIF-1α的表達(dá)，小膠質(zhì)細(xì)胞的炎癥細(xì)胞因子IL-8和TNF-α表達(dá)量減少；⑥體外氧糖剝奪模型中，，小膠質(zhì)細(xì)胞自噬增加；⑦體外氧糖剝奪模型中，阻斷HIF-1α的表達(dá)，小膠質(zhì)細(xì)胞自噬減少；⑧體外氧糖剝奪模型中，抑制自噬及Beclin1蛋白，小膠質(zhì)細(xì)胞的死亡率減少，存活率增加；促進(jìn)自噬，小膠質(zhì)細(xì)胞的死亡率增加，存活率減少；⑨體內(nèi)小鼠大腦中動脈栓塞模型中，缺血區(qū)的小膠質(zhì)細(xì)胞自噬增加，小鼠神經(jīng)功能受損；自噬抑制劑3-MA能減輕缺血區(qū)的小膠質(zhì)細(xì)胞自噬及小鼠神經(jīng)功能缺損情況。 研究結(jié)論：腦缺血缺氧環(huán)境誘導(dǎo)小膠質(zhì)細(xì)胞HIF-1α表達(dá)上調(diào)，并通過Beclin1信號通路啟動自噬途徑，導(dǎo)致了細(xì)胞的炎癥和死亡效應(yīng)，并進(jìn)一步加重了神經(jīng)功能損傷。干擾自噬途徑，可以抑制小膠質(zhì)細(xì)胞的炎癥和死亡效應(yīng)，為缺血性腦卒中提供了理想的腦保護(hù)策略。
[Abstract]:Ischemic stroke is a neurological disorder caused by interruption of cerebral blood flow. The incidence of ischemic stroke is high in acute cerebrovascular diseases, and has a high disability rate. It has seriously endangered human health. More than half of the survivors are accompanied by varying degrees of neurological disability. With the gradual increase of the elderly population in China, the number of ischemic stroke patients will further increase. Therefore, ischemic stroke has become the number one enemy of human health. It is of great scientific significance and clinical value to study the pathogenesis and treatment strategy of ischemic stroke.
Vascular nerve network theory, a new concept for the pathogenesis and treatment strategy of ischemic stroke, has been proposed. It is believed that vascular endothelial cells, neurons, glial cells and peripheral extracellular matrix constitute the vascular nerve unit complex. The theory holds that ischemic stroke is not a simple vascular event, nor a single neurological event, but a process of interaction and dialogue between blood vessels and nerves, between parenchymal cells and matrix, and of mutual regulation and balance. Neurons and cell components should be valued and protected rather than simply protecting certain cells. Previous studies on ischemic stroke have focused on neurons and vascular endothelial cells, but lack of in-depth study of glial cells. After stroke, it plays an important role in injury.
Microglia, like normal cells, can sense the stimulation of hypoxic signals under hypoxic conditions, activate the corresponding signaling pathways, activate the expression of corresponding genes, adapt to the harmful effects of hypoxic microenvironment on cells, and maintain the stability of the cellular environment. When cells are stimulated by hypoxic signals, a variety of genes are induced to express. Intermediate hypoxia inducible factor-1 (HIF-1) plays an important role in hypoxic adaptation. It has been shown that HIF-1 can activate downstream related genes rapidly in hypoxic environment, regulate intracellular energy metabolism and oxygen utilization, and play an important role in organs or local tissues. HIF-1 can play an important role in hypoxic adaptation, such as nutritional deficiency, injury, etc. Under harmful environment, especially under hypoxia stress, cells can regulate the expression of downstream genes accurately and precisely by activating the molecular switch of HIF-1, and maintain basic energy metabolism.
Autophagy is a special phenomenon that organisms retain in their evolutionary process. It is essentially a lysosomal degradation pathway in the process of cell survival, development, differentiation and metabolism. Its main role is to protect the stability of the body's internal environment and avoid being influenced by various external factors. Recent studies have shown that autophagy is not only fine-grained but also fine-grained. In addition to the homeostasis of cellular environment, it is also an important molecular mechanism involved in the regulation of many genes and the occurrence of many diseases, such as metabolic diseases, heart diseases, genetic diseases, tumors, inflammation and neurological diseases.
Then what are the pathophysiological characteristics of microglia in hypoxic and cerebral ischemia environment? How is the expression of HIF-1a in microglia? Is microglia autophagy occurring? What are the specific mechanisms of its occurrence and regulation? Does interfering with the above-mentioned links have a protective effect on the neurological function of cerebral ischemia? To confirm these doubts, we designed a series of experiments in this study to clarify the effect of HIF-1a expression on microglia autophagy and its biological significance in hypoxic and ischemic environment.
