缺氧誘導(dǎo)因子及其調(diào)節(jié)基因在化學(xué)缺氧預(yù)處理保護(hù)中的作用研究
發(fā)布時間：2018-08-08 13:54
【摘要】： 缺氧與許多臨床疾病有關(guān),也是高原、高空、深水等特殊環(huán)境中常見的一種致病因素。雖然不同類型細(xì)胞對缺氧的敏感性不同,但是,隨著缺氧程度和持續(xù)時間的增加,最終都會引起細(xì)胞代謝和功能障礙,甚至導(dǎo)致細(xì)胞死亡。神經(jīng)細(xì)胞強(qiáng)烈依賴有氧代謝進(jìn)行能量供應(yīng),對缺氧特別敏感,因而容易因?yàn)槿毖跏艿綋p傷。尋找促進(jìn)神經(jīng)元對低氧產(chǎn)生耐受的機(jī)制,對臨床相關(guān)疾病的預(yù)防和治療有非常重要的意義。近年來有研究顯示,缺氧預(yù)處理可以保護(hù)神經(jīng)元對后繼發(fā)生的嚴(yán)重缺氧或其它致死性應(yīng)激產(chǎn)生耐受,因此,缺氧預(yù)處理的神經(jīng)保護(hù)作用受到越來越多學(xué)者的關(guān)注。盡管多種機(jī)制和分子被報(bào)道可能參與缺氧預(yù)處理的神經(jīng)保護(hù)作用[1],但是這些分子最終怎樣發(fā)揮保護(hù)作用還不確定,沒有一個清晰系統(tǒng)的預(yù)適應(yīng)機(jī)制受到普遍認(rèn)可。缺氧誘導(dǎo)因子可被多種損傷因素誘導(dǎo),也可調(diào)控許多信號途徑,是介導(dǎo)缺氧信號與眾多缺氧誘導(dǎo)基因轉(zhuǎn)錄激活的一個重要調(diào)控因子,可通過使細(xì)胞適應(yīng)低氧環(huán)境,發(fā)揮促進(jìn)細(xì)胞生存的重要作用。有關(guān)缺氧誘導(dǎo)因子及其調(diào)控基因/產(chǎn)物功能及作用的研究已經(jīng)成為當(dāng)前的熱點(diǎn)和前沿[2, 3]。針對缺氧誘導(dǎo)因子及其調(diào)節(jié)基因/產(chǎn)物在缺氧預(yù)處理中的作用和機(jī)制,目前有許多有爭議的報(bào)道[4, 5]。如有研究顯示,HIF-1的表達(dá)增加在缺氧預(yù)處理中可能具有非常重要的作用。然而,亦有文獻(xiàn)報(bào)道缺氧預(yù)處理通過抑制HIF-1活性產(chǎn)生保護(hù)作用。還有報(bào)道顯示,缺氧預(yù)處理不依賴HIF-1的途徑產(chǎn)生保護(hù)作用。因而,缺氧誘導(dǎo)因子及其調(diào)節(jié)基因/產(chǎn)物在缺氧預(yù)處理中的作用仍不清楚,有待進(jìn)一步研究。CoCl2是常用的缺氧模擬化合物,CoCl2預(yù)處理可產(chǎn)生與缺氧預(yù)處理相似的神經(jīng)保護(hù)作用[6]。本課題通過分化的神經(jīng)型SH-SY5Y細(xì)胞建立CoCl2化學(xué)缺氧預(yù)處理的神經(jīng)型細(xì)胞保護(hù)模型,對缺氧誘導(dǎo)因子1、2及其調(diào)節(jié)基因VEGF、GLUT-1、EPO、LDH-A等在CoCl2預(yù)處理中的作用進(jìn)行初步的探討,并通過應(yīng)用重組人VEGF和可抑制VEGF活性的VEGF抗體,對缺氧誘導(dǎo)基因產(chǎn)物VEGF的缺氧保護(hù)作用進(jìn)行研究。主要的實(shí)驗(yàn)方法、結(jié)果和結(jié)論如下: 方法 1.人神經(jīng)母細(xì)胞瘤SH-SY5Y常規(guī)培養(yǎng)于含15 %胎牛血清(FCS)的MEM/F12(1:1體積比)培養(yǎng)液,10μmol/L維甲酸(RA)誘導(dǎo)細(xì)胞分化3-7d后用于實(shí)驗(yàn)。 2.分化的SH-SY5Y細(xì)胞隨機(jī)分為對照組、化學(xué)缺氧預(yù)處理組(細(xì)胞先用50μmol/LCoCl2預(yù)處理3 h,換液后常氧培養(yǎng)1 h,然后在2%的低氧孵箱內(nèi)缺氧28 h)、缺氧組(2%的低氧孵箱內(nèi)缺氧28 h)。或?qū)⒎只腟H-SY5Y細(xì)胞隨機(jī)分為對照組C (常氧培養(yǎng));化學(xué)缺氧預(yù)處理組HP(細(xì)胞先用75μmol/L CoCl2預(yù)處理1.5 h,換液后常氧培養(yǎng)3 h,然后用250μmol/LCoCl2化學(xué)缺氧24 h);化學(xué)缺氧組H(250μmol/LCoCl2化學(xué)缺氧24 h)。通過乳酸脫氫酶釋放率測定、MTT細(xì)胞活力測定判斷上述各組細(xì)胞的損傷程度。 3. RT-PCR測細(xì)胞的VEGF、GLUT-1、EPO、LDH-A的mRNA表達(dá)。 4.用Western Blotting法測細(xì)胞VEGF的蛋白表達(dá)。 5.