预适应理论抗缺氧脑损伤效应实验研究
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摘要
研究背景
缺氧/缺血性脑损伤是神经系统常见病、多发病,不仅影响患者肢体运动功能和高级认知功能,而且具有较高致残率和死亡率,严重威胁人类健康和降低生活质量,给家庭和社会带来沉重的经济和精神负担。缺氧/缺血性脑损伤的脑保护治疗尤为重要,与患者预后密切相关。由于目前缺氧/缺血性脑损伤的脑保护治疗方法或措施疗效有限,所以脑保护治疗是国内外研究的热点、难点问题。预适应理论已经证实具有脑保护作用,相应机制已初步明确。缺氧/低氧/缺血预适应(Hypoxic/Ischemic Preconditioning, HPC/IPC)和药理性预适应(Pharmacological Preconditioning, PPC)都属于预适应理论范畴,如何达到预适应理论临床应用即增加其临床可操作性是转化医学亟待解决的问题。基于低氧预适应理论发明专利(名称：心脑血管保健治疗仪,发明人：吉训明等,专利号：200710176701)的问世为预适应理论抗缺氧/缺血性脑损伤即转化医学研究带来了动力和曙光。在预适应理论抗缺氧/缺血性脑损伤效应方面,我们既往研究发现,整体低氧预适应对小鼠急性脑梗死致脑缺血性损伤具有保护作用,仍存在预适应理论临床应用困难；为进一步增加临床可操作性,我们初步证实低氧预适应小鼠脑匀浆液提取液对大鼠鼠胚海马神经元缺氧复氧后神经细胞具有保护作用。故在前期研究基础之上,本实验研究将继续探讨预适应理论抗缺氧损伤效应,包括以下两方面内容：(1)低氧预适应Wistar大鼠脑脊液、脑匀浆提取液和血浆对原代培养的Wistar大鼠乳鼠海马神经元氧糖剥夺损伤的作用及相关机制；(2)腺苷药理性预适应对原代培养的Wistar大鼠乳鼠海马神经元氧糖剥夺损伤的作用及相关机制。以期通过本研究,明确预适应理论抗缺氧脑损伤效应,并为其转化医学研究奠定理论基础。
研究目的
1、探讨低氧预适应Wistar大鼠脑脊液、脑匀浆提取液和血浆对原代培养的Wistar大鼠乳鼠海马神经元氧糖剥夺损伤的作用及相关机制。
2、探讨腺苷药理性预适应对原代培养的Wistar大鼠乳鼠海马神经元氧糖剥夺损伤的作用及相关机制。
研究方法
1、实验选用200-250g成年健康Wistar大鼠,雌雄不拘,进行低氧预适应模型复制并分别提取脑脊液、脑组织和血液,制成无菌脑脊液、脑匀浆提取液和血浆；出生24h内的Wistar大鼠乳鼠,取其海马组织进行神经元原代培养,然后进行氧糖剥夺培养。
2、将培养的神经细胞随机分为正常对照组(N组)、单纯氧糖剥夺组(OGD组)、正常脑脊液/脑匀浆提取液/血浆干预+氧糖剥夺组(CSF/BH/BP+OGD组)和低氧预适应脑脊液/脑匀浆提取液/血浆干预+氧糖剥夺组(HPC+OGD组),用含Na2S2O4(1 mmol/L)的无糖Earle’s液复制神经元氧糖剥夺损伤模型,通过比色法比较低氧预适应和正常脑脊液、脑匀浆和血浆中SOD和MDA含量；采用AnnexinV/PI双染及免疫荧光染色法检测细胞凋亡及凋亡相关蛋白表达状况。
3、将原代培养的Wistar大鼠乳鼠海马神经元随机分为正常对照组(N组)、单纯氧糖剥夺组(OGD组)、低浓度腺苷预适应+氧糖剥夺作用(ADL+OGD组)、中浓度腺苷预适应+氧糖剥夺作用(ADM+OGD组)和高浓度腺苷预适应+氧糖剥夺作用(ADH+OGD组),采用AnexinV/PI双染及免疫荧光染色法,观察低、中、高三种腺苷浓度预处理对神经元氧糖剥夺损伤后凋亡和凋亡相关蛋白表达的影响。
研究结果
1、与正常大鼠脑脊液相比,低氧预适应大鼠脑脊液SOD含量升高(P<0.01),MDA含量降低(P<0.05)；与正常大鼠脑匀浆提取液相比,低氧预适应大鼠脑匀浆提取液SOD含量明显升高(P<0.01),MDA含量降低(P<0.01)；与正常大鼠血浆相比,低氧预适应大鼠血浆SOD含量升高(P<0.01),MDA含量显著降低(P<0.01)。
2、激光共聚焦显微镜检测AnnexinV/PI双染结果显示：N组偶有细胞凋亡, OGD组出现大量凋亡神经元。正常大鼠脑脊液(P<0.01)和低氧预适应脑脊液(P<0.01)对神经元氧糖剥夺损伤均有保护作用,后者作用更强(P<0.01);正常大鼠脑匀浆提取液对损伤神经元有微弱保护作用(P<0.05)低氧预适应大鼠脑匀浆提取液对损伤神经元有显著保护作用(P<0.01)；正常大鼠血浆能保护神经抗氧糖剥夺损伤(P<0.01),低氧预适应大鼠血浆能更大程度上减少损伤性神经元凋亡(P<0.01)。流式细胞仪检测与以上结果一致。
3、Bcl-2蛋白和Bax蛋白表达检测发现：N组仅有极少量Bcl-2蛋白和Bax蛋白表达,OGD组有极少量Bcl-2蛋白表达和大量Bax蛋白表达。正常大鼠脑脊液和低氧预适应脑脊液均能显著增加Bcl-2蛋白表达(P<0.01),减少Bax蛋白表达(P<0.01),升高Bcl-2/Bax比率；正常大鼠脑匀浆提取液能在一定程度上增加Bcl-2蛋白表达(P<0.05),显著减低Bax蛋白表达(P<0.01),与正常大鼠脑匀浆提取液相比,低氧预适应大鼠脑匀浆提取液能显著增加Bcl-2蛋白表达(P<0.01),进一步降低Bax蛋白表达(P<0.05)；正常大鼠血浆可增加Bcl-2蛋白表达(P<0.05),对Bax蛋白表达无影响(P=0.063),低氧预适应血浆不但能更大程度上增强Bcl-2蛋白表达(P<0.01),还能明显降低Bax蛋白表达(P<0.01)
4、腺苷预适应海马神经检测发现：与单纯氧糖剥夺组相比,低剂量腺苷预处理不能产生神经元氧糖剥夺损伤保护作用；中剂量腺苷预处理能减少氧糖剥夺诱导的神经元凋亡数量,增加Bcl-2蛋白表达(P<0.01),减低Bax蛋白表达(P<0.01)；高剂量腺苷预适应具有更显著的细胞保护作用,能进一步提高Bcl-2/Bax比率。
研究结论
1、低氧预适应能诱导大鼠脑脊液、脑匀浆提取液和血浆SOD活性升高和MDA含量降低。
2、低氧预适应大鼠脑脊液、脑匀浆提取液和血浆能不同程度对抗神经元氧糖剥夺损伤诱导的细胞凋亡,该作用可能是通过降低氧化应激水平,上调Bcl-2蛋白表达和减少Bax蛋白表达实现的。
3、中、高剂量的腺苷预适应能诱导神经元缺氧糖耐受性的提高,通过升高Bc1-2/Bax比率减少神经元凋亡,起到抗缺氧脑损伤效应。
BACKGROUD
Stroke is associated with high morbidity and mortality.Its prevalence is continuously increasing worldwide. Acute cerebrovascular disease is one of the first three top causes of the death and the first cause of disability in the world.Among the total,the incidence of cerebral infarction is coming the. first,with percentage of 80%. Cerebral infarction causes death and disability, which bring albatross to their family and the society.Hypoxia can lead to various kinds of reactions in animal or human bodies to compensate for hypoxic influence. But the extent to which animal or human bodies fight against hypoxia,namely tolerate hypoxia is limited.Brain is the most vulnerable organ in animal and human bodies and the main limiting factor influencing tolerance of the bodies to hypoxia. In the central nerve system, injuries induced by hypoxia/ischemia