【摘要】：目的:大腦對(duì)缺氧最為敏感,缺氧可導(dǎo)致腦水腫和神經(jīng)細(xì)胞受損,嚴(yán)重影響大腦功能。對(duì)于不同程度的乏氧性缺氧(hypoxic hypoxia),大腦將表現(xiàn)出不同程度的神經(jīng)功能紊亂,如急性重度缺氧將引起頭痛、煩躁、驚厥、昏迷甚至死亡;慢性中度缺氧將引起疲勞、嗜睡、注意力不集中、記憶力下降等癥狀;而輕度及早期乏氧性缺氧發(fā)生時(shí)由于腦血管及星形膠質(zhì)細(xì)胞系統(tǒng)的代償性低氧保護(hù)反應(yīng),可為神經(jīng)元提供充分的氧供和能量底物,以維持大腦的正常生理功能,因此,研究輕度缺氧條件下中樞神經(jīng)系統(tǒng)(central nervous system,CNS)代償性低氧保護(hù)機(jī)制,對(duì)于乏氧性缺氧腦損傷疾病的預(yù)防和治療具有重要意義。CNS結(jié)構(gòu)與功能錯(cuò)綜復(fù)雜,神經(jīng)血管單元(neurovascular units,NVU)是其最小結(jié)構(gòu)與功能單位,該模型主要由腦微血管、星形膠質(zhì)細(xì)胞和神經(jīng)元共同構(gòu)造組成。星形膠質(zhì)細(xì)胞處于NVU物質(zhì)轉(zhuǎn)運(yùn)與信息傳遞的中心,充當(dāng)神經(jīng)元與微血管之間的紐帶與橋梁作用。當(dāng)星形膠質(zhì)細(xì)胞感受到氧供減少后,能夠啟動(dòng)代償性低氧保護(hù)反應(yīng),包括減少葡萄糖有氧氧化、增加無氧糖酵解,降低自身氧耗、促進(jìn)葡萄糖與乳酸轉(zhuǎn)運(yùn)、釋放血管新生因子等,是維持神經(jīng)元正常生理功能的重要代償機(jī)制。根據(jù)NVU結(jié)構(gòu)的特點(diǎn),腦微血管內(nèi)皮細(xì)胞(brain microvascular endothelial cell,BMEC)緊臨血液,可最先感知血氧分壓降低,其后才是星形膠質(zhì)細(xì)胞和神經(jīng)元。因此如能在星形膠質(zhì)細(xì)胞感受到氧供減少前啟動(dòng)代償性低氧保護(hù)反應(yīng),對(duì)于維持神經(jīng)元細(xì)胞的正常生理功能無疑具有更為重要的生理意義。在缺氧發(fā)生時(shí),BMEC最先感受到血氧分壓降低,并引起一系列的血管系統(tǒng)代償性低氧保護(hù)反應(yīng),而最重要也是最快速的代償方式是通過舒張血管的作用增加局部組織供氧量,此過程是內(nèi)皮細(xì)胞釋放一氧化氮(nitric oxide,NO),并擴(kuò)散至血管平滑肌細(xì)胞,通過活化可溶性鳥苷酸環(huán)化酶(s GC)的方式啟動(dòng)舒張血管作用,而內(nèi)皮細(xì)胞釋放的NO能否繼續(xù)擴(kuò)散至其他鄰近細(xì)胞,并發(fā)揮相應(yīng)的生理功能與效應(yīng),有待進(jìn)一步探索。NO是重要的細(xì)胞信使分子,參與機(jī)體多種生理或病理過程,在機(jī)體心血管系統(tǒng)、神經(jīng)系統(tǒng)和免疫系統(tǒng)中發(fā)揮重要的生物學(xué)作用。研究表明NO除了具有舒張血管的作用外,還參與低氧誘導(dǎo)因子-1α(hypoxia-inducible factor-1α,HIF-1α)的穩(wěn)定性調(diào)控,通過干預(yù)脯氨酰羥化酶(PHD)綁定Fe2+而阻斷PHD酶的活性,從而阻斷HIF-1α的降解,還可通過PI3K/Akt/m TOR信號(hào)通路誘導(dǎo)HIF-1α表達(dá)上調(diào)。鑒于NO分子可調(diào)控HIF-1α穩(wěn)定性與表達(dá)水平,并且是重要的內(nèi)源性細(xì)胞信使分子,無疑是理想的低氧信號(hào)傳導(dǎo)分子,因此我們將NO分子作為重要的非氧氣的調(diào)節(jié)因素與信息分子進(jìn)行低氧信號(hào)傳導(dǎo)的研究對(duì)象。既然輕度缺氧時(shí),BMEC釋放的NO能夠作為低氧信號(hào)擴(kuò)散至血管平滑肌細(xì)胞產(chǎn)生舒張血管的作用,那么BMEC釋放的NO能否繼續(xù)擴(kuò)散至鄰近的星形膠質(zhì)細(xì)胞,并在其未感知缺氧時(shí)通過HIF-1α啟動(dòng)代償性低氧保護(hù)反應(yīng)?有待研究證實(shí)。基于上述分析,本研究擬分離培養(yǎng)大鼠原代腦星形膠質(zhì)細(xì)胞和原代BMEC,探索腦星形膠質(zhì)細(xì)胞未感受缺氧的氧氣閾值濃度,并在此缺氧條件下研究外源性給藥產(chǎn)生和共培養(yǎng)BMEC源性釋放的NO預(yù)處理能否阻斷腦星形膠質(zhì)細(xì)胞HIF-1α蛋白的降解,以及對(duì)葡萄糖代謝與無氧糖酵解、葡萄糖和乳酸轉(zhuǎn)運(yùn)、血管新生等相關(guān)基因的轉(zhuǎn)錄和表達(dá)調(diào)控;同時(shí)對(duì)照清除NO或抑制NO生成,并結(jié)合運(yùn)用RNAi技術(shù)干擾HIF-1α基因轉(zhuǎn)錄,探索NO-低氧信號(hào)如何介導(dǎo)腦星形膠質(zhì)細(xì)胞的低氧保護(hù)反應(yīng),以期初步闡明BMEC源性NO介導(dǎo)的低氧信號(hào)傳導(dǎo)啟動(dòng)腦星形膠質(zhì)細(xì)胞低氧保護(hù)反應(yīng)的作用機(jī)制,為進(jìn)一步研究乏氧性缺氧腦損傷疾病的致病機(jī)制與干預(yù)保護(hù)措施奠定基礎(chǔ)。方法:第一部分大鼠腦星形膠質(zhì)細(xì)胞、腦微血管內(nèi)皮細(xì)胞的原代分離培養(yǎng)與鑒定1.分離培養(yǎng)原代腦星形膠質(zhì)細(xì)胞及細(xì)胞鑒定:采用一步酶消化及振蕩純化的方法分離培養(yǎng)大鼠大腦星形膠質(zhì)細(xì)胞;免疫熒光染色法檢測(cè)GFAP的表達(dá)用于腦星形膠質(zhì)細(xì)胞的鑒定;2.