分化的SH-SY5Y細(xì)胞隨機(jī)分為對照組、化學(xué)缺氧預(yù)處理組(細(xì)胞先用50μmol/LCoCl2預(yù)處理3 h,換液后常氧培養(yǎng)1 h,然后在2%的低氧孵箱內(nèi)缺氧28 h)、化學(xué)缺氧預(yù)處理+VEGF抗體組(細(xì)胞加40μg/ml VEGF單克隆抗體,其余同化學(xué)缺氧預(yù)處理組)、缺氧組(2%的低氧孵箱內(nèi)缺氧28 h)、缺氧+VEGF組(細(xì)胞加100 ng/ml VEGF純品,其余同缺氧組)。通過MTT比色法測各組細(xì)胞的活力。 6.用Western Blotting法測細(xì)胞HIF-1α、HIF-2α蛋白表達(dá)。 結(jié)果 1.化學(xué)缺氧預(yù)處理組細(xì)胞較化學(xué)缺氧組細(xì)胞活力顯著增高(P0.05),乳酸脫氫酶釋放率顯著減少(P0.05)。2.化學(xué)缺氧預(yù)處理組細(xì)胞較常壓缺氧組細(xì)胞活力顯著增高(P0.05),乳酸脫氫酶釋放率顯著減少(P0.01)。 3.化學(xué)缺氧預(yù)處理組細(xì)胞GLUT-1、EPO mRNA表達(dá)顯著高于缺氧組(P0.05),VEGFmRNA表達(dá)顯著高于缺氧組(P0.01),LDH-AmRNA表達(dá)在兩組之間沒有顯著性差異。 4.化學(xué)缺氧預(yù)處理組細(xì)胞VEGF蛋白表達(dá)顯著高于缺氧組(P0.01)。 5. MTT細(xì)胞活力測定顯示40μg/mlVEGF單克隆抗體可抑制化學(xué)缺氧預(yù)處理的保護(hù)作用,而100 ng/ml重組人VEGF可模擬化學(xué)缺氧預(yù)處理組的保護(hù)作用。 6.化學(xué)缺氧預(yù)處理組細(xì)胞HIF-2α蛋白表達(dá)顯著高于缺氧組(P0.01),HIF-1α蛋白表達(dá)在兩組之間沒有顯著性差異。 結(jié)論 1.通過乳酸脫氫酶釋放率和MTT分析,建立了CoCl2化學(xué)缺氧預(yù)處理抵抗CoCl2化學(xué)缺氧的神經(jīng)型細(xì)胞模型。 2.通過乳酸脫氫酶釋放率和MTT分析,建立了CoCl2化學(xué)缺氧預(yù)處理保護(hù)神經(jīng)型細(xì)胞對常壓缺氧產(chǎn)生耐受的預(yù)處理模型。 3. CoCl2化學(xué)缺氧預(yù)處理抵抗常壓缺氧的效果較CoCl2化學(xué)缺氧預(yù)處理抵抗CoCl2化學(xué)缺氧的效果更強(qiáng)。 4.化學(xué)缺氧預(yù)處理可能通過促進(jìn)缺氧調(diào)節(jié)基因GLUT-1、EPO、VEGF的表達(dá)而產(chǎn)生保護(hù)作用。 5. VEGF在化學(xué)缺氧預(yù)處理中具有非常重要的保護(hù)作用。 6.化學(xué)缺氧預(yù)處理可能通過HIF-2,而不是HIF-1,促進(jìn)包括VEGF在內(nèi)的缺氧調(diào)節(jié)基因表達(dá)產(chǎn)生耐缺氧的保護(hù)作用。
[Abstract]:Hypoxia is associated with many clinical diseases. It is also a common pathogenic factor in special environments such as high altitude, high altitude and deep water. Although the sensitivity of different types of cells to hypoxia is different, it will eventually cause cell metabolism and dysfunction and even cell death with the increase of hypoxia and duration. Nerve cells are strong. It is dependent on oxygen metabolism to supply energy and is particularly sensitive to hypoxia, so it is easily damaged by hypoxia. Finding a mechanism to promote the tolerance of neurons to hypoxia is of great significance for the prevention and treatment of clinical related diseases. In recent years, studies have shown that hypoxia preconditioning can protect neurons from subsequent occurrence. Hypoxia or other fatal stress is tolerated, so the neuroprotective effect of hypoxic preconditioning is concerned by more and more scholars. Although a variety of mechanisms and molecules are reported to be involved in the