大鼠大脑中动脉缺血再灌注损伤后膜铁转运辅助蛋白的表达变化研究及作用探讨
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摘要
第一部分：大鼠大脑中动脉缺血再灌注模型的建立
脑血管疾病已成为目前人类的第三大重要的死亡原因之一，特别是发病率、致残率高,治愈率低,尤其是大面积、基底节区脑梗死对人类危害更大,因此对该类疾病的防治显得非常重要,可为临床实行超早期治疗与康复护理训练打下基础。因此就需要能制作出可靠的、能重复使用的、能模拟临床病理生理变化的动物模型。近年来人们越来越多的使用线栓法制作大鼠局造性缺血性模型,在于它能近于模拟人类大脑中动脉脑缺血病理过程,从而达到科研的目的。人类最为常见的缺血类型就是大脑中动脉梗塞,所以在实验中动物大脑中动脉栓塞模型应用的也就最多,参照Zealonga线栓法,并加以改进,在实验中使用聚乙烯醇处理后的兔须作为线栓制作MCAO模型,好处在于处理后线栓遇血发生膨胀,可使线栓能够充满所需要栓塞的血管,使血流完全中断,进而提高了模型制作的成功率；使用颈总动脉(CCA)及颈外动脉(ECA)两种不同的进线方法,通过比较神经肌肉运动功能评分、前庭功能评分、脑梗死体积等,进而得出两种制作方法的优缺点。
目的
用两种进线法建立Wistar大鼠大脑中动脉局灶性脑缺血模型,比较这两种方法的优劣,以观察模型的可靠性,为早期诊治缺血性脑血管病提供理论依据。
方法
选取健康清洁级Wistar大鼠70只。具体分组如下：对照组(5只)、假手术组(5只)、颈总动脉(CCA)进线组(30只)和颈外动脉(ECA)进线组(30只),后两组按照处死时间分6个亚组,即缺血再灌注后4小时、8小时、16小时、32小时、64小时、128小时,6个亚组,每亚组5只大鼠,建立大鼠脑缺血再灌注损伤动物模型。参照Zealonga的线栓法,加以改进,使用兔须替代尼龙线,经聚乙烯醇处理后分别由颈总动脉(CCA)和颈外动脉(ECA)进线制作栓塞模型。缺血90分钟后,向外抽拔线栓实现再灌注,然后在再灌注后4小时、8小时、16小时、32小时、64小时、128小时联合应用神经肌肉运动功能评分法、前庭运动功能评分法评价大鼠运动功能,3天后处死大鼠,测定大鼠大脑梗塞体积并进行比较两种方法的优缺点。
结果
颈总动脉进线法和颈外动脉进线法神经肌肉功能评分、前庭功能评分脑梗死体积分别为4.1±0.645和5.1±0.546、2.6±0.576和3.6±0.519、22.9%±4.6%和30.5%±3.5%,两法比较神经肌肉功能评分及前庭功能评分差异有统计学意义(p<0.05)；两法脑梗死体积相比差异亦有统计学意义(p<0.05)。
结论
两种方法制作的大鼠脑缺血模型的均出现神经功能障碍,脑组织缺血梗死表现。与颈总动脉进线法相比,颈外动脉进线法大鼠神经行为学评分高、脑梗死体积大、成功率高,模型稳定性好。
第二部分：脑缺血再灌注损伤后膜铁转运辅助蛋白的表达与变化研究及作用探讨
铁是生命体内不可缺少的微量元素,同样又是人体中最丰富的过渡金属。最近研究发现铁的功能远比人们想象的重要。铁在人脑中分布广泛,但这种分布并不是均匀的。人类神经系统的各种细胞中几乎都有铁的分布,如神经元、少突触胶质细胞、小胶质细胞和一部分星形胶质细胞都含量较多,其中含铁量最多的是少突胶质细胞。脑内铁具有极其重要的作用,维持铁的稳态对机体非常重要,众多的研究表明急性脑缺血后机体各器官会出现铁代谢紊乱,铁负荷过重可介导一系列的病理生理变化,从而诱发各种疾病。
研究证明,铁蛋白(transferrin, Tf)和转铁蛋白受体(transferrin receptor, T:fR)是参与铁在脑组织中吸收的两种途径的主要蛋白。研究发现铁在脑细胞中的代谢机制与外周代谢机制大不相同,且前者代谢及平衡机制更为复杂,已有研究证明,铁代谢的主要途径是转铁蛋白受体介导的细胞转铁蛋白—fe(Tf-fe)参与的途径,另外新发现的铁代谢蛋白如膜铁转运蛋白l(Ferroportin1 FP1)、二价金属转运体(DMT 1)等也参与了脑的铁代谢过程。膜铁转运辅助蛋白(Hephaestin, Hp)是研究性连贫血(sex-linked anemic, sla)小鼠时发现的一种新的铁转运相关蛋白。该蛋白的表达有明显的组织特异性,其中小肠高度表达。研究表明在小鼠和大鼠的大脑皮层、纹状体、脉络丛、海马、黑质等脑区也有Hp mRNA和蛋白的丰富表达,最近有报道人类Hp在肠道神经系统以及胰腺β细胞也有表达。Hp与铜蓝蛋白(Ceruloplasmin, CP)属同一家族,也是一含铜的亚铁氧化酶,能将有毒的二价铁氧化成无毒的三价铁。到目前为止,已有报道脑缺血后Hephaestin表达的变化,本研究目的是用不同种类的大鼠在不同的缺血再灌注时段观察Hephaestin的表达变化。
目的
探讨大鼠脑缺血再灌注后Hephaestin在大脑皮层、纹状体表达的动态变化与再灌注时间的关系,初步探讨Hephaestin在脑铁代谢中的作用。
方法
选取40只健康清洁级Wistar大鼠,随机分为8组,每组5只。第一组(假手术组)仅分离颈总动脉和颈外动脉及颈内动脉,并不进行插线；第二组为对照组,不做任何处理；第三组为缺血/再灌注组,依据再灌注后4小时、8小时、16小时、32小时、64小时、128小时不同时间点分为六个亚组；建立大鼠脑缺血再灌注损伤动物模型,免疫组化染色,通过Leica Q550IW系统进行图像分析,计算出损伤后不同时点大鼠大脑皮层、纹状体区平均光密度值。
结果
正常大鼠的大脑皮层、纹状体都有Hephaestin表达,缺血侧大脑皮层、纹状体再灌注后8小时表达明显增加并持续到再灌注后64小时,表达高峰出现在再灌注16至32小时期间(P<0.05),至128小时表达较正常对照组有明显减少((P<0.05)。
结论
大鼠脑组织中Hephaestin'的表达在急性脑缺血再灌注后出现明显的变化,研究表明,在脑缺血再灌注16-32小时后表达最为明显,128小时后表达程度达到低谷。Hephaestin在急性脑缺血的病理生理变化中可能发挥着重要的作用,Hephaestin的表达可能与大鼠缺血的大脑组织损伤程度及脑缺血后脑组织中铁水平变化有密切关系。
PartⅠThe establishment of the middle cerebral artery occlusionmodel model