分離培養(yǎng)原代腦微血管內(nèi)皮細(xì)胞及細(xì)胞鑒定:采用兩步酶消化、梯度密度離心及嘌呤霉素純化的方法分離培養(yǎng)大鼠大腦微血管內(nèi)皮細(xì)胞;免疫熒光染色法檢測(cè)v WF相關(guān)抗原的表達(dá)用于腦微血管內(nèi)皮細(xì)胞的鑒定。第二部分外源性NO啟動(dòng)輕度缺氧星形膠質(zhì)細(xì)胞低氧保護(hù)反應(yīng)的實(shí)驗(yàn)研究1.不同氧氣濃度條件對(duì)原代腦星形膠質(zhì)細(xì)胞HIF-1α蛋白水平的影響:Western blot檢測(cè)21%、9%、7%、5%、3%和1%氧氣濃度條件下星形膠質(zhì)細(xì)胞HIF-1α蛋白水平,以獲得星形膠質(zhì)細(xì)胞未感受缺氧的氧氣閾值濃度;2.5%O_2輕度缺氧條件下,DETA(NO供體)對(duì)原代腦星形膠質(zhì)細(xì)胞HIF-1α蛋白水平的影響:Western blot檢測(cè)DETA不同劑量處理和不同處理時(shí)間后腦星形膠質(zhì)細(xì)胞HIF-1α蛋白水平;3.5%O_2輕度缺氧條件下,DETA對(duì)原代腦星形膠質(zhì)細(xì)胞葡萄糖代謝相關(guān)酶基因、葡萄糖和乳酸轉(zhuǎn)運(yùn)體蛋白基因、血管內(nèi)皮生長(zhǎng)因子(VEGF)基因轉(zhuǎn)錄水平,VEGF蛋白表達(dá)水平和乳酸(LAC)釋放水平的影響:RT-q PCR檢測(cè)DETA處理后腦星形膠質(zhì)細(xì)胞PDK1、LDHA、LDHB、HK1、HK2、GLUT1、GLUT3、MCT1、MCT2、MCT4、VEGF基因轉(zhuǎn)錄水平;Western blot檢測(cè)DETA處理后腦星形膠質(zhì)細(xì)胞VEGF蛋白水平,酶促動(dòng)力學(xué)比色法檢測(cè)培養(yǎng)液LAC釋放水平;4.干擾原代腦星形膠質(zhì)細(xì)胞HIF-1α基因轉(zhuǎn)錄后,DETA對(duì)5%O_2輕度缺氧條件下腦星形膠質(zhì)細(xì)胞葡萄糖代謝相關(guān)酶基因、葡萄糖和乳酸轉(zhuǎn)運(yùn)體蛋白基因、VEGF基因轉(zhuǎn)錄水平,VEGF蛋白表達(dá)水平和LAC釋放水平的影響:采用HIF-1α特異性si RNA干擾原代腦星形膠質(zhì)細(xì)胞HIF-1α基因轉(zhuǎn)錄,RT-q PCR和Western blot鑒定細(xì)胞轉(zhuǎn)染效率;RT-q PCR檢測(cè)DETA處理后腦星形膠質(zhì)細(xì)胞PDK1、LDHA、HK1、HK2、GLUT1、MCT4、VEGF基因轉(zhuǎn)錄水平;Western blot檢測(cè)DETA處理后星形膠質(zhì)細(xì)胞VEGF蛋白水平,酶促動(dòng)力學(xué)比色法檢測(cè)培養(yǎng)液LAC釋放水平。第三部分腦微血管內(nèi)皮細(xì)胞源性NO啟動(dòng)輕度缺氧星形膠質(zhì)細(xì)胞低氧保護(hù)反應(yīng)的實(shí)驗(yàn)研究1.5%O_2輕度缺氧條件下,原代腦微血管內(nèi)皮細(xì)胞和原代腦星形膠質(zhì)細(xì)胞NO的釋放水平:熒光分析法分別檢測(cè)腦微血管內(nèi)皮細(xì)胞和腦星形膠質(zhì)細(xì)胞培養(yǎng)液中Nitrite/Nitrate水平;2.5%O_2輕度缺氧條件下,腦微血管內(nèi)皮細(xì)胞源性NO對(duì)原代腦星形膠質(zhì)細(xì)胞HIF-1α蛋白水平的影響:采用Transwell培養(yǎng)板進(jìn)行原代腦微血管內(nèi)皮細(xì)胞與原代腦星形膠質(zhì)細(xì)胞共培養(yǎng),Western blot檢測(cè)共培養(yǎng)后腦星形膠質(zhì)細(xì)胞HIF-1α蛋白水平;3.5%O_2輕度缺氧條件下,腦微血管內(nèi)皮細(xì)胞源性NO對(duì)原代腦星形膠質(zhì)細(xì)胞葡萄糖代謝相關(guān)酶基因、葡萄糖和乳酸轉(zhuǎn)運(yùn)體蛋白基因、VEGF基因轉(zhuǎn)錄水平,VEGF蛋白表達(dá)水平和LAC釋放水平的影響:采用Transwell培養(yǎng)板進(jìn)行原代腦微血管內(nèi)皮細(xì)胞與原代腦星形膠質(zhì)細(xì)胞共培養(yǎng);RT-q PCR檢測(cè)腦星形膠質(zhì)細(xì)胞PDK1、LDHA、HK1、HK2、GLUT1、MCT4、VEGF基因轉(zhuǎn)錄水平;Western blot檢測(cè)原代腦星形膠質(zhì)細(xì)胞VEGF蛋白水平;酶促動(dòng)力學(xué)比色法檢測(cè)培養(yǎng)液LAC釋放水平。結(jié)果:第一部分:1.獲得原代培養(yǎng)的大鼠大腦星形膠質(zhì)細(xì)胞,純度為98.4%;2.獲得原代培養(yǎng)的大鼠腦微血管內(nèi)皮細(xì)胞,純度為95.2%。第二部分:1.與常氧條件(21%O_2)相比,1~3%氧氣濃度條件下,原代腦星形膠質(zhì)細(xì)胞HIF-1α蛋白水平顯著升高,而5~9%氧氣濃度條件下,HIF-1α蛋白水平無顯著性變化,提示5%O_2氧氣濃度條件是原代腦星形膠質(zhì)細(xì)胞未感受缺氧的臨界濃度,我們將此低氧條件定義本實(shí)驗(yàn)研究的輕度缺氧條件;2.5%O_2輕度缺氧條件下,1.0 m M DETA(NO供體)預(yù)處理12 h能夠誘導(dǎo)原代腦星形膠質(zhì)細(xì)胞HIF-1α蛋白水平上調(diào),但能被NO清除劑抑制;3.5%O_2輕度缺氧條件下,DETA預(yù)處理后原代腦星形膠質(zhì)細(xì)胞PDK1、LDHA、HK1、HK2、GLUT1、GLUT3、MCT4、VEGF基因轉(zhuǎn)錄水平顯著升高,VEGF蛋白水平和LAC釋放水平顯著升高;4.5%O_2輕度缺氧條件下,干擾原代腦星形膠質(zhì)細(xì)胞HIF-1α基因轉(zhuǎn)錄能夠抑制DETA上調(diào)PDK1、LDHA、HK1、HK2、GLUT1、GLUT3、MCT4、VEGF基因轉(zhuǎn)錄,抑制VEGF蛋白表達(dá)和LAC釋放。