affect the health of human beings seriously. However,in recent years, investigations have showed that the tolerance of the bodies to hypoxia can be enhanced through such measures as hypoxic/ischemic preconditioning(HPC) and pharmacological preconditioning(PPC). Both HPC and PPC are category of precondition theory.Researches have indicated that HPC and PPC could improve the hypoxic tolerance of animals and in vitro cells.The problem is how to apply it to clinical application.The invention of cardia-cerebral vessels health care meter(Pat-number:200710176701) is the first step of clinical application.On the base of our prophase work,in this experimental we will investigate the effects and mechanisms of HPC and PPC on cultured hippocampal neurons.
OBJECTIVES
Firstly, to investigate the effect and imaginabale mechanisms of cerebrospinal fluid, brain homogenate and blood plasma of hypoxic-preconditioned Wistar rat on apoptosis of hippocampal neurons. Secondly, to study the effect and possible mechanisms of adenosine preconditioning on apoptosis of hippocampal neurons.
METHODS
1.Adult healthy Witar rats were exposed to hypoxia cabin for 3 hours to copy the hypoxic-preconditioning model, taking the cerebrospinal fluid, brain tissue and blood, dealing them to obtain aseptic cerebrospinal fluid, brain homogenate extract and blood plasma, to determine the SOD and MDA content of them.Cultured hippocampal neurons randomized into normal control group, oxygen-glucose deficiency group, normal cerebrospinal fluid/brain homogenate extrat/blood plasma and oxygen-glucose deficiency group,hypoxic- recondition-ned cerebrospinal fluid/brain homogenate extract/blood plasma and oxygen-glucose deficiency group. Neurons apoptosis was induced by oxygen-glucose deficiency Earle's solution. Apoptosis was studied by using method of AnnexinV/PI double staining. The expression of apoptosis related genes bcl-2 and bax were analized by quantitative immunofluorescence staining.
2.Cultured hippocampal neurons randomized into normal control group, oxygen-glucose deficiency group, low-does adenosine and oxygen-glucose deficiency group, middle-does adenosine and oxygen- glucose deficiency group, high-does adenosine and oxygen-glucose deficiency group, Apoptosis was studied by using method of AnexinV/PI double staining. The expression of apoptosis related genes bcl-2 and bax were analized by quantitative immunofl-uorescence staining.
RESULTS
1.The content of SOD in hypoxic-preconditioned cerebrospinal fluid was increased (P<0.01)and content of MDA was decreased(P<0.05)compard with normal cerebrospinal fluid; The content of SOD in hypoxic-preconditioned brain homogenate was increased(P<0.01)and content of MDA was decreased(P< 0.01)compard with normal brain homogenate; The content of SOD in hypoxic-preconditioned blood plasma was increased(P<0.01)and content of MDA was decreased(P<0.01)compard with normal blood plasma.
2. AnnexinV/PI double staining results showed that apoptotic cells were observed by chance in N group,and a devil of apoptotic cells in OGD group.Both normal and hypoxic-preconditioned CSF could protected cultured hippocampal neurons from oxygen- glucose deficiency(P<0.01),the later one has stronger effects(P<0.01);Normal brain homogenate could protect neurons weakly(P< 0.05),and hypoxic- preconditioned brain homogenate defended neurons obviously(P<0.01); Both normal and hypoxic-preconditioned blood plasma could protected oxygen- glucose deficiency neurons(P<0.01),the later one has greater conservation(P<0.01).
