促红细胞生成素对创伤性脑损伤神经保护的实验研究
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摘要
本研究在建立稳定可靠的颅脑液压损伤模型的基础上，从EPO对TBI大鼠神经细胞凋亡及其相关基因表达的作用及TBI大鼠认知功能的影响等方面说明外源性EPO在TBI大鼠模型中的神经保护作用。
第一部分促红细胞生成素及受体在TBI大鼠模型的表达
目的：研究在创伤性颅脑损伤大鼠模型中损伤周围脑组织EPO及其受体的表达。内容和方法：78只SD大鼠随机分为对照组6只，假手术组36只，液压冲击脑创伤模型组36只，根据处死时间，分为6、24 h和3、5、7、14d 6个时相点，每个时相点假手术组和脑损伤组各处死6只大鼠，取损伤周围脑组织。利用实时荧光定量PCR和Western Blot方法检测EPO、EPOR的mRNA表达和蛋白表达。
结果：EPO在外伤后24h内即可升高，到第3d达到高峰，并可维持2d左右，至伤后第7d开始下降，于伤后14d基本恢复至伤前水平；而EPOR于伤后24h到达高峰，至外伤后14d表达量仍可维持较高的水平。
结论：TBI后24h即可促进内源性EPO及其受体的表达，但二者的表达具有不一致性，且EPO相对EPOR表达具有短暂性。
第二部分促红细胞生成素对创伤性神经细胞损伤凋亡作用的体内研究
目的：探讨EPO对TBI大鼠神经细胞调控凋亡通路，以阐明其作用机制。内容和方法：72只SD大鼠随机分为六组：对照组12只；假手术组12只；TBI组12只；TBI+EPO组12只；TBI+EPO+DMSO组12只；TBI+EPO+LY294002组12只。治疗组腹腔注射EPO 5000u／Kg，Q8H；并分组脑室给予LY294002，以阻断PI-3K通路。于外伤后2d处死大鼠，行Western Blot检测EPO及pAKT的蛋白表达，行免疫组化检测凋亡相关蛋白表达及TUNEL观察凋亡指数。
结果：Western Blot检测发现治疗组EPO表达明显上调，且LY294002可以阻断PI-3K信号传导通路，下调pAKT的表达，而载体组对pAKT的表达无明显影响。免疫组化检测发现EPO可以明显抑制BAX、Caspase 3的表达，从而抑制凋亡；而LY294002干预组BAX、Caspase 3的表达量明显上调，TUNEL示凋亡指数增高。结论：EPO可以抑制TBI大鼠神经细胞的凋亡，阻断PI3K信号传导通路后，凋亡指数明显增高。
第三部分促红细胞生成素对创伤性大鼠认知功能影响的实验研究
目的：探讨EPO对TBI大鼠认知功能的影响。
内容和方法：48只SD大鼠随机分为4组：对照组12只，假手术组12只，液压冲击脑创伤模型(TBI)组12只，脑创伤促红细胞生成素(EPO+TBI)治疗组12只，TBI+EPO组于外伤后立即腹腔注射EPO(5000U／Kg，Q8H，连续2d)；TBI组在同一时间点给予相同剂量的生理盐水腹腔注射。于外伤后8d Morris水迷宫检测认知功能，后处死大鼠灌注，行BDNF免疫组化检测。
结果：在定位航行实验中，各组大鼠寻找平台的潜伏期都随实验进程越来越短，且各组大鼠游泳速度无差别。而潜伏期的结果表明对照组及假手术组时间最短，外伤组最长，EPO治疗组居于两者之间。提示EPO治疗改善大鼠的定位航行实验表现。空间搜索实验表明：：对照组大鼠在平台所在象限停留的时间较长，外伤组在此象限游泳的时间明显短于对照组，EPO治疗介于两者之间，提示EPO治疗大鼠创伤后参考记忆有明显恢复。免疫组化结果表明，EPO可明显上调BDNF的表达，提示EPO可能通过促进BDNF的表达来改善外伤后大鼠的认知功能
结论：液压冲击造成的颅脑损伤可损害大鼠的认知功能，外源性给予EPO可以使大鼠在Morris水迷宫的检测结果明显提高，提示EPO可以促进大鼠的空间学习记忆能力；与外伤组相比，EPO治疗组中大鼠的BDNF蛋白的表达明显上调，具有统计学差异，说明外源性EPO通过促进BDNF的表达，从而改善TBI大鼠的认知能力。
On the basis of stable and reliable TBI models,we studied the expression oferythropoietin and its receptor around the injured brain tissue in the rats aftertraumatic brain injury;then,by the means of Western blot,immunohistochemisty andTUNEL,the apoptosis index and related apotosis gene expression were tested;at last,it was certified that effect of erythropoietin on the TBI rats' cognitive function.