第三部分:1.5%O_2輕度缺氧條件下,原代腦星形膠質(zhì)細(xì)胞產(chǎn)生和釋放的NO較少(86±41 n M Nitrite/Nitrate),而原代腦微血管內(nèi)皮細(xì)胞能夠釋放一定量的NO(832±83 n M Nitrite/Nitrate,與0.8 m M DETA釋放的Nitrite/Nitrate峰值濃度相當(dāng)),但能被NOS抑制劑抑制;2.5%O_2輕度缺氧條件下,原代腦微血管內(nèi)皮細(xì)胞與原代腦星形膠質(zhì)細(xì)胞共培養(yǎng)后能夠誘導(dǎo)腦星形膠質(zhì)細(xì)胞HIF-1α蛋白水平的上調(diào),但能被NOS抑制劑抑制;3.5%O_2輕度缺氧條件下,原代腦微血管內(nèi)皮細(xì)胞與原代腦星形膠質(zhì)細(xì)胞共培養(yǎng)后能夠上調(diào)腦星形膠質(zhì)細(xì)胞PDK1、LDHA、HK1、HK2、GLUT1、MCT4、VEGF基因轉(zhuǎn)錄,上調(diào)VEGF蛋白表達(dá)和LAC釋放,但能被NOS抑制劑抑制。結(jié)論:在5%O_2輕度缺氧條件下,體外培養(yǎng)的原代大鼠腦星形膠質(zhì)細(xì)胞未感受到低氧,而此低氧條件下給予NO供體藥物和共培養(yǎng)腦微血管內(nèi)皮細(xì)胞均能夠使腦星形膠質(zhì)細(xì)胞感受到低氧,并上調(diào)HIF-1α下游的與無氧糖酵解、葡萄糖和乳酸轉(zhuǎn)運(yùn)、血管新生等低氧代償作用相關(guān)的酶或因子的基因轉(zhuǎn)錄或表達(dá);對(duì)照清除NO或抑制NO生成,以及干擾HIF-1α基因轉(zhuǎn)錄的結(jié)果表明,NO通過阻斷HIF-1α的降解,上調(diào)其下游的基因轉(zhuǎn)錄或表達(dá)。綜上所述,輕度缺氧時(shí)原代大鼠腦微血管內(nèi)皮細(xì)胞釋放的NO可作為低氧信號(hào),擴(kuò)散傳導(dǎo)至腦星形膠質(zhì)細(xì)胞,并通過HIF-1α啟動(dòng)腦星形膠質(zhì)細(xì)胞的低氧代償性保護(hù)反應(yīng)。本研究豐富了NO作為內(nèi)源性細(xì)胞信號(hào)分子的效應(yīng)機(jī)制,闡釋了一種輕度缺氧時(shí)神經(jīng)血管單元細(xì)胞間低氧信號(hào)的傳導(dǎo)途徑,為進(jìn)一步研究乏氧性缺氧腦損傷疾病的致病機(jī)制與干預(yù)保護(hù)措施奠定了基礎(chǔ)。
[Abstract]:Objective: the brain is most sensitive to hypoxia. Hypoxia can lead to brain edema and nerve cell damage, which seriously affect the brain function. For different degrees of hypoxia (hypoxic hypoxia), the brain will show different degrees of nerve dysfunction, such as acute severe anoxia, which will lead to headache, irritability, convulsion, coma and even death; chronic moderate deficiency. Oxygen will cause symptoms such as fatigue, lethargy, concentration of attention, and decline in memory; while mild and early anoxic hypoxia can provide sufficient oxygen supply and energy substrates for neurons to maintain normal physiological functions of the brain and study mild hypoxia at the time of hypoxic hypoxia. Under conditions, the compensatory hypoxic protection mechanism of the central nervous system (CNS) is of great significance for the prevention and treatment of hypoxic hypoxic brain damage. The.CNS structure and function are complex, and the neurovascular units (NVU) is the smallest structure and function unit. The model is mainly from the brain microvessel and the star. Astrocytes and neurons are constructed together. Astrocytes are located at the center of NVU material transport and information transfer, acting as a link between neurons and microvessels. When the astrocytes feel the oxygen supply is reduced, it can start compensatory hypoxic protection reverse, including reducing glucose oxygen oxidation and increasing no oxygen. Oxygen glycolysis, reduce oxygen consumption, promote the transport of glucose and lactic acid, release angiogenesis factors and so on. It is an important compensatory mechanism to maintain normal physiological function of