neuroprotective effect of hypoxic preconditioning [1], how these molecules are ultimately protected is uncertain and no clear system is predefined. The adaptation mechanism is universally recognized. Hypoxia inducible factor can be induced by a variety of damage factors and can also regulate many signal pathways. It is an important regulator to mediate the activation of hypoxia signal and many hypoxia induced genes. It can play an important role in promoting cell survival by adapting the cells to the hypoxia environment. The research on the function and function of the genes / products of their regulatory genes / products has become the current hot and frontier [2. 3]. has a number of controversial reports on the role and mechanism of hypoxia inducible factor and its regulatory gene / product in anoxic preconditioning. 5]., such as [4, has shown that the increase of HIF-1 expression may have no effect in anoxic preconditioning. However, it is also reported that hypoxia preconditioning has protective effects on HIF-1 activity. It is also reported that hypoxia preconditioning does not depend on the HIF-1 pathway. Therefore, the role of hypoxia inducible factor and its regulatory gene / product in anoxia preconditioning remains unclear, and.CoCl2 needs to be further studied. It is a common anoxic analogue compound, CoCl2 preconditioning can produce neuroprotective effect similar to hypoxic preconditioning [6]., a neurotype protective model of CoCl2 chemical anoxia preconditioning was established through differentiated neural SH-SY5Y cells, and the hypoxic inducible factor 1,2 and its regulatory gene VEGF, GLUT-1, EPO, LDH-A, etc. were pretreated by CoCl2 The role in the study was preliminarily discussed, and the anoxia protective effect of hypoxia induced gene product VEGF was studied by the application of recombinant human VEGF and the VEGF antibody that could inhibit the activity of VEGF. The main experimental methods, results and conclusions were as follows:
Method
The 1. human neuroblastoma SH-SY5Y was routinely cultured on the MEM/F12 (1:1 volume ratio) culture medium containing 15% fetal bovine serum (FCS), and 10 mu mol/L retinoic acid (RA) induced the cells to differentiate into 3-7d and was used for the experiment.