Part One:The expression of erythropoietin and its receptor in the rats followingtraumatic brain injury.
Objective:The part was aimed to study the expression of erythropoietin and itsreceptor around the injured brain tissue in the rats after traumatic brain injury.
Methods:78 SD rats were randomly divided into 3 groups including controlgroup(six rats),sham group(thirty-six rats) and fluid percussion injurygroup(thirty-six rats).According to the time of death,the latter two groups weredivided into six subgroups which were 6 hours,twenty-four hours,three days,fivedays,seven days and fourteen days after traumatic brain injury (TBI).Six rats weresentenced to death in the sham and TBI group at each time point.It test the messengerRNA and protein of erythropoietin and its receptor of the brain tissue surrounding theinjuries by the methods of real-time PCR and Western Blot respectively.
Results:EPO and its receptor mRNA expressed in normal control and sham groupat each time point were just a small amount.Real-time PCR and Western Blotindicated that EPO mRNA and protein expressions increased at 6h,peaked at 3d,which sustained two days,and gradually declined to 7 d,returned to normal level at14d.EPOR mRNA and protein expressions in the TBI group began to increase at 6hafter injury,peaked at 24h,sustained to 14d.
Conclusion:EPOR expression was significantly upregulated at least for 14 daysafter traumatic brain injury,while EPO mRNA and its protein were only shortlyelevated.The protective mechanism of EPO on central nervous system is possiblyrelated to the markedly increased expression of erythropoietin receptor after TBI.
Part Two:The effect of erythropoietin on apoptosis of nerve cells in the TBI modelsin Vivo.
Objective:The part was aimed to evaluate the effect of erythropoietin on apoptosis inthe TBI model,so as to conclude the neuroprotection mechanism of erythropoietin.
Methods:76 SD rats was randomly divided into six groups including control,sham, TBI,EPO,EPO+DMSO and EPO+LY294002 group,each group had twelve rats.Thelatter three groups were injected with erythropoietin (5000u/Kg,Q8h) by the way ofintraperitoneal.Meanwhile,the latter two groups were injected with DMSO orLY294002 through intraventricule in order to block PI3K pathway.After 2 days,allrats were sentenced to death,in order to test the protein of EPO and pAKT byWestern Blot,and the protein of apoptosis by immunohistochemisty,and apoptosisindex by TUNEL.
Results:it is indicated in Western Blot that the erythropoietin treatment can promotethe expression of its protein,while downregulated the expression of pAKT.It isindicated in immunohistochemisty that EPO can inhibit the expression of BAX andCaspase-3,thus reduce apoptosis,while LY294002 can block the PI3K pathway andupregulated BAX and Caspase-3,so the apoptosis index was more than the EPOtreatment group.
Conclusion:EPO can inhibit the apoptosis in the TBI model,when it is blocked withthe PI-3K pathway,which increased the apoptosis index.
Part Three:The effect on cognitive function of erythropoietin in the rats aftertraumatic brain injury.
Objective:The part was aimed to evaluate the effect on cognitive function oferythropoietin.
Methods:48 SD rats were randomly divided into four groups including control,sham,TBI and EPO+TBI group,each have twelve rats.The last group were injected witherythropoietin immediately after injury,the others were injected with saline at thesame time.After eight days,we used the Morris Water Maze to evaluate the referencememory and expression of BDNF by immunohistochemisty.
Results:In the navigation experiment,the latent period that all the rats found theplatform was shorter and shorter,and there is no difference in speed,while the controland sham groups' latent period was the shortest,and the TBI group's was longest,EPO can improve the latent period.Meanwhile,it can promote the expression ofBDNF.