neurons. According to the characteristics of NVU structure, brain microvascular endothelial cell (BMEC) is closely associated with blood. It is astrocytes and neurons. Therefore, it is undoubtedly of more important physiological significance to maintain the normal physiological function of neuron cells before the oxygen supply is reduced in astrocytes. In the case of hypoxia, BMEC first feels the reduction of blood oxygen pressure and causes a series of blood vessels. The system compensatory hypoxic protection reaction, and the most important and fastest way of compensation is the increase of local tissue oxygen supply through the role of diastolic blood vessels. This process is that endothelial cells release nitric oxide (nitric oxide, NO) and spread to vascular smooth muscle cells and activate diastolic blood by activating soluble guanosine cyclase (s GC). Whether or not the NO can continue to spread to other adjacent cells and play the corresponding physiological functions and effects, it is necessary to further explore.NO as an important cell messenger, participate in various physiological or pathological processes of the body, and play an important biological role in the body's cardiovascular system, the nervous system and the immune system. In addition to the effect of diastolic blood vessels, NO also participates in the regulation of the stability of the hypoxia inducible factor -1 alpha (hypoxia-inducible factor-1 a, HIF-1 alpha), blocking the activity of the PHD enzyme by interfering with the binding of Fe2+ to the prolyyl hydroxylase (PHD) and blocking the degradation of the HIF-1 alpha, and can also induce the expression of the HIF-1 alpha by PI3K/Akt/m TOR signal pathway. In view of the fact that NO molecules can regulate the stability and expression level of HIF-1 alpha and are important endogenous cell messenger molecules, it is undoubtedly an ideal hypoxia signal transduction molecule, so we use NO molecules as an important regulator of non oxygen and information molecules to carry out the study of hypoxia signal transduction. Since mild hypoxia, BMEC release NO can be used as a hypoxic signal to spread to vascular smooth muscle cells to produce diastolic blood vessels, then can the NO released by BMEC continue to spread to adjacent astrocytes and activate compensatory hypoxic protection by HIF-1 alpha in the absence of hypoxia? Primary astrocytes and primary BMEC were used to explore the oxygen threshold concentration in the brain astrocytes, and the study of NO preconditioning for exogenous drug delivery and co culture of BMEC derived release under this hypoxia condition could block the degradation of HIF-1 alpha protein in astrocytes, as well as glucose metabolism and