The contents of this study were as follows: 1) The survival rate of microglia was measured by PI and MTT at different time points of 0-48 hours after oxygen-glucose deprivation in vitro; 2) The expression of inflammatory cytokines IL-8 and TNF-alpha in microglia was detected by RT-PCR and ELISA at different time points of 0-48 hours after oxygen-glucose deprivation in vitro. Western blot was used to detect the expression of HIF-1a in microglia at different time points of 0-48 hours with oxygen-glucose deprivation model. In the model of glucose deprivation, the expression of HIF-1a was blocked by 2ME, YC-1 and RNA of HIF-1a respectively, and the expression of inflammatory cytokines IL-8 and TNF-alpha in microglia was detected by ELISA. _Oxygen-glucose deprivation model was used in vitro, and the autophagy correlation of microglia was detected by Western blot at different time points of 0-48 hours. The expression of protein LC3, the expression of GFP-LC3 plasmid in microglia was observed by laser confocal microscopy, the autophagy-related condition of microglia was analyzed by MDC and acridine orange staining, the autophagy vesicle formation of microglia was detected by electron microscopy, and_Oxyglucose was used in vitro. In the deprivation model, HIF-1a inhibitors 2ME, YC-1 and HIF-1a RNA were used to block the expression of HIF-1a respectively, and the effects of HIF-1a on microglia autophagy were detected by these methods. _Oxygen-glucose deprivation model was used in vitro, autophagy inhibitors 3-MA and BafA1, and accelerator Rapa were used to treat microglia respectively to detect the survival of microglia. Rate; The survival rate of microglia was detected by using the RNAi of autophagy key regulatory protein Beclin1; _The middle cerebral artery embolization model was established in mice to detect the autophagy of microglia in ischemic area and the neurological function of mice; _The middle cerebral artery embolization model was established in mice, and autophagy inhibitor 3-M was injected into lateral ventricle. A, to detect autophagy, microglia autophagy and neurological function in mice after inhibition of autophagy.
Our results are as follows: 1) In the oxygen-glucose deprivation model in vitro, the mortality of microglia gradually increased and the survival rate gradually decreased with the prolongation of time; 2) In the oxygen-glucose deprivation model in vitro, the expression of inflammatory cytokines IL-8 and TNF-a gradually increased with the prolongation of time; 3) In the oxygen-glucose deprivation model in vitro, the expression of inflammatory cytokines IL-8 and TNF-a gradually increased. Blocking the expression of HIF-1a resulted in a decrease in microglial mortality and an increase in survival rate. _Blocking the expression of HIF-1a resulted in a decrease in the expression of inflammatory cytokines IL-8 and TNF-alpha. _Increased microglial autophagy in the oxygen-glucose deprivation model in vitro and_Blocking HIF-1 in the oxygen-glucose deprivation model in vitro. _in vitro oxygen-glucose deprivation model, inhibition of autophagy and Beclin-1 protein reduced the mortality of microglia and increased the survival rate; promote autophagy, microglia mortality increased, the survival rate decreased; _in vivo mouse middle cerebral artery embolism model, the ischemic area of microglia autophagy Autophagy inhibitor 3-MA can alleviate microglia autophagy and neurological deficit in ischemic area.
Conclusion: HIF-1a expression in microglia is up-regulated by hypoxic-ischemic environment, and autophagy pathway is activated by Beclin-1 signaling pathway, which leads to inflammation and death of microglia, and further aggravates neurological impairment. It provides ideal brain protection strategy.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R743.3
本文編號：2227101
[Abstract]:Ischemic stroke is a neurological disorder caused by interruption of cerebral blood flow. The incidence of ischemic stroke is high in acute cerebrovascular diseases, and has a high disability rate. It has seriously endangered human health. More than half of the survivors are accompanied by varying degrees of neurological disability. With the gradual increase of the elderly population in China, the number of ischemic stroke patients will further increase. Therefore, ischemic stroke has become the number one enemy of human health. It is of great scientific significance and clinical value to study the pathogenesis and treatment strategy of ischemic stroke.
Vascular nerve network theory, a new concept for the pathogenesis and treatment strategy of ischemic stroke, has been proposed. It is believed that vascular endothelial cells, neurons, glial cells and peripheral extracellular matrix constitute the vascular nerve unit complex. The theory holds that ischemic stroke is not a simple vascular event, nor a single neurological event, but a process of interaction and dialogue between blood vessels and nerves, between parenchymal cells and matrix, and of mutual regulation and balance. Neurons and cell components should be valued and protected rather than simply protecting certain cells. Previous studies on ischemic stroke have focused on neurons and vascular endothelial cells, but lack of in-depth study of glial cells. After stroke, it plays an important role in injury.
Microglia, like normal cells, can sense the stimulation of hypoxic signals under hypoxic conditions, activate the corresponding signaling pathways, activate the expression of corresponding genes, adapt to the harmful effects of hypoxic microenvironment on cells, and maintain the stability of the cellular environment. When cells are stimulated by hypoxic signals, a variety of genes are induced to express. Intermediate hypoxia inducible factor-1 (HIF-1) plays an important role in hypoxic adaptation. It has been shown that HIF-1 can activate downstream related genes rapidly in hypoxic environment, regulate intracellular energy metabolism and oxygen utilization, and play an important role in organs or local tissues. HIF-1 can play an important role in hypoxic adaptation, such as nutritional deficiency, injury, etc. Under harmful environment, especially under hypoxia stress, cells can regulate the expression of downstream genes accurately and precisely by activating the molecular switch of HIF-1, and maintain basic energy metabolism.