Conclusion:TBI can damage the cognitive function of rats,while exogenous EPOcan improve the rats' cognitive function and upregulated the expression of BDNF.
第一部分促红细胞生成素及受体在TBI大鼠模型的表达
目的：研究在创伤性颅脑损伤大鼠模型中损伤周围脑组织EPO及其受体的表达。内容和方法：78只SD大鼠随机分为对照组6只，假手术组36只，液压冲击脑创伤模型组36只，根据处死时间，分为6、24 h和3、5、7、14d 6个时相点，每个时相点假手术组和脑损伤组各处死6只大鼠，取损伤周围脑组织。利用实时荧光定量PCR和Western Blot方法检测EPO、EPOR的mRNA表达和蛋白表达。
结果：EPO在外伤后24h内即可升高，到第3d达到高峰，并可维持2d左右，至伤后第7d开始下降，于伤后14d基本恢复至伤前水平；而EPOR于伤后24h到达高峰，至外伤后14d表达量仍可维持较高的水平。
结论：TBI后24h即可促进内源性EPO及其受体的表达，但二者的表达具有不一致性，且EPO相对EPOR表达具有短暂性。
第二部分促红细胞生成素对创伤性神经细胞损伤凋亡作用的体内研究
目的：探讨EPO对TBI大鼠神经细胞调控凋亡通路，以阐明其作用机制。内容和方法：72只SD大鼠随机分为六组：对照组12只；假手术组12只；TBI组12只；TBI+EPO组12只；TBI+EPO+DMSO组12只；TBI+EPO+LY294002组12只。治疗组腹腔注射EPO 5000u／Kg，Q8H；并分组脑室给予LY294002，以阻断PI-3K通路。于外伤后2d处死大鼠，行Western Blot检测EPO及pAKT的蛋白表达，行免疫组化检测凋亡相关蛋白表达及TUNEL观察凋亡指数。
结果：Western Blot检测发现治疗组EPO表达明显上调，且LY294002可以阻断PI-3K信号传导通路，下调pAKT的表达，而载体组对pAKT的表达无明显影响。免疫组化检测发现EPO可以明显抑制BAX、Caspase 3的表达，从而抑制凋亡；而LY294002干预组BAX、Caspase 3的表达量明显上调，TUNEL示凋亡指数增高。结论：EPO可以抑制TBI大鼠神经细胞的凋亡，阻断PI3K信号传导通路后，凋亡指数明显增高。
第三部分促红细胞生成素对创伤性大鼠认知功能影响的实验研究
目的：探讨EPO对TBI大鼠认知功能的影响。
内容和方法：48只SD大鼠随机分为4组：对照组12只，假手术组12只，液压冲击脑创伤模型(TBI)组12只，脑创伤促红细胞生成素(EPO+TBI)治疗组12只，TBI+EPO组于外伤后立即腹腔注射EPO(5000U／Kg，Q8H，连续2d)；TBI组在同一时间点给予相同剂量的生理盐水腹腔注射。于外伤后8d Morris水迷宫检测认知功能，后处死大鼠灌注，行BDNF免疫组化检测。
结果：在定位航行实验中，各组大鼠寻找平台的潜伏期都随实验进程越来越短，且各组大鼠游泳速度无差别。而潜伏期的结果表明对照组及假手术组时间最短，外伤组最长，EPO治疗组居于两者之间。提示EPO治疗改善大鼠的定位航行实验表现。空间搜索实验表明：：对照组大鼠在平台所在象限停留的时间较长，外伤组在此象限游泳的时间明显短于对照组，EPO治疗介于两者之间，提示EPO治疗大鼠创伤后参考记忆有明显恢复。免疫组化结果表明，EPO可明显上调BDNF的表达，提示EPO可能通过促进BDNF的表达来改善外伤后大鼠的认知功能
结论：液压冲击造成的颅脑损伤可损害大鼠的认知功能，外源性给予EPO可以使大鼠在Morris水迷宫的检测结果明显提高，提示EPO可以促进大鼠的空间学习记忆能力；与外伤组相比，EPO治疗组中大鼠的BDNF蛋白的表达明显上调，具有统计学差异，说明外源性EPO通过促进BDNF的表达，从而改善TBI大鼠的认知能力。
On the basis of stable and reliable TBI models,we studied the expression oferythropoietin and its receptor around the injured brain tissue in the rats aftertraumatic brain injury;then,by the means of Western blot,immunohistochemisty andTUNEL,the apoptosis index and related apotosis gene expression were tested;at last,it was certified that effect of erythropoietin on the TBI rats' cognitive function.