anaerobic glycolysis, The transcriptional and expression regulation of glucose and lactic acid transport, angiogenesis and other related genes, simultaneously control NO or inhibit NO production, and combine with RNAi technology to interfere with HIF-1 alpha gene transcription, explore how NO- hypoxia signal mediates the hypoxic protective response of astrocytes in brain astrocytes, in order to clarify the hypoxia signal conduction mediated by BMEC derived NO. The mechanism of activating the hypoxic protective reaction of astrocytes in the brain was initiated to lay the foundation for further study on the pathogenesis and protective measures of hypoxic hypoxic brain injury. Methods: the first part of the rat brain astrocytes, the primary separation culture and identification of the cerebral microvascular endothelial cells were isolated and cultured for the isolation and culture of the primary astroglia. Cell and cell identification: the rat brain astrocytes were isolated and cultured with one step enzyme digestion and oscillatory purification. Immunofluorescence staining was used to detect the expression of GFAP in the identification of astrocytes. 2. the isolation and culture of primary cerebral microvascular endothelial cells and cell identification: two step enzyme digestion, gradient density centrifugation and purinamycin Purified rat brain microvascular endothelial cells were isolated and cultured; immunofluorescence staining was used to detect the expression of V WF related antigen in the identification of cerebral microvascular endothelial cells. Second the experimental study on the initiation of hypoxic protective reaction of mild hypoxic astrocytes by exogenous NO; 1. different oxygen concentration conditions for the primary astroglia Effect of cellular HIF-1 alpha protein level: Western blot detection of astrocytes HIF-1 alpha protein level in 21%, 9%, 7%, 5%, 3% and 1% oxygen concentrations to obtain oxygen threshold concentration in astrocytes without hypoxia; the effect of DETA (NO donor) on the level of HIF-1 a protein of primary astrocytes under mild 2.5%O_2: West Ern blot was used to detect the HIF-1 alpha protein level of astrocytes in DETA after different doses of treatment and different processing time; DETA gene, glucose and lactic acid transporter gene, VEGF gene transcription level, VEGF protein table under mild hypoxia in 3.5%O_2 RT-q PCR detection of PDK1, LDHA, LDHB, HK1, HK2, GLUT1, GLUT3, LDHB, GLUT1, GLUT3, RT-q PCR The effect of DETA on glucose metabolism related enzyme gene, glucose and lactic acid transporter gene, VEGF gene transcriptional level, VEGF protein expression level and LAC release level in the brain astrocytes of primary astrocytes after HIF-1 