Nowadays,the cerebral vascular disease is becoming the third most common death in our country. The disease is a severe disease with high morta-lity and disability,and the low cure rate. Eespecially,alarge area, basal nuclei cerebral infarction has caused very serious infulence to the work and life of the patients.So it is very important to prevention and cure these disease, it can help to early treatment and convalescence in the clinical.So the reliable and reroducible animal models of cerebral ischemia is seriously needed,because it can simulate clinical pathophysiology process.In rencent years,the focal ischemic model is frequently used, it can simulate the process of clinical pathophysiology,thereby it can get the purpose of scientific research,and middle cerebral artery occlusion is the most application in the experimentation. And the model was modified by method of Zealonga.In the experiment,the rabbit beards which was dealt with by polyvinyl alcohol was used to make MCAO. The polyvinyl alcohol was inflation when meet the rat's blood,so it could fit different rat with different body weight and different thickness blood vessel,and the blood flow was blocked more completely. Simultaneously,it can elevate the achievement ratio of the model. The common carotid artery embolism method and the external carotid artery embolism method was used,and through the nerve muscle score, vestibular function score, cerebral infarction volum,to compare the superiority of the two method.
Objective
to make focal cerebral ischumic model of middle cerebral artery in Wistar rats by two methods, and to compare the methods of occlusion of common carotid artery and external carotid artery. And to offer the theory gist for diagnosis and treatment of the ischemic cerebrovascular disease
Method
Here to selective 70 clearing Wistar rat, and the embolism reperfusion model was divided randomly into 4 groups, control group(5), sham operation group(5), common carotid artery group(30), external carotid artery group(30), and the last two groups were divided randomly into 6 subsections, every subsections has 5 rats,that is 4h,8h,16h,32h,64h,128h after ischemic reper-fusion.The ischemic refusion model was established.The middle artery ischemia model of Wistar rats were made by the modified method of Zealonga.In the experiment,the rabbit beards which was dealt with by polyvinyl alcohol was used to make MCAO via the methods of common carotid artery and external carotid artery. The reperfusion was done after 90min of ischemic.The nerve muscle score method and the vestibule motor function score method was used to score the rat's motor function after 4hour,8hour,16hour,32hour,64hour and 128hour of reperfusion.After 3days,the rats were put to death,and the infarction volume was deterined and compared.