Autophagy is a special phenomenon that organisms retain in their evolutionary process. It is essentially a lysosomal degradation pathway in the process of cell survival, development, differentiation and metabolism. Its main role is to protect the stability of the body's internal environment and avoid being influenced by various external factors. Recent studies have shown that autophagy is not only fine-grained but also fine-grained. In addition to the homeostasis of cellular environment, it is also an important molecular mechanism involved in the regulation of many genes and the occurrence of many diseases, such as metabolic diseases, heart diseases, genetic diseases, tumors, inflammation and neurological diseases.
Then what are the pathophysiological characteristics of microglia in hypoxic and cerebral ischemia environment? How is the expression of HIF-1a in microglia? Is microglia autophagy occurring? What are the specific mechanisms of its occurrence and regulation? Does interfering with the above-mentioned links have a protective effect on the neurological function of cerebral ischemia? To confirm these doubts, we designed a series of experiments in this study to clarify the effect of HIF-1a expression on microglia autophagy and its biological significance in hypoxic and ischemic environment.
The contents of this study were as follows: 1) The survival rate of microglia was measured by PI and MTT at different time points of 0-48 hours after oxygen-glucose deprivation in vitro; 2) The expression of inflammatory cytokines IL-8 and TNF-alpha in microglia was detected by RT-PCR and ELISA at different time points of 0-48 hours after oxygen-glucose deprivation in vitro. Western blot was used to detect the expression of HIF-1a in microglia at different time points of 0-48 hours with oxygen-glucose deprivation model. In the model of glucose deprivation, the expression of HIF-1a was blocked by 2ME, YC-1 and RNA of HIF-1a respectively, and the expression of inflammatory cytokines IL-8 and TNF-alpha in microglia was detected by ELISA. _Oxygen-glucose deprivation model was used in vitro, and the autophagy correlation of microglia was detected by Western blot at different time points of 0-48 hours. The expression of protein LC3, the expression of GFP-LC3 plasmid in microglia was observed by laser confocal microscopy, the autophagy-related condition of microglia was analyzed by MDC and acridine orange staining, the autophagy vesicle formation of microglia was detected by electron microscopy, and_Oxyglucose was used in vitro. In the deprivation model, HIF-1a inhibitors 2ME, YC-1 and HIF-1a RNA were used to block the expression of HIF-1a respectively, and the effects of HIF-1a on microglia autophagy were detected by these methods. _Oxygen-glucose deprivation model was used in vitro, autophagy inhibitors 3-MA and BafA1, and accelerator Rapa were used to treat microglia respectively to detect the survival of microglia. Rate; The survival rate of microglia was detected by using the RNAi of autophagy key regulatory protein Beclin1; _The middle cerebral artery embolization model was established in mice to detect the autophagy of microglia in ischemic area and the neurological function of mice; _The middle cerebral artery embolization model was established in mice, and autophagy inhibitor 3-M was injected into lateral ventricle. A, to detect autophagy, microglia autophagy and neurological function in mice after inhibition of autophagy.
Our results are as follows: 1) In the oxygen-glucose deprivation model in vitro, the mortality of microglia gradually increased and the survival rate gradually decreased with the prolongation of time; 2) In the oxygen-glucose deprivation model in vitro, the expression of inflammatory cytokines IL-8 and TNF-a gradually increased with the prolongation of time; 3) In the oxygen-glucose deprivation model in vitro, the expression of inflammatory cytokines IL-8 and TNF-a gradually increased. Blocking the expression of HIF-1a resulted in a decrease in microglial mortality and an increase in survival rate. _Blocking the expression of HIF-1a resulted in a decrease in the expression of inflammatory cytokines IL-8 and TNF-alpha. _Increased microglial autophagy in the oxygen-glucose deprivation model in vitro and_Blocking HIF-1 in the oxygen-glucose deprivation model in vitro. _in vitro oxygen-glucose deprivation model, inhibition of autophagy and Beclin-1 protein reduced the mortality of microglia and increased the survival rate; promote autophagy, microglia mortality increased, the survival rate decreased; _in vivo mouse middle cerebral artery embolism model, the ischemic area of microglia autophagy Autophagy inhibitor 3-MA can alleviate microglia autophagy and neurological deficit in ischemic area.
Conclusion: HIF-1a expression in microglia is up-regulated by hypoxic-ischemic environment, and autophagy pathway is activated by Beclin-1 signaling pathway, which leads to inflammation and death of microglia, and further aggravates neurological impairment. It provides ideal brain protection strategy.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R743.3
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相關(guān)期刊論文 前3條
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