alpha gene transcription: HIF-1 alpha specific Si RNA interfering with the original brain astrocytes. HIF-1 alpha gene transcription of glial cells, RT-q PCR and Western blot identification of cell transfection efficiency, RT-q PCR detection of PDK1, LDHA, HK1, HK2, and transcriptional level after DETA treated astrocytes An experimental study on the initiation of hypoxic protective reaction of mild hypoxic astrocytes in the third part of cerebral microvascular endothelial cell derived NO, the release level of NO in primary cerebral microvascular endothelial cells and primary brain astrocytes under mild hypoxic conditions in 1.5%O_2: fluorescence analysis for the detection of cerebral microvascular endothelial cells and astrocytes respectively Nitrite/Nitrate level in the culture medium; the effect of NO on the level of HIF-1 alpha protein of primary brain astrocytes under mild hypoxic 2.5%O_2: Transwell culture plate was used to co culture the primary cerebral microvascular endothelial cells and the primary astrocytes, and the Western blot was used to detect the astrocytes after the culture of the brain astrocytes. The level of cell HIF-1 alpha protein; the effect of NO on glucose metabolism related enzyme gene, glucose and lactic acid transporter gene, VEGF gene transcription level, VEGF protein expression level and LAC release level of primary brain astrocytes under mild hypoxic condition of 3.5%O_2: using Transwell culture plate for primary generation Cerebral microvascular endothelial cells were co cultured with primary astrocytes; RT-q PCR was used to detect astrocytes PDK1, LDHA, HK1, HK2, GLUT1, MCT4, and VEGF transcriptional level; Western blot was used to detect the level of the primary astrocyte VEGF protein; enzyme kinetics colorimetric assay was used to detect the release level. Results: the first part: 1. obtained the original The purity of the cultured rat brain astrocytes was 98.4%; 2. obtained the primary cultured rat brain microvascular endothelial cells, and the purity was 95.2%. second: 1. compared with the normal oxygen condition (21%O_2), the level of HIF-1 alpha protein in the primary astrocytes was significantly increased under the 1~3% oxygen concentration condition, and the HIF-1 alpha protein under the 5~9% oxygen concentration condition. There is no significant change in level, suggesting that the 5%O_2 oxygen concentration condition is the critical concentration of the primary brain astrocytes in the absence of hypoxia. We define this hypoxic condition as a mild hypoxic condition. Under mild hypoxia, 1 m M DETA (NO donor) preconditioning 12 h can induce primary astrocyte HIF-1 alpha protein. The level was up, but could be suppressed by NO scavenger. Under mild