2. differentiated SH-SY5Y cells were randomly divided into control group, chemical anoxia preconditioning group (cells pretreated first with 50 mu mol/LCoCl2 3 h, oxygen culture 1 h after replacement, and 28 h in 2% hypoxia incubator), hypoxia group (2% hypoxic incubator 28 h). Or the differentiated SH-SY5Y cells were randomly divided into control group C (normal oxygen culture); chemical hypoxia preconditioning The treatment group HP (the cells first treated 1.5 h with 75 mu CoCl2, 3 h after the change of liquid oxygen, then 250 micron mol/LCoCl2 chemical anoxia 24 h), and the chemical anoxic group H (250 u mol/LCoCl2 chemical hypoxia 24 h). The damage degree of the cells was determined by the determination of the lactate dehydrogenase release rate and the activity of MTT cells.
3. RT-PCR was used to measure the expression of VEGF, GLUT-1, EPO and LDH-A in cells.
4. the protein expression of VEGF was measured by Western Blotting.
5. differentiated SH-SY5Y cells were randomly divided into control group, chemical anoxia preconditioning group (cells pretreated first 3 h with 50 mu mol/LCoCl2, 1 h after changing liquid oxygen, and 28 h in 2% hypoxia incubator), chemical anoxia pretreatment of +VEGF antibody group (cell plus 40 mu g/ml VEGF monkline antibody, other chemical anoxic preconditioning group), hypoxia group (2%) Hypoxia incubator hypoxia 28 hours, hypoxia + VEGF group (cells plus 100 ng / ml pure VEGF, the rest with hypoxia group). MTT colorimetric method was used to measure the cell viability.
6. the expression of HIF-1 and HIF-2 HIF-2 protein was measured by Western Blotting.
Result
1. the cell viability of the chemical anoxic preconditioning group was significantly higher than that in the chemical anoxic group (P0.05), and the release rate of lactate dehydrogenase was significantly decreased (P0.05) in the.2. chemical anoxic preconditioning group, the cell viability was significantly higher than that in the normal hypoxia group (P0.05), and the release rate of lactate dehydrogenase was significantly decreased (P0.01).
3. the expression of GLUT-1 and EPO mRNA in the chemical anoxic preconditioning group was significantly higher than that in the hypoxia group (P0.05), and the expression of VEGFmRNA was significantly higher than that in the hypoxia group (P0.01). There was no significant difference in the expression of LDH-AmRNA between the two groups.
4. the expression of VEGF protein in chemical hypoxia preconditioning group was significantly higher than that in hypoxia group (P0.01).
5. MTT cell viability assay showed that 40 g/mlVEGF monoclonal antibodies could inhibit the protective effect of chemical hypoxia preconditioning, while 100 ng/ml recombinant human VEGF could mimic the protective effect of chemical anoxia preconditioning group.
6. The expression of HIF-2 alpha protein in the chemical hypoxia preconditioning group was significantly higher than that in the hypoxia group (P 0.01). There was no significant difference in the expression of HIF-1 alpha protein between the two groups.
conclusion
1. By analyzing the release rate of lactate dehydrogenase and MTT, a neuronal cell model of CoCl2 chemical hypoxia preconditioning was established.
2. By analyzing the release rate of lactate dehydrogenase and MTT, a pretreatment model of CoCl2 chemical hypoxia preconditioning was established to protect neuronal cells from hypoxia.
3. CoCl2 chemical hypoxia preconditioning is more effective than CoCl2 chemical hypoxia preconditioning in resisting atmospheric hypoxia.