3. The expression of apoptosis related genes Bcl-2 and Bax, which were analized by quantitative immunofluorescence, were both discovered seldomly in N group.Bcl-2 was up-regulated in groups,which cultured by normal CSF(P< 0.01), brain homogenate(P<0.05)and blood plasma(P<0.05), compared with OGD group,and hypoxic-preconditioned CSF(P<0.01), brain homogenate(P< 0.01)and blood plasma(P<0.01),compared with normal CSF/brain homogenate/ blood plasma group.At the same time, Bax was down-regulated in groups,which cultured by normal CSF(P<0.01), brain homogenate(P<0.01)compared with OGD group,and hypoxic-preconditioned CSF(P<0.01), brain homogenate(P< 0.05)and blood plasma(P<0.01), compared with normal CSF/brain homogenate/ blood plasma group. There were no significant difference on the expression of Bax between normal blood plasma and OGD group and OGD only group.
4.Adenosine preconditioning detectection resulted that low-does adenosine preconditioning had no conservation on OGD hippocampal neurons.Middle-does adenosine preconditioning could protecte neurons obviously, increasing the express of Bcl-2 protein and decreasing the express of Bax protein compared with OGD group.high-does adenosine preconditioning could protecte neurons and advancing the rate of Bcl-2/Bax more respectively compared with Middle-does adenosine preconditioning and OGD group.
CONCLUSIONS
1.hypoxic-preconditioning increase the activity of SOD and decrease the content of MDA in cerebrospinal fluid, brain homogenate and blood plasma.
2.hypoxic-preconditioned CSF, brain homogenate and blood plasma could protected hippocampal neurons from OGD injury, degrade the rate of apoptosis, up-regulate the express of Bcl-2 and down-regulate the expression of Bax.
3.Middle-does and high-does adenosine could enhance the hypoxic tolerance of hippocampal neurons,and advancing of the rate of Bcl-2/Bax played an important role in inhibiting apoptosis.
缺氧/缺血性脑损伤是神经系统常见病、多发病,不仅影响患者肢体运动功能和高级认知功能,而且具有较高致残率和死亡率,严重威胁人类健康和降低生活质量,给家庭和社会带来沉重的经济和精神负担。缺氧/缺血性脑损伤的脑保护治疗尤为重要,与患者预后密切相关。由于目前缺氧/缺血性脑损伤的脑保护治疗方法或措施疗效有限,所以脑保护治疗是国内外研究的热点、难点问题。预适应理论已经证实具有脑保护作用,相应机制已初步明确。缺氧/低氧/缺血预适应(Hypoxic/Ischemic Preconditioning, HPC/IPC)和药理性预适应(Pharmacological Preconditioning, PPC)都属于预适应理论范畴,如何达到预适应理论临床应用即增加其临床可操作性是转化医学亟待解决的问题。基于低氧预适应理论发明专利(名称：心脑血管保健治疗仪,发明人：吉训明等,专利号：200710176701)的问世为预适应理论抗缺氧/缺血性脑损伤即转化医学研究带来了动力和曙光。在预适应理论抗缺氧/缺血性脑损伤效应方面,我们既往研究发现,整体低氧预适应对小鼠急性脑梗死致脑缺血性损伤具有保护作用,仍存在预适应理论临床应用困难；为进一步增加临床可操作性,我们初步证实低氧预适应小鼠脑匀浆液提取液对大鼠鼠胚海马神经元缺氧复氧后神经细胞具有保护作用。故在前期研究基础之上,本实验研究将继续探讨预适应理论抗缺氧损伤效应,包括以下两方面内容：(1)低氧预适应Wistar大鼠脑脊液、脑匀浆提取液和血浆对原代培养的Wistar大鼠乳鼠海马神经元氧糖剥夺损伤的作用及相关机制；(2)腺苷药理性预适应对原代培养的Wistar大鼠乳鼠海马神经元氧糖剥夺损伤的作用及相关机制。以期通过本研究,明确预适应理论抗缺氧脑损伤效应,并为其转化医学研究奠定理论基础。
研究目的
1、探讨低氧预适应Wistar大鼠脑脊液、脑匀浆提取液和血浆对原代培养的Wistar大鼠乳鼠海马神经元氧糖剥夺损伤的作用及相关机制。
2、探讨腺苷药理性预适应对原代培养的Wistar大鼠乳鼠海马神经元氧糖剥夺损伤的作用及相关机制。
研究方法
1、实验选用200-250g成年健康Wistar大鼠,雌雄不拘,进行低氧预适应模型复制并分别提取脑脊液、脑组织和血液,制成无菌脑脊液、脑匀浆提取液和血浆；出生24h内的Wistar大鼠乳鼠,取其海马组织进行神经元原代培养,然后进行氧糖剥夺培养。
2、将培养的神经细胞随机分为正常对照组(N组)、单纯氧糖剥夺组(OGD组)、正常脑脊液/脑匀浆提取液/血浆干预+氧糖剥夺组(CSF/BH/BP+OGD组)和低氧预适应脑脊液/脑匀浆提取液/血浆干预+氧糖剥夺组(HPC+OGD组),用含Na2S2O4(1 mmol/L)的无糖Earle’s液复制神经元氧糖剥夺损伤模型,通过比色法比较低氧预适应和正常脑脊液、脑匀浆和血浆中SOD和MDA含量；采用AnnexinV/PI双染及免疫荧光染色法检测细胞凋亡及凋亡相关蛋白表达状况。
3、将原代培养的Wistar大鼠乳鼠海马神经元随机分为正常对照组(N组)、单纯氧糖剥夺组(OGD组)、低浓度腺苷预适应+氧糖剥夺作用(ADL+OGD组)、中浓度腺苷预适应+氧糖剥夺作用(ADM+OGD组)和高浓度腺苷预适应+氧糖剥夺作用(ADH+OGD组),采用AnexinV/PI双染及免疫荧光染色法,观察低、中、高三种腺苷浓度预处理对神经元氧糖剥夺损伤后凋亡和凋亡相关蛋白表达的影响。
研究结果
1、与正常大鼠脑脊液相比,低氧预适应大鼠脑脊液SOD含量升高(P<0.01),MDA含量降低(P<0.05)；与正常大鼠脑匀浆提取液相比,低氧预适应大鼠脑匀浆提取液SOD含量明显升高(P<0.01),MDA含量降低(P<0.01)；与正常大鼠血浆相比,低氧预适应大鼠血浆SOD含量升高(P<0.01),MDA含量显著降低(P<0.01)。
2、激光共聚焦显微镜检测AnnexinV/PI双染结果显示：N组偶有细胞凋亡, OGD组出现大量凋亡神经元。正常大鼠脑脊液(P<0.01)和低氧预适应脑脊液(P<0.01)对神经元氧糖剥夺损伤均有保护作用,后者作用更强(P<0.01);正常大鼠脑匀浆提取液对损伤神经元有微弱保护作用(P<0.05)低氧预适应大鼠脑匀浆提取液对损伤神经元有显著保护作用(P<0.01)；正常大鼠血浆能保护神经抗氧糖剥夺损伤(P<0.01),低氧预适应大鼠血浆能更大程度上减少损伤性神经元凋亡(P<0.01)。流式细胞仪检测与以上结果一致。
3、Bcl-2蛋白和Bax蛋白表达检测发现：N组仅有极少量Bcl-2蛋白和Bax蛋白表达,OGD组有极少量Bcl-2蛋白表达和大量Bax蛋白表达。正常大鼠脑脊液和低氧预适应脑脊液均能显著增加Bcl-2蛋白表达(P<0.01),减少Bax蛋白表达(P<0.01),升高Bcl-2/Bax比率；正常大鼠脑匀浆提取液能在一定程度上增加Bcl-2蛋白表达(P<0.05),显著减低Bax蛋白表达(P<0.01),与正常大鼠脑匀浆提取液相比,低氧预适应大鼠脑匀浆提取液能显著增加Bcl-2蛋白表达(P<0.01),进一步降低Bax蛋白表达(P<0.05)；正常大鼠血浆可增加Bcl-2蛋白表达(P<0.05),对Bax蛋白表达无影响(P=0.063),低氧预适应血浆不但能更大程度上增强Bcl-2蛋白表达(P<0.01),还能明显降低Bax蛋白表达(P<0.01)
4、腺苷预适应海马神经检测发现：与单纯氧糖剥夺组相比,低剂量腺苷预处理不能产生神经元氧糖剥夺损伤保护作用；中剂量腺苷预处理能减少氧糖剥夺诱导的神经元凋亡数量,增加Bcl-2蛋白表达(P<0.01),减低Bax蛋白表达(P<0.01)；高剂量腺苷预适应具有更显著的细胞保护作用,能进一步提高Bcl-2/Bax比率。
研究结论
1、低氧预适应能诱导大鼠脑脊液、脑匀浆提取液和血浆SOD活性升高和MDA含量降低。
2、低氧预适应大鼠脑脊液、脑匀浆提取液和血浆能不同程度对抗神经元氧糖剥夺损伤诱导的细胞凋亡,该作用可能是通过降低氧化应激水平,上调Bcl-2蛋白表达和减少Bax蛋白表达实现的。
3、中、高剂量的腺苷预适应能诱导神经元缺氧糖耐受性的提高,通过升高Bc1-2/Bax比率减少神经元凋亡,起到抗缺氧脑损伤效应。
BACKGROUD
Stroke is associated with high morbidity and mortality.Its prevalence is continuously increasing worldwide. Acute cerebrovascular disease is one of the first three top causes of the death and the first cause of disability in the world.Among the total,the incidence of cerebral infarction is coming the. first,with percentage of 80%. Cerebral infarction causes death and disability, which bring albatross to their family and the society.Hypoxia can lead to various kinds of reactions in animal or human bodies to compensate for hypoxic influence. But the extent to which animal or human bodies fight against hypoxia,namely tolerate hypoxia is limited.Brain is the most vulnerable organ in animal and human bodies and the main limiting factor influencing tolerance of the bodies to hypoxia. In the central nerve system, injuries induced by hypoxia/ischemia affect the health of human beings seriously. However,in recent years, investigations have showed that the tolerance of the bodies to hypoxia can be enhanced through such measures as hypoxic/ischemic preconditioning(HPC) and pharmacological preconditioning(PPC). Both HPC and PPC are category of precondition theory.Researches have indicated that HPC and PPC could improve the hypoxic tolerance of animals and in vitro cells.The problem is how to apply it to clinical application.The invention of cardia-cerebral vessels health care meter(Pat-number:200710176701) is the first step of clinical application.On the base of our prophase work,in this experimental we will investigate the effects and mechanisms of HPC and PPC on cultured hippocampal neurons.
OBJECTIVES
Firstly, to investigate the effect and imaginabale mechanisms of cerebrospinal fluid, brain homogenate and blood plasma of hypoxic-preconditioned Wistar rat on apoptosis of hippocampal neurons. Secondly, to study the effect and possible mechanisms of adenosine preconditioning on apoptosis of hippocampal neurons.
METHODS
1.Adult healthy Witar rats were exposed to hypoxia cabin for 3 hours to copy the hypoxic-preconditioning model, taking the cerebrospinal fluid, brain tissue and blood, dealing them to obtain aseptic cerebrospinal fluid, brain homogenate extract and blood plasma, to determine the SOD and MDA content of them.Cultured hippocampal neurons randomized into normal control group, oxygen-glucose deficiency group, normal cerebrospinal fluid/brain homogenate extrat/blood plasma and oxygen-glucose deficiency group,hypoxic- recondition-ned cerebrospinal fluid/brain homogenate extract/blood plasma and oxygen-glucose deficiency group. Neurons apoptosis was induced by oxygen-glucose deficiency Earle's solution. Apoptosis was studied by using method of AnnexinV/PI double staining. The expression of apoptosis related genes bcl-2 and bax were analized by quantitative immunofluorescence staining.
2.Cultured hippocampal neurons randomized into normal control group, oxygen-glucose deficiency group, low-does adenosine and oxygen-glucose deficiency group, middle-does adenosine and oxygen- glucose deficiency group, high-does adenosine and oxygen-glucose deficiency group, Apoptosis was studied by using method of AnexinV/PI double staining. The expression of apoptosis related genes bcl-2 and bax were analized by quantitative immunofl-uorescence staining.
RESULTS
1.The content of SOD in hypoxic-preconditioned cerebrospinal fluid was increased (P<0.01)and content of MDA was decreased(P<0.05)compard with normal cerebrospinal fluid; The content of SOD in hypoxic-preconditioned brain homogenate was increased(P<0.01)and content of MDA was decreased(P< 0.01)compard with normal brain homogenate; The content of SOD in hypoxic-preconditioned blood plasma was increased(P<0.01)and content of MDA was decreased(P<0.01)compard with normal blood plasma.
2. AnnexinV/PI double staining results showed that apoptotic cells were observed by chance in N group,and a devil of apoptotic cells in OGD group.Both normal and hypoxic-preconditioned CSF could protected cultured hippocampal neurons from oxygen- glucose deficiency(P<0.01),the later one has stronger effects(P<0.01);Normal brain homogenate could protect neurons weakly(P< 0.05),and hypoxic- preconditioned brain homogenate defended neurons obviously(P<0.01); Both normal and hypoxic-preconditioned blood plasma could protected oxygen- glucose deficiency neurons(P<0.01),the later one has greater conservation(P<0.01).
3. The expression of apoptosis related genes Bcl-2 and Bax, which were analized by quantitative immunofluorescence, were both discovered seldomly in N group.Bcl-2 was up-regulated in groups,which cultured by normal CSF(P< 0.01), brain homogenate(P<0.05)and blood plasma(P<0.05), compared with OGD group,and hypoxic-preconditioned CSF(P<0.01), brain homogenate(P< 0.01)and blood plasma(P<0.01),compared with normal CSF/brain homogenate/ blood plasma group.At the same time, Bax was down-regulated in groups,which cultured by normal CSF(P<0.01), brain homogenate(P<0.01)compared with OGD group,and hypoxic-preconditioned CSF(P<0.01), brain homogenate(P< 0.05)and blood plasma(P<0.01), compared with normal CSF/brain homogenate/ blood plasma group. There were no significant difference on the expression of Bax between normal blood plasma and OGD group and OGD only group.
4.Adenosine preconditioning detectection resulted that low-does adenosine preconditioning had no conservation on OGD hippocampal neurons.Middle-does adenosine preconditioning could protecte neurons obviously, increasing the express of Bcl-2 protein and decreasing the express of Bax protein compared with OGD group.high-does adenosine preconditioning could protecte neurons and advancing the rate of Bcl-2/Bax more respectively compared with Middle-does adenosine preconditioning and OGD group.
CONCLUSIONS
1.hypoxic-preconditioning increase the activity of SOD and decrease the content of MDA in cerebrospinal fluid, brain homogenate and blood plasma.
2.hypoxic-preconditioned CSF, brain homogenate and blood plasma could protected hippocampal neurons from OGD injury, degrade the rate of apoptosis, up-regulate the express of Bcl-2 and down-regulate the expression of Bax.
3.Middle-does and high-does adenosine could enhance the hypoxic tolerance of hippocampal neurons,and advancing of the rate of Bcl-2/Bax played an important role in inhibiting apoptosis.
引文
[1]牛敬忠,杨明峰,岳伟.低氧预适应增强小鼠学习记忆能力[J].泰山医学院学报,2006,27(8)：694-696.
[2]吕国蔚,史美棠,李凌等.急性重复性缺氧对小鼠耐受性的影响及其机制的初步探讨[J].中国病理生理杂志,1992,8(4)：425-428.
[3]张颜波,吕国蔚,杨明峰等.低氧预适应小鼠海马Bcl-2表达和Caspase-3活性的变化[J].中华神经医学杂志,2007,40(8)：553-555.
[4]刘亮,吕国蔚.缺氧预适应小鼠脑匀浆去蛋白液对缺氧突触体膜的保护作用.中国神经科学杂志,2001,17(4)：373-375.
[5]VannucciRC,Towfighi J,Vannucci SJ.Hypoxic preconditioning and hypoxia-ischemic brain damdge in the immature rat:pathologic and metab- olic corrclatcs[J]. JNcurochem,1998,71(3):1215-1220.
[6]杨明峰,岳伟,张伟等.大鼠鼠胚海马神经细胞原代培养及神经元鉴定[J].泰山医学院学报,2006,27(7)：614-616.
[7]Fodoroffs.Cell tissue and organ culture in neuron-biology. Academic ress, 1979,191-213.
[8]Kerr JF, Wyllie AH, Currie A R. A basic biological phenomenon with wide rang implications in tissue Kinetics. Br J Cancer,1972,26:239.
[9]Thompson C B. Apoptosis in the pathogenesis and the treatment of disease [J]. Science,1995,267:1456.
[10]Nitatori T,Sato N,Waguri S,et al.Delayed neuronal death in the CA1 pyramidal cell layer of the gerbil hippocampus following transient isehemia is apoptosis.J Neurosei,1995,15(2):1001-1011.
[11]Guegan C, Boutin H, Boudry C, et al. Apoptosis death in cortical neurons of mice subjected to focal ischemia. C R Acad Sci 1996,319:875-885.