hypoxia, 3.5%O_2, LDHA, HK1, HK2, GLUT1, GLUT3, MCT4, VEGF gene transcriptional level increased significantly after DETA pretreatment, and the level of VEGF protein and the release level increased significantly. Gene transcription can inhibit DETA up regulation of PDK1, LDHA, HK1, HK2, GLUT1, GLUT3, MCT4, VEGF gene transcription, inhibition of VEGF protein expression and LAC release. The third part: under the mild hypoxia conditions, the generation and release of primary brain astrocytes are less (86 + 41), and the primary cerebral microvascular endothelial cells can release a certain amount. NO (832 + 83 n M Nitrite/Nitrate, equivalent to the peak concentration of Nitrite/Nitrate released by 0.8 m M DETA), but can be inhibited by NOS inhibitors; under mild hypoxic condition, the primary brain microvascular endothelial cells co cultured with the primary astrocytes can induce the up regulation of astrocyte HIF-1 alpha protein level, but can be suppressed by NOS. Preparation inhibition; under mild hypoxic condition of 3.5%O_2, primary cerebral microvascular endothelial cells co cultured with primary astrocytes can up regulate astrocyte PDK1, LDHA, HK1, HK2, GLUT1, MCT4, VEGF gene transcription, up regulation of VEGF protein expression and LAC release, but can be inhibited by NOS inhibitor. Conclusion: under mild hypoxia conditions, in vitro, in vitro The cultured primary rat astrocytes did not feel hypoxic, and the NO donor drugs and co cultured brain microvascular endothelial cells under this hypoxic condition could make the brain astrocytes feel hypoxic, and up regulation of HIF-1 alpha downstream and anaerobic glycolysis, glucose and lactic acid transport, angiogenesis and other low oxygen compensatory effects The transcriptional or expression of the gene of the enzyme or factor; the results of eliminating NO or inhibiting NO production, and interfering with the transcription of HIF-1 a gene indicate that NO can up regulate the transcription or expression of the downstream genes by blocking the degradation of HIF-1 a. To sum up, the NO released by the primary rat cerebral microvascular endothelial cells in mild hypoxia can be used as a hypoxic signal and diffusion conduction. This study enriches the effect mechanism of NO as an endogenous cell signal molecule, and explains the conduction pathway of the hypoxia signal in the neurons of the neurovascular unit at the time of mild hypoxia, in order to further study the hypoxic hypoxic brain damage disease, and further study the disease of hypoxic hypoxic brain damage. The pathogenesis and intervention measures laid the foundation.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R742

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