4. chemical hypoxic preconditioning may play a protective role by promoting hypoxia to regulate the expression of GLUT-1, EPO and VEGF.
5. VEGF plays an important role in chemical hypoxia preconditioning.
6. Chemical hypoxia preconditioning may induce hypoxia-tolerant protective effect through HIF-2, not HIF-1, and promote hypoxia-regulated gene expression including VEGF.
【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】：2007
【分類號】：R363
本文編號：2171990
[Abstract]:Hypoxia is associated with many clinical diseases. It is also a common pathogenic factor in special environments such as high altitude, high altitude and deep water. Although the sensitivity of different types of cells to hypoxia is different, it will eventually cause cell metabolism and dysfunction and even cell death with the increase of hypoxia and duration. Nerve cells are strong. It is dependent on oxygen metabolism to supply energy and is particularly sensitive to hypoxia, so it is easily damaged by hypoxia. Finding a mechanism to promote the tolerance of neurons to hypoxia is of great significance for the prevention and treatment of clinical related diseases. In recent years, studies have shown that hypoxia preconditioning can protect neurons from subsequent occurrence. Hypoxia or other fatal stress is tolerated, so the neuroprotective effect of hypoxic preconditioning is concerned by more and more scholars. Although a variety of mechanisms and molecules are reported to be involved in the neuroprotective effect of hypoxic preconditioning [1], how these molecules are ultimately protected is uncertain and no clear system is predefined. The adaptation mechanism is universally recognized. Hypoxia inducible factor can be induced by a variety of damage factors and can also regulate many signal pathways. It is an important regulator to mediate the activation of hypoxia signal and many hypoxia induced genes. It can play an important role in promoting cell survival by adapting the cells to the hypoxia environment. The research on the function and function of the genes / products of their regulatory genes / products has become the current hot and frontier [2. 3]. has a number of controversial reports on the role and mechanism of hypoxia inducible factor and its regulatory gene / product in anoxic preconditioning. 5]., such as [4, has shown that the increase of HIF-1 expression may have no effect in anoxic preconditioning. However, it is also reported that hypoxia preconditioning has protective effects on HIF-1 activity. It is also reported that hypoxia preconditioning does not depend on the HIF-1 pathway. Therefore, the role of hypoxia inducible factor and its regulatory gene / product in anoxia preconditioning remains unclear, and.CoCl2 needs to be further studied. It is a common anoxic analogue compound, CoCl2 preconditioning can produce neuroprotective effect similar to hypoxic preconditioning [6]., a neurotype protective model of CoCl2 chemical anoxia preconditioning was established through differentiated neural SH-SY5Y cells, and the hypoxic inducible factor 1,2 and its regulatory gene VEGF, GLUT-1, EPO, LDH-A, etc. were pretreated by CoCl2 The role in the study was preliminarily discussed, and the anoxia protective effect of hypoxia induced gene product VEGF was studied by the application of recombinant human VEGF and the VEGF antibody that could inhibit the activity of VEGF. The main experimental methods, results and conclusions were as follows:
Method
The 1. human neuroblastoma SH-SY5Y was routinely cultured on the MEM/F12 (1:1 volume ratio) culture medium containing 15% fetal bovine serum (FCS), and 10 mu mol/L retinoic acid (RA) induced the cells to differentiate into 3-7d and was used for the experiment.
2. differentiated SH-SY5Y cells were randomly divided into control group, chemical anoxia preconditioning group (cells pretreated first with 50 mu mol/LCoCl2 3 h, oxygen culture 1 h after replacement, and 28 h in 2% hypoxia incubator), hypoxia group (2% hypoxic incubator 28 h). Or the differentiated SH-SY5Y cells were randomly divided into control group C (normal oxygen culture); chemical hypoxia preconditioning The treatment group HP (the cells first treated 1.5 h with 75 mu CoCl2, 3 h after the change of liquid oxygen, then 250 micron mol/LCoCl2 chemical anoxia 24 h), and the chemical anoxic group H (250 u mol/LCoCl2 chemical hypoxia 24 h). The damage degree of the cells was determined by the determination of the lactate dehydrogenase release rate and the activity of MTT cells.
3. RT-PCR was used to measure the expression of VEGF, GLUT-1, EPO and LDH-A in cells.
4. the protein expression of VEGF was measured by Western Blotting.
5. differentiated SH-SY5Y cells were randomly divided into control group, chemical anoxia preconditioning group (cells pretreated first 3 h with 50 mu mol/LCoCl2, 1 h after changing liquid oxygen, and 28 h in 2% hypoxia incubator), chemical anoxia pretreatment of +VEGF antibody group (cell plus 40 mu g/ml VEGF monkline antibody, other chemical anoxic preconditioning group), hypoxia group (2%) Hypoxia incubator hypoxia 28 hours, hypoxia + VEGF group (cells plus 100 ng / ml pure VEGF, the rest with hypoxia group). MTT colorimetric method was used to measure the cell viability.
6. the expression of HIF-1 and HIF-2 HIF-2 protein was measured by Western Blotting.
Result
1. the cell viability of the chemical anoxic preconditioning group was significantly higher than that in the chemical anoxic group (P0.05), and the release rate of lactate dehydrogenase was significantly decreased (P0.05) in the.2. chemical anoxic preconditioning group, the cell viability was significantly higher than that in the normal hypoxia group (P0.05), and the release rate of lactate dehydrogenase was significantly decreased (P0.01).
3. the expression of GLUT-1 and EPO mRNA in the chemical anoxic preconditioning group was significantly higher than that in the hypoxia group (P0.05), and the expression of VEGFmRNA was significantly higher than that in the hypoxia group (P0.01). There was no significant difference in the expression of LDH-AmRNA between the two groups.
4. the expression of VEGF protein in chemical hypoxia preconditioning group was significantly higher than that in hypoxia group (P0.01).
5. MTT cell viability assay showed that 40 g/mlVEGF monoclonal antibodies could inhibit the protective effect of chemical hypoxia preconditioning, while 100 ng/ml recombinant human VEGF could mimic the protective effect of chemical anoxia preconditioning group.
6. The expression of HIF-2 alpha protein in the chemical hypoxia preconditioning group was significantly higher than that in the hypoxia group (P 0.01). There was no significant difference in the expression of HIF-1 alpha protein between the two groups.
conclusion
1. By analyzing the release rate of lactate dehydrogenase and MTT, a neuronal cell model of CoCl2 chemical hypoxia preconditioning was established.
2. By analyzing the release rate of lactate dehydrogenase and MTT, a pretreatment model of CoCl2 chemical hypoxia preconditioning was established to protect neuronal cells from hypoxia.
3. CoCl2 chemical hypoxia preconditioning is more effective than CoCl2 chemical hypoxia preconditioning in resisting atmospheric hypoxia.
4. chemical hypoxic preconditioning may play a protective role by promoting hypoxia to regulate the expression of GLUT-1, EPO and VEGF.
5. VEGF plays an important role in chemical hypoxia preconditioning.
6. Chemical hypoxia preconditioning may induce hypoxia-tolerant protective effect through HIF-2, not HIF-1, and promote hypoxia-regulated gene expression including VEGF.
【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】：2007
【分類號】：R363
【引證文獻(xiàn)】
相關(guān)碩士學(xué)位論文 前2條
1 桂靜;磷酸肌酸鈉對缺氧窒息幼鼠腦組織中NO、CaM水平及HIF-1amRNA表達(dá)的影響[D];鄭州大學(xué);2012年
2 劉月梅;磷酸肌酸鈉對窒息缺氧幼鼠腎組織HIF-1α和VEGF表達(dá)的影響[D];鄭州大學(xué);2013年
，本文編號：2171990
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