[12]尹琰,寿伟璋.流式细胞术AnnexinV细胞凋亡检测方法探讨.东南大学学报,2003,22：169.
[13]Span L P,Pennings AH,Vierwinden G,et al.The dynamic process of apoptosis analyzed by flow cytometry usingAnnexinV/propidium iodide and amodified in situ end abeling technique.Cytometry,2002,47 (1):24
[14]莫简.医用自由基生物学导论[M].北京：人民卫生出版社,1989,81-83.
[15]Jarasch ED, Bruder G, Hei HV, et al. Significance of xanthine oxidase in capillary endotheliacell[J]. Acta Pysiol Scand Suppl,1986,548:39.
[16]Lewen A,Matz P,Chan PH.Free radical pathways in CNS injury[J].J Neurotrauma,2000,17 (10):871-890.
[17]吴其夏,余应年,卢建.新编病理生理学[M].第1版.北京：中国协和医科大学出版社,1999,174-208.
[18]Takahashi G, SakuraiM, Abe K, et al. MCI-186 prevents spinal cord damage and affects enzyme levels of nitric oxide synthase and Cu/Zn superoxide dismutase after transient ischemia in rabbits[J]. J Thorac Cardiovasc Surg,2003,12.6:1461.
[19]刘庆华,邵书丽.细胞凋亡.解剖学进展,1999：5(3)：127-130
[20]Hanada M, Aim-SemPe C, Sato T, et al.Structure-function analysis of Bel-2 protein.Identification of conserved domains important for homodimeriza-tion with Bel-2 and heterodimerization with Bax.J Biol Chem,1995,270 (20):11962-11969.
[21]Hockenbery DM, Oltvai ZN,Yin XM. Bcl-2 function in an antioxidant pathyway to prevent apoptosis. Cell 1993,75:241-251
[22]Tsujimono Y,Shimizu S, Eguchi Y. Bcl-2 and Bcl-x1 block apoptosis as well as necrosis:possible involvement of common mediators in apoptotic and necrotic signal ransudation pathways. Leukemia 1997,3:380-382.
[23]Oltvai ZN, Milliman C L, Kormeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homology, Ax, that accelerates programmed cell death. Cell 1993,74:609-619
[24]Reed J C. Bcl-2 and the regulation of programmed cell death. J Cell'Biol 1994,17:260-263.
[25]Groc L,Bezin L,JiangH,et al. Bax, Bcl-2,and cyclin expression and apoptosis in rat substantia nigra during development [J].Neurosci Lett, 2001,306:198-202.
[1]牛敬忠,杨明峰,张颜波等.蛋白激酶C抑制剂灯盏花素对低氧预适应小鼠海马NO/cGMP信号转导系统的影响[J].泰山医学院学报,2008,29(1)：5-7.
[2]张颜波,吕国蔚,牛敬忠等.低氧预适应小鼠皮层Bcl-2表达和Caspase-3活性的变化[J].生理学报,2008,60(2)：49-253.
[3]杨明峰,张颜波,牛敬忠等.大鼠鼠胚海马神经元缺氧复氧致细胞凋亡模型的建立[J].泰山医学院学报,2008,29(1)：949-951.
[4]吕国蔚,史美棠,李凌等.急性重复性缺氧对小鼠耐受性的影响及其机制的初步探讨[J].中国病理生理杂志,1992,8(4)：425-428
[5]董苍转,吕国蔚.缺氧预适应小鼠脑匀浆提取液对PC12细胞的影响[J].首都医科大学学报,2001,22(2)97-99.
[6]李宏,高文祥,高钰琪等.缺氧预适应小鼠脑匀浆提取液对Hep G2细胞缺氧耐受性的影响[J].河南师范大学学报,2007,35(4)：127-130.
[1]吕国蔚,崔秀玉,赵兰峰等.低氧预适应的脑机制[J].中国应用生理性杂志,2004,20(1)：98-103.
[2]Stenzel-Poore M P,Stevens S L,Simon R P.Genomics of preconditioning. Stroke,2004,35(11 Suppl 1):2683-2686.
[3]Stenzel-Poore M P,Stevens S L,Xiong Z G,et al.Effect of ischemic preconditioning on genomic response to cerebral ischemia:similarity to neuroprotective strategies in hibernation and hypoxia-tolerant states.Lancet, 2003,362(9389):1028-1037.
[4]邵国,苏燕,张彦波等.缺氧预适应小鼠海马组织HIF-1α表达的变化.中国神经疾病杂志,2007,33(8)：489-491.
[5]Kawahara N,Wang Y,Mukasa A,et al.Genome-wide gene expression analysis for induced ischemic tolerance and delayed neuronal death following transient global ischemia in rats.J Cereb Blood Flow Metab,2004,24(2): 212-223.
[6]SmithV,PttigrwLC,SpalkyRM.et al.Metyrapone,an inhibitor of glucocorticoid pordution, reduces brain injury induced by focal and global ischemia and seizures.J Cereb Blood Flow Metab,1996,16(4):585.
[7]郑大明,周宏君,杨惠吟等.脑缺血预适应血清C反应蛋白、D-二聚体及纤维蛋白原的变化[J].广东医学,2007,28(8)：1312-1313.
[8]方忠林,谢志泉.缺血预适应对SD大鼠缺血/再灌注血清TNF-α,IL-8和心肌IL-10水平的影响[J].心脏杂志,2008,20(5)：517-520.
[9]Ren C,Gao X, Steinberg GK,et al. Limb remote-preconditioning protects against focal ischemia in rats and contradicts the dogma of therapeutic time windows for preconditioning[J]. Neuroscience,2008,151:1099-1103.
[10]罗琼,袁恒杰,吴艳娜等.无创性延迟肢体缺血预适应减轻大鼠脑缺血/再灌注损伤[J].基础医学与临床,2009,(6)：603-606.