Result
The nerve muscle scorings in common carotid artery group and external carotid artery were 4.1±0.645 and 5.1±0.546, respectively. The scorings between the two methods was significantly different (p<0.05).The vestibule motor function scorings of the two methods were 2.6±0.576 and 3.6±0.519 respectively, The scorings between the two methods was significantly different too, (P<0.05). The infarction volume of the two methods was 22.9%±4.6% and 30.5%±3.5%, and also there was significantly different in the infarction volume between the two groups, (p<0.05).
Conclusion
Both the common carotid artery group and external carotid artery were appeared nerve functional impairment and ischemic infarction of brain tissue. Compared with the common carotid artery occlusion group, the neurological behavior scoring of external carotid artery group was higher, the infarction volume was larger, and the stability was better.
PARTⅡImpression and effectiveness of Hephaestin after middle cerebral artery ischemis-reperfusion in rats
Iron is the sine qua non trace meter in all life,also it is the most affluent transition metal in the human body.The original study discovers that the importance of function of iron that people to know more than people can imagine. There is iron in the every parts of brain,but the distribution is not uniformity.Iron reside in many cell of nervous system,such as neurone, microglia, oligodendrocyte and part of astrocyte,and the oligodendrocyte has the more iron than any other part of brain.The iron in the brain to rise to get up very important physiological effectiveness,it is electron transporter and the important matter of mitochondrion oxidizing reaction and oxygen transport.It participate the synthesize of DNA,RNA, protein and myelin,and it also participate the development and genesis of nerve myelin sheath. Meanwhile,it is indispensable in the synthesis of neurotransmitter,such as dopamine and so on.It is very important to maintain the homeostasis of organism.More and more study has discovered that the homeostasis disequilibrium after acute cerebral ischemia.The redundant redundant iron can mediate a series of patho- physio logy course,and can induce various kinds of disease.
Generally,the absorption of iron in the brain tissue via the classic transferrin and transferrin receptor. But recentlt's study shows that the mechanism to take in iron in brain is more complicated than periphery,and the equilibrium mechanism of iron in brain is more complicated than out brain.Besides the main path of transferrin that is mediated by transferrin receptor,the new discovered iron metabolism proteinum such as DMT 1 and Fpl also participate iron metabolism in brain.Hephaestin is a kind of new iron conveying associated protein,it was discovered when studying sex-linked anemic mouse.The expression of hephaestin has conspicuous tissue specificity, the expression in intestinum is height.The experimental result indicated that the expression of Hephaestin mRNA and proteinum were abundance in cerebral cortex, hippocampus, choroid plexus, corpora striata and substantia nigra of rat and mouse.And the latest report say that the people's Hp also expresses in the nervous system of intestinal tract and theβcells of abdominal salivary gland. Hephaestin and Ceruloplasmin are in the same family,which is a kind of ferrous oxidase of cuprine, it can transform the noxious bivalence iron into atoxigenic Ferric.By far,it has reported the changes of expression of Hephaestin after cerebral ischemia of rat.The objective of this study is to observe diversity the diversity law of Hephaestin in different rat and in different time of ischemia reperfusion,and to approach the role in tissue of acute cerebral ischemia and hypoxia.
Objective
To study the dynamic state changes of Hephaestin in cerebral cortex, corpus striatum and hippocampus after cerebral ischemia reperfusion injury and the relationship between it and reperfusion time. Preliminary discussion on the effectiveness in brain iron metabolism of Hephaestin.
Method
Here to selective 40 clearing Wistar rat, and the embolism reperfusion model was divided randomly into 8 groups, control group(5), sham operation group(5), ischemic reperfusion group(30), and the last groups were divided randomly into 6 subsections,every subsections has 5 rats,that is 4h,8h,16h,32h, 64h,128h after ischemic reperfusion.The ischemic refusion model was established, immunohistochemistry staining, and the Leica Q550IW system was used to analyze the picture,the mean optical density values in cerebral cortex, corpus striatum was worked out in different reperfusion time.
Result
Hephaestin was expressed in cerebral cortex,corpus striatum in normal rat.After 8 hours of cerebral ischemia reperfusion,the expression of Hephaestin was increasing obviously,and it could last out 64 hours after reperfusion.At 16 to 32 hours after reperfusion it get to peak(P<0.05),and at 128 hours it was diminished greatly (P<0.05).
Conclusion
The change of expression of hephaestin in the cortex and striatum during acute cerebral ischemia and reperfusion was greatly.From the study we can see the expression of Hephaestin was most obviously after reperfusion 16hour to 32hour,and after reperfusion 128hour the expression appeared the lowest leval. and the change may play an important role in the pathphysiology after acute cerebral ischemic.It is possible that the expression of Hephaestin is closely related to the degree of injury of brain tissue and the level of iron in the cerebral tissue after cerebral injury.
脑血管疾病已成为目前人类的第三大重要的死亡原因之一，特别是发病率、致残率高,治愈率低,尤其是大面积、基底节区脑梗死对人类危害更大,因此对该类疾病的防治显得非常重要,可为临床实行超早期治疗与康复护理训练打下基础。因此就需要能制作出可靠的、能重复使用的、能模拟临床病理生理变化的动物模型。近年来人们越来越多的使用线栓法制作大鼠局造性缺血性模型,在于它能近于模拟人类大脑中动脉脑缺血病理过程,从而达到科研的目的。人类最为常见的缺血类型就是大脑中动脉梗塞,所以在实验中动物大脑中动脉栓塞模型应用的也就最多,参照Zealonga线栓法,并加以改进,在实验中使用聚乙烯醇处理后的兔须作为线栓制作MCAO模型,好处在于处理后线栓遇血发生膨胀,可使线栓能够充满所需要栓塞的血管,使血流完全中断,进而提高了模型制作的成功率；使用颈总动脉(CCA)及颈外动脉(ECA)两种不同的进线方法,通过比较神经肌肉运动功能评分、前庭功能评分、脑梗死体积等,进而得出两种制作方法的优缺点。
目的
用两种进线法建立Wistar大鼠大脑中动脉局灶性脑缺血模型,比较这两种方法的优劣,以观察模型的可靠性,为早期诊治缺血性脑血管病提供理论依据。
方法
选取健康清洁级Wistar大鼠70只。具体分组如下：对照组(5只)、假手术组(5只)、颈总动脉(CCA)进线组(30只)和颈外动脉(ECA)进线组(30只),后两组按照处死时间分6个亚组,即缺血再灌注后4小时、8小时、16小时、32小时、64小时、128小时,6个亚组,每亚组5只大鼠,建立大鼠脑缺血再灌注损伤动物模型。参照Zealonga的线栓法,加以改进,使用兔须替代尼龙线,经聚乙烯醇处理后分别由颈总动脉(CCA)和颈外动脉(ECA)进线制作栓塞模型。缺血90分钟后,向外抽拔线栓实现再灌注,然后在再灌注后4小时、8小时、16小时、32小时、64小时、128小时联合应用神经肌肉运动功能评分法、前庭运动功能评分法评价大鼠运动功能,3天后处死大鼠,测定大鼠大脑梗塞体积并进行比较两种方法的优缺点。
结果
颈总动脉进线法和颈外动脉进线法神经肌肉功能评分、前庭功能评分脑梗死体积分别为4.1±0.645和5.1±0.546、2.6±0.576和3.6±0.519、22.9%±4.6%和30.5%±3.5%,两法比较神经肌肉功能评分及前庭功能评分差异有统计学意义(p<0.05)；两法脑梗死体积相比差异亦有统计学意义(p<0.05)。
结论
两种方法制作的大鼠脑缺血模型的均出现神经功能障碍,脑组织缺血梗死表现。与颈总动脉进线法相比,颈外动脉进线法大鼠神经行为学评分高、脑梗死体积大、成功率高,模型稳定性好。
第二部分：脑缺血再灌注损伤后膜铁转运辅助蛋白的表达与变化研究及作用探讨
铁是生命体内不可缺少的微量元素,同样又是人体中最丰富的过渡金属。最近研究发现铁的功能远比人们想象的重要。铁在人脑中分布广泛,但这种分布并不是均匀的。人类神经系统的各种细胞中几乎都有铁的分布,如神经元、少突触胶质细胞、小胶质细胞和一部分星形胶质细胞都含量较多,其中含铁量最多的是少突胶质细胞。脑内铁具有极其重要的作用,维持铁的稳态对机体非常重要,众多的研究表明急性脑缺血后机体各器官会出现铁代谢紊乱,铁负荷过重可介导一系列的病理生理变化,从而诱发各种疾病。
研究证明,铁蛋白(transferrin, Tf)和转铁蛋白受体(transferrin receptor, T:fR)是参与铁在脑组织中吸收的两种途径的主要蛋白。研究发现铁在脑细胞中的代谢机制与外周代谢机制大不相同,且前者代谢及平衡机制更为复杂,已有研究证明,铁代谢的主要途径是转铁蛋白受体介导的细胞转铁蛋白—fe(Tf-fe)参与的途径,另外新发现的铁代谢蛋白如膜铁转运蛋白l(Ferroportin1 FP1)、二价金属转运体(DMT 1)等也参与了脑的铁代谢过程。膜铁转运辅助蛋白(Hephaestin, Hp)是研究性连贫血(sex-linked anemic, sla)小鼠时发现的一种新的铁转运相关蛋白。该蛋白的表达有明显的组织特异性,其中小肠高度表达。研究表明在小鼠和大鼠的大脑皮层、纹状体、脉络丛、海马、黑质等脑区也有Hp mRNA和蛋白的丰富表达,最近有报道人类Hp在肠道神经系统以及胰腺β细胞也有表达。Hp与铜蓝蛋白(Ceruloplasmin, CP)属同一家族,也是一含铜的亚铁氧化酶,能将有毒的二价铁氧化成无毒的三价铁。到目前为止,已有报道脑缺血后Hephaestin表达的变化,本研究目的是用不同种类的大鼠在不同的缺血再灌注时段观察Hephaestin的表达变化。
目的
探讨大鼠脑缺血再灌注后Hephaestin在大脑皮层、纹状体表达的动态变化与再灌注时间的关系,初步探讨Hephaestin在脑铁代谢中的作用。
方法
选取40只健康清洁级Wistar大鼠,随机分为8组,每组5只。第一组(假手术组)仅分离颈总动脉和颈外动脉及颈内动脉,并不进行插线；第二组为对照组,不做任何处理；第三组为缺血/再灌注组,依据再灌注后4小时、8小时、16小时、32小时、64小时、128小时不同时间点分为六个亚组；建立大鼠脑缺血再灌注损伤动物模型,免疫组化染色,通过Leica Q550IW系统进行图像分析,计算出损伤后不同时点大鼠大脑皮层、纹状体区平均光密度值。
结果
正常大鼠的大脑皮层、纹状体都有Hephaestin表达,缺血侧大脑皮层、纹状体再灌注后8小时表达明显增加并持续到再灌注后64小时,表达高峰出现在再灌注16至32小时期间(P<0.05),至128小时表达较正常对照组有明显减少((P<0.05)。
结论
大鼠脑组织中Hephaestin'的表达在急性脑缺血再灌注后出现明显的变化,研究表明,在脑缺血再灌注16-32小时后表达最为明显,128小时后表达程度达到低谷。Hephaestin在急性脑缺血的病理生理变化中可能发挥着重要的作用,Hephaestin的表达可能与大鼠缺血的大脑组织损伤程度及脑缺血后脑组织中铁水平变化有密切关系。
PartⅠThe establishment of the middle cerebral artery occlusionmodel model
Nowadays,the cerebral vascular disease is becoming the third most common death in our country. The disease is a severe disease with high morta-lity and disability,and the low cure rate. Eespecially,alarge area, basal nuclei cerebral infarction has caused very serious infulence to the work and life of the patients.So it is very important to prevention and cure these disease, it can help to early treatment and convalescence in the clinical.So the reliable and reroducible animal models of cerebral ischemia is seriously needed,because it can simulate clinical pathophysiology process.In rencent years,the focal ischemic model is frequently used, it can simulate the process of clinical pathophysiology,thereby it can get the purpose of scientific research,and middle cerebral artery occlusion is the most application in the experimentation. And the model was modified by method of Zealonga.In the experiment,the rabbit beards which was dealt with by polyvinyl alcohol was used to make MCAO. The polyvinyl alcohol was inflation when meet the rat's blood,so it could fit different rat with different body weight and different thickness blood vessel,and the blood flow was blocked more completely. Simultaneously,it can elevate the achievement ratio of the model. The common carotid artery embolism method and the external carotid artery embolism method was used,and through the nerve muscle score, vestibular function score, cerebral infarction volum,to compare the superiority of the two method.
Objective
to make focal cerebral ischumic model of middle cerebral artery in Wistar rats by two methods, and to compare the methods of occlusion of common carotid artery and external carotid artery. And to offer the theory gist for diagnosis and treatment of the ischemic cerebrovascular disease
Method
Here to selective 70 clearing Wistar rat, and the embolism reperfusion model was divided randomly into 4 groups, control group(5), sham operation group(5), common carotid artery group(30), external carotid artery group(30), and the last two groups were divided randomly into 6 subsections, every subsections has 5 rats,that is 4h,8h,16h,32h,64h,128h after ischemic reper-fusion.The ischemic refusion model was established.The middle artery ischemia model of Wistar rats were made by the modified method of Zealonga.In the experiment,the rabbit beards which was dealt with by polyvinyl alcohol was used to make MCAO via the methods of common carotid artery and external carotid artery. The reperfusion was done after 90min of ischemic.The nerve muscle score method and the vestibule motor function score method was used to score the rat's motor function after 4hour,8hour,16hour,32hour,64hour and 128hour of reperfusion.After 3days,the rats were put to death,and the infarction volume was deterined and compared.
Result
The nerve muscle scorings in common carotid artery group and external carotid artery were 4.1±0.645 and 5.1±0.546, respectively. The scorings between the two methods was significantly different (p<0.05).The vestibule motor function scorings of the two methods were 2.6±0.576 and 3.6±0.519 respectively, The scorings between the two methods was significantly different too, (P<0.05). The infarction volume of the two methods was 22.9%±4.6% and 30.5%±3.5%, and also there was significantly different in the infarction volume between the two groups, (p<0.05).
Conclusion
Both the common carotid artery group and external carotid artery were appeared nerve functional impairment and ischemic infarction of brain tissue. Compared with the common carotid artery occlusion group, the neurological behavior scoring of external carotid artery group was higher, the infarction volume was larger, and the stability was better.
PARTⅡImpression and effectiveness of Hephaestin after middle cerebral artery ischemis-reperfusion in rats
Iron is the sine qua non trace meter in all life,also it is the most affluent transition metal in the human body.The original study discovers that the importance of function of iron that people to know more than people can imagine. There is iron in the every parts of brain,but the distribution is not uniformity.Iron reside in many cell of nervous system,such as neurone, microglia, oligodendrocyte and part of astrocyte,and the oligodendrocyte has the more iron than any other part of brain.The iron in the brain to rise to get up very important physiological effectiveness,it is electron transporter and the important matter of mitochondrion oxidizing reaction and oxygen transport.It participate the synthesize of DNA,RNA, protein and myelin,and it also participate the development and genesis of nerve myelin sheath. Meanwhile,it is indispensable in the synthesis of neurotransmitter,such as dopamine and so on.It is very important to maintain the homeostasis of organism.More and more study has discovered that the homeostasis disequilibrium after acute cerebral ischemia.The redundant redundant iron can mediate a series of patho- physio logy course,and can induce various kinds of disease.
Generally,the absorption of iron in the brain tissue via the classic transferrin and transferrin receptor. But recentlt's study shows that the mechanism to take in iron in brain is more complicated than periphery,and the equilibrium mechanism of iron in brain is more complicated than out brain.Besides the main path of transferrin that is mediated by transferrin receptor,the new discovered iron metabolism proteinum such as DMT 1 and Fpl also participate iron metabolism in brain.Hephaestin is a kind of new iron conveying associated protein,it was discovered when studying sex-linked anemic mouse.The expression of hephaestin has conspicuous tissue specificity, the expression in intestinum is height.The experimental result indicated that the expression of Hephaestin mRNA and proteinum were abundance in cerebral cortex, hippocampus, choroid plexus, corpora striata and substantia nigra of rat and mouse.And the latest report say that the people's Hp also expresses in the nervous system of intestinal tract and theβcells of abdominal salivary gland. Hephaestin and Ceruloplasmin are in the same family,which is a kind of ferrous oxidase of cuprine, it can transform the noxious bivalence iron into atoxigenic Ferric.By far,it has reported the changes of expression of Hephaestin after cerebral ischemia of rat.The objective of this study is to observe diversity the diversity law of Hephaestin in different rat and in different time of ischemia reperfusion,and to approach the role in tissue of acute cerebral ischemia and hypoxia.
Objective
To study the dynamic state changes of Hephaestin in cerebral cortex, corpus striatum and hippocampus after cerebral ischemia reperfusion injury and the relationship between it and reperfusion time. