缺氧诱导因子及其调节基因在化学缺氧预处理保护中的作用研究
发布时间：2018-08-08 13:54
【摘要】： 缺氧与许多临床疾病有关,也是高原、高空、深水等特殊环境中常见的一种致病因素。虽然不同类型细胞对缺氧的敏感性不同,但是,随着缺氧程度和持续时间的增加,最终都会引起细胞代谢和功能障碍,甚至导致细胞死亡。神经细胞强烈依赖有氧代谢进行能量供应,对缺氧特别敏感,因而容易因为缺氧受到损伤。寻找促进神经元对低氧产生耐受的机制,对临床相关疾病的预防和治疗有非常重要的意义。近年来有研究显示,缺氧预处理可以保护神经元对后继发生的严重缺氧或其它致死性应激产生耐受,因此,缺氧预处理的神经保护作用受到越来越多学者的关注。尽管多种机制和分子被报道可能参与缺氧预处理的神经保护作用[1],但是这些分子最终怎样发挥保护作用还不确定,没有一个清晰系统的预适应机制受到普遍认可。缺氧诱导因子可被多种损伤因素诱导,也可调控许多信号途径,是介导缺氧信号与众多缺氧诱导基因转录激活的一个重要调控因子,可通过使细胞适应低氧环境,发挥促进细胞生存的重要作用。有关缺氧诱导因子及其调控基因/产物功能及作用的研究已经成为当前的热点和前沿[2, 3]。针对缺氧诱导因子及其调节基因/产物在缺氧预处理中的作用和机制,目前有许多有争议的报道[4, 5]。如有研究显示,HIF-1的表达增加在缺氧预处理中可能具有非常重要的作用。然而,亦有文献报道缺氧预处理通过抑制HIF-1活性产生保护作用。还有报道显示,缺氧预处理不依赖HIF-1的途径产生保护作用。因而,缺氧诱导因子及其调节基因/产物在缺氧预处理中的作用仍不清楚,有待进一步研究。CoCl2是常用的缺氧模拟化合物,CoCl2预处理可产生与缺氧预处理相似的神经保护作用[6]。本课题通过分化的神经型SH-SY5Y细胞建立CoCl2化学缺氧预处理的神经型细胞保护模型,对缺氧诱导因子1、2及其调节基因VEGF、GLUT-1、EPO、LDH-A等在CoCl2预处理中的作用进行初步的探讨,并通过应用重组人VEGF和可抑制VEGF活性的VEGF抗体,对缺氧诱导基因产物VEGF的缺氧保护作用进行研究。主要的实验方法、结果和结论如下: 方法 1.人神经母细胞瘤SH-SY5Y常规培养于含15 %胎牛血清(FCS)的MEM/F12(1:1体积比)培养液,10μmol/L维甲酸(RA)诱导细胞分化3-7d后用于实验。 2.分化的SH-SY5Y细胞随机分为对照组、化学缺氧预处理组(细胞先用50μmol/LCoCl2预处理3 h,换液后常氧培养1 h,然后在2%的低氧孵箱内缺氧28 h)、缺氧组(2%的低氧孵箱内缺氧28 h)。或将分化的SH-SY5Y细胞随机分为对照组C (常氧培养);化学缺氧预处理组HP(细胞先用75μmol/L CoCl2预处理1.5 h,换液后常氧培养3 h,然后用250μmol/LCoCl2化学缺氧24 h);化学缺氧组H(250μmol/LCoCl2化学缺氧24 h)。通过乳酸脱氢酶释放率测定、MTT细胞活力测定判断上述各组细胞的损伤程度。 3. RT-PCR测细胞的VEGF、GLUT-1、EPO、LDH-A的mRNA表达。 4.用Western Blotting法测细胞VEGF的蛋白表达。 5.分化的SH-SY5Y细胞随机分为对照组、化学缺氧预处理组(细胞先用50μmol/LCoCl2预处理3 h,换液后常氧培养1 h,然后在2%的低氧孵箱内缺氧28 h)、化学缺氧预处理+VEGF抗体组(细胞加40μg/ml VEGF单克隆抗体,其余同化学缺氧预处理组)、缺氧组(2%的低氧孵箱内缺氧28 h)、缺氧+VEGF组(细胞加100 ng/ml VEGF纯品,其余同缺氧组)。通过MTT比色法测各组细胞的活力。 6.用Western Blotting法测细胞HIF-1α、HIF-2α蛋白表达。 结果 1.化学缺氧预处理组细胞较化学缺氧组细胞活力显著增高(P0.05),乳酸脱氢酶释放率显著减少(P0.05)。2.化学缺氧预处理组细胞较常压缺氧组细胞活力显著增高(P0.05),乳酸脱氢酶释放率显著减少(P0.01)。 3.化学缺氧预处理组细胞GLUT-1、EPO mRNA表达显著高于缺氧组(P0.05),VEGFmRNA表达显著高于缺氧组(P0.01),LDH-AmRNA表达在两组之间没有显著性差异。 4.化学缺氧预处理组细胞VEGF蛋白表达显著高于缺氧组(P0.01)。 5. MTT细胞活力测定显示40μg/mlVEGF单克隆抗体可抑制化学缺氧预处理的保护作用,而100 ng/ml重组人VEGF可模拟化学缺氧预处理组的保护作用。 6.化学缺氧预处理组细胞HIF-2α蛋白表达显著高于缺氧组(P0.01),HIF-1α蛋白表达在两组之间没有显著性差异。 结论 1.通过乳酸脱氢酶释放率和MTT分析,建立了CoCl2化学缺氧预处理抵抗CoCl2化学缺氧的神经型细胞模型。 2.通过乳酸脱氢酶释放率和MTT分析,建立了CoCl2化学缺氧预处理保护神经型细胞对常压缺氧产生耐受的预处理模型。 3. CoCl2化学缺氧预处理抵抗常压缺氧的效果较CoCl2化学缺氧预处理抵抗CoCl2化学缺氧的效果更强。 4.化学缺氧预处理可能通过促进缺氧调节基因GLUT-1、EPO、VEGF的表达而产生保护作用。 5. VEGF在化学缺氧预处理中具有非常重要的保护作用。 6.化学缺氧预处理可能通过HIF-2,而不是HIF-1,促进包括VEGF在内的缺氧调节基因表达产生耐缺氧的保护作用。
[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.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R363
本文编号：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.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R363
【引证文献】
相关硕士学位论文 前2条
1 桂静;磷酸肌酸钠对缺氧窒息幼鼠脑组织中NO、CaM水平及HIF-1amRNA表达的影响[D];郑州大学;2012年
2 刘月梅;磷酸肌酸钠对窒息缺氧幼鼠肾组织HIF-1α和VEGF表达的影响[D];郑州大学;2013年
，本文编号：2171990
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