Part One:The expression of erythropoietin and its receptor in the rats followingtraumatic brain injury.
Objective:The part was aimed to study the expression of erythropoietin and itsreceptor around the injured brain tissue in the rats after traumatic brain injury.
Methods:78 SD rats were randomly divided into 3 groups including controlgroup(six rats),sham group(thirty-six rats) and fluid percussion injurygroup(thirty-six rats).According to the time of death,the latter two groups weredivided into six subgroups which were 6 hours,twenty-four hours,three days,fivedays,seven days and fourteen days after traumatic brain injury (TBI).Six rats weresentenced to death in the sham and TBI group at each time point.It test the messengerRNA and protein of erythropoietin and its receptor of the brain tissue surrounding theinjuries by the methods of real-time PCR and Western Blot respectively.
Results:EPO and its receptor mRNA expressed in normal control and sham groupat each time point were just a small amount.Real-time PCR and Western Blotindicated that EPO mRNA and protein expressions increased at 6h,peaked at 3d,which sustained two days,and gradually declined to 7 d,returned to normal level at14d.EPOR mRNA and protein expressions in the TBI group began to increase at 6hafter injury,peaked at 24h,sustained to 14d.
Conclusion:EPOR expression was significantly upregulated at least for 14 daysafter traumatic brain injury,while EPO mRNA and its protein were only shortlyelevated.The protective mechanism of EPO on central nervous system is possiblyrelated to the markedly increased expression of erythropoietin receptor after TBI.
Part Two:The effect of erythropoietin on apoptosis of nerve cells in the TBI modelsin Vivo.
Objective:The part was aimed to evaluate the effect of erythropoietin on apoptosis inthe TBI model,so as to conclude the neuroprotection mechanism of erythropoietin.
Methods:76 SD rats was randomly divided into six groups including control,sham, TBI,EPO,EPO+DMSO and EPO+LY294002 group,each group had twelve rats.Thelatter three groups were injected with erythropoietin (5000u/Kg,Q8h) by the way ofintraperitoneal.Meanwhile,the latter two groups were injected with DMSO orLY294002 through intraventricule in order to block PI3K pathway.After 2 days,allrats were sentenced to death,in order to test the protein of EPO and pAKT byWestern Blot,and the protein of apoptosis by immunohistochemisty,and apoptosisindex by TUNEL.
Results:it is indicated in Western Blot that the erythropoietin treatment can promotethe expression of its protein,while downregulated the expression of pAKT.It isindicated in immunohistochemisty that EPO can inhibit the expression of BAX andCaspase-3,thus reduce apoptosis,while LY294002 can block the PI3K pathway andupregulated BAX and Caspase-3,so the apoptosis index was more than the EPOtreatment group.
Conclusion:EPO can inhibit the apoptosis in the TBI model,when it is blocked withthe PI-3K pathway,which increased the apoptosis index.
Part Three:The effect on cognitive function of erythropoietin in the rats aftertraumatic brain injury.
Objective:The part was aimed to evaluate the effect on cognitive function oferythropoietin.
Methods:48 SD rats were randomly divided into four groups including control,sham,TBI and EPO+TBI group,each have twelve rats.The last group were injected witherythropoietin immediately after injury,the others were injected with saline at thesame time.After eight days,we used the Morris Water Maze to evaluate the referencememory and expression of BDNF by immunohistochemisty.
Results:In the navigation experiment,the latent period that all the rats found theplatform was shorter and shorter,and there is no difference in speed,while the controland sham groups' latent period was the shortest,and the TBI group's was longest,EPO can improve the latent period.Meanwhile,it can promote the expression ofBDNF.
Conclusion:TBI can damage the cognitive function of rats,while exogenous EPOcan improve the rats' cognitive function and upregulated the expression of BDNF.
引文
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