[11]袁恒杰,罗琼,朱学慧等.无创性延迟肢体缺血预适应对大鼠脑缺血再灌注皮层的保护作用[J].基础研究,2009,14(2)：144-149.
[12]Kitagawa K,Matsumoto M,Tagaya M,et al.Ischemic tolerance phenomenon found in the brain[J].Brain Res,1990,528:21-27.
[1]Lazar HL,Bao Y,Rivers S,et al.Pretreatment with angiotensin- converting enzyme inhibitors attenuates ischemia-reperfusion injury[J]. Ann Thorac Surg,2002,73(5):1522-1527.
[2]Wolfrum S,Richardt G,Dominiak P, et al. Apstatin,a selective inhi-bitor of aminopeptidase Preduces myocardial infarct size by a kinin-dependent pathway[J]. B r J Pharmacol,2001,134(2):370-374.
[3]Schwarz ER,Montino H, Flcischhauer J,et al.Angiotensin Ⅱ receptor antagonist EXP 3174 reduces infarct size comparable with enalaprilat and augments preconditi- oning in the pig heart [J]. Cardiovasc Drug Ther, 1997,11(5):687-695.
[4]Jacobson KA.Introduction to adenosine receptors as therapeutic targets. Handb Exp Pharmacol 2009,(193):1-24.
[5]陈霞平,杜士明,王启斌等.腺苷促进体外培养神经干细胞增殖作用研究[J].医药导报,2008,27(8)：911-914.
[6]张伟丽,吕国蔚.急性重复缺氧对小鼠脑组织腺苷及其Al受体的影响[J].中国应用生理学杂志,1997,13(2)：114-117.
[7]刘亮,吕国蔚.缺氧预适应小鼠脑匀浆去蛋白液对缺氧突触体膜的保护作用.中国神经科学杂志,2001,17(4)：373-375.
[8]Kis B, Rajapakse N C, Snipes J A, et al. Diazoxide induces delayd pre-conditioning in cultured rat cortical neurons. J Neurochim,2003,87:969-980.
[2]吕国蔚,史美棠,李凌等.急性重复性缺氧对小鼠耐受性的影响及其机制的初步探讨[J].中国病理生理杂志,1992,8(4)：425-428.
[3]张颜波,吕国蔚,杨明峰等.低氧预适应小鼠海马Bcl-2表达和Caspase-3活性的变化[J].中华神经医学杂志,2007,40(8)：553-555.
[4]刘亮,吕国蔚.缺氧预适应小鼠脑匀浆去蛋白液对缺氧突触体膜的保护作用.中国神经科学杂志,2001,17(4)：373-375.
[5]VannucciRC,Towfighi J,Vannucci SJ.Hypoxic preconditioning and hypoxia-ischemic brain damdge in the immature rat:pathologic and metab- olic corrclatcs[J]. JNcurochem,1998,71(3):1215-1220.
[6]杨明峰,岳伟,张伟等.大鼠鼠胚海马神经细胞原代培养及神经元鉴定[J].泰山医学院学报,2006,27(7)：614-616.
[7]Fodoroffs.Cell tissue and organ culture in neuron-biology. Academic ress, 1979,191-213.
[8]Kerr JF, Wyllie AH, Currie A R. A basic biological phenomenon with wide rang implications in tissue Kinetics. Br J Cancer,1972,26:239.
[9]Thompson C B. Apoptosis in the pathogenesis and the treatment of disease [J]. Science,1995,267:1456.
[10]Nitatori T,Sato N,Waguri S,et al.Delayed neuronal death in the CA1 pyramidal cell layer of the gerbil hippocampus following transient isehemia is apoptosis.J Neurosei,1995,15(2):1001-1011.
[11]Guegan C, Boutin H, Boudry C, et al. Apoptosis death in cortical neurons of mice subjected to focal ischemia. C R Acad Sci 1996,319:875-885.
[12]尹琰,寿伟璋.流式细胞术AnnexinV细胞凋亡检测方法探讨.东南大学学报,2003,22：169.
[13]Span L P,Pennings AH,Vierwinden G,et al.The dynamic process of apoptosis analyzed by flow cytometry usingAnnexinV/propidium iodide and amodified in situ end abeling technique.Cytometry,2002,47 (1):24
[14]莫简.医用自由基生物学导论[M].北京：人民卫生出版社,1989,81-83.
[15]Jarasch ED, Bruder G, Hei HV, et al. Significance of xanthine oxidase in capillary endotheliacell[J]. Acta Pysiol Scand Suppl,1986,548:39.
[16]Lewen A,Matz P,Chan PH.Free radical pathways in CNS injury[J].J Neurotrauma,2000,17 (10):871-890.
[17]吴其夏,余应年,卢建.新编病理生理学[M].第1版.北京：中国协和医科大学出版社,1999,174-208.
[18]Takahashi G, SakuraiM, Abe K, et al. MCI-186 prevents spinal cord damage and affects enzyme levels of nitric oxide synthase and Cu/Zn superoxide dismutase after transient ischemia in rabbits[J]. J Thorac Cardiovasc Surg,2003,12.6:1461.
[19]刘庆华,邵书丽.细胞凋亡.解剖学进展,1999：5(3)：127-130
[20]Hanada M, Aim-SemPe C, Sato T, et al.Structure-function analysis of Bel-2 protein.Identification of conserved domains important for homodimeriza-tion with Bel-2 and heterodimerization with Bax.J Biol Chem,1995,270 (20):11962-11969.
[21]Hockenbery DM, Oltvai ZN,Yin XM. Bcl-2 function in an antioxidant pathyway to prevent apoptosis. Cell 1993,75:241-251
[22]Tsujimono Y,Shimizu S, Eguchi Y. Bcl-2 and Bcl-x1 block apoptosis as well as necrosis:possible involvement of common mediators in apoptotic and necrotic signal ransudation pathways. Leukemia 1997,3:380-382.
[23]Oltvai ZN, Milliman C L, Kormeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homology, Ax, that accelerates programmed cell death. Cell 1993,74:609-619
[24]Reed J C. Bcl-2 and the regulation of programmed cell death. J Cell'Biol 1994,17:260-263.
[25]Groc L,Bezin L,JiangH,et al. Bax, Bcl-2,and cyclin expression and apoptosis in rat substantia nigra during development [J].Neurosci Lett, 2001,306:198-202.
[1]牛敬忠,杨明峰,张颜波等.蛋白激酶C抑制剂灯盏花素对低氧预适应小鼠海马NO/cGMP信号转导系统的影响[J].泰山医学院学报,2008,29(1)：5-7.
[2]张颜波,吕国蔚,牛敬忠等.低氧预适应小鼠皮层Bcl-2表达和Caspase-3活性的变化[J].生理学报,2008,60(2)：49-253.
[3]杨明峰,张颜波,牛敬忠等.大鼠鼠胚海马神经元缺氧复氧致细胞凋亡模型的建立[J].泰山医学院学报,2008,29(1)：949-951.
[4]吕国蔚,史美棠,李凌等.急性重复性缺氧对小鼠耐受性的影响及其机制的初步探讨[J].中国病理生理杂志,1992,8(4)：425-428
[5]董苍转,吕国蔚.缺氧预适应小鼠脑匀浆提取液对PC12细胞的影响[J].首都医科大学学报,2001,22(2)97-99.
[6]李宏,高文祥,高钰琪等.缺氧预适应小鼠脑匀浆提取液对Hep G2细胞缺氧耐受性的影响[J].河南师范大学学报,2007,35(4)：127-130.
[1]吕国蔚,崔秀玉,赵兰峰等.低氧预适应的脑机制[J].中国应用生理性杂志,2004,20(1)：98-103.
[2]Stenzel-Poore M P,Stevens S L,Simon R P.Genomics of preconditioning. Stroke,2004,35(11 Suppl 1):2683-2686.
[3]Stenzel-Poore M P,Stevens S L,Xiong Z G,et al.Effect of ischemic preconditioning on genomic response to cerebral ischemia:similarity to neuroprotective strategies in hibernation and hypoxia-tolerant states.Lancet, 2003,362(9389):1028-1037.
[4]邵国,苏燕,张彦波等.缺氧预适应小鼠海马组织HIF-1α表达的变化.中国神经疾病杂志,2007,33(8)：489-491.
[5]Kawahara N,Wang Y,Mukasa A,et al.Genome-wide gene expression analysis for induced ischemic tolerance and delayed neuronal death following transient global ischemia in rats.J Cereb Blood Flow Metab,2004,24(2): 212-223.
[6]SmithV,PttigrwLC,SpalkyRM.et al.Metyrapone,an inhibitor of glucocorticoid pordution, reduces brain injury induced by focal and global ischemia and seizures.J Cereb Blood Flow Metab,1996,16(4):585.
[7]郑大明,周宏君,杨惠吟等.脑缺血预适应血清C反应蛋白、D-二聚体及纤维蛋白原的变化[J].广东医学,2007,28(8)：1312-1313.
[8]方忠林,谢志泉.缺血预适应对SD大鼠缺血/再灌注血清TNF-α,IL-8和心肌IL-10水平的影响[J].心脏杂志,2008,20(5)：517-520.
[9]Ren C,Gao X, Steinberg GK,et al. Limb remote-preconditioning protects against focal ischemia in rats and contradicts the dogma of therapeutic time windows for preconditioning[J]. Neuroscience,2008,151:1099-1103.
[10]罗琼,袁恒杰,吴艳娜等.无创性延迟肢体缺血预适应减轻大鼠脑缺血/再灌注损伤[J].基础医学与临床,2009,(6)：603-606.
[11]袁恒杰,罗琼,朱学慧等.无创性延迟肢体缺血预适应对大鼠脑缺血再灌注皮层的保护作用[J].基础研究,2009,14(2)：144-149.
[12]Kitagawa K,Matsumoto M,Tagaya M,et al.Ischemic tolerance phenomenon found in the brain[J].Brain Res,1990,528:21-27.
[1]Lazar HL,Bao Y,Rivers S,et al.Pretreatment with angiotensin- converting enzyme inhibitors attenuates ischemia-reperfusion injury[J]. Ann Thorac Surg,2002,73(5):1522-1527.
[2]Wolfrum S,Richardt G,Dominiak P, et al. Apstatin,a selective inhi-bitor of aminopeptidase Preduces myocardial infarct size by a kinin-dependent pathway[J]. B r J Pharmacol,2001,134(2):370-374.
[3]Schwarz ER,Montino H, Flcischhauer J,et al.Angiotensin Ⅱ receptor antagonist EXP 3174 reduces infarct size comparable with enalaprilat and augments preconditi- oning in the pig heart [J]. Cardiovasc Drug Ther, 1997,11(5):687-695.
[4]Jacobson KA.Introduction to adenosine receptors as therapeutic targets. Handb Exp Pharmacol 2009,(193):1-24.
[5]陈霞平,杜士明,王启斌等.腺苷促进体外培养神经干细胞增殖作用研究[J].医药导报,2008,27(8)：911-914.
[6]张伟丽,吕国蔚.急性重复缺氧对小鼠脑组织腺苷及其Al受体的影响[J].中国应用生理学杂志,1997,13(2)：114-117.
[7]刘亮,吕国蔚.缺氧预适应小鼠脑匀浆去蛋白液对缺氧突触体膜的保护作用.中国神经科学杂志,2001,17(4)：373-375.
[8]Kis B, Rajapakse N C, Snipes J A, et al. Diazoxide induces delayd pre-conditioning in cultured rat cortical neurons. J Neurochim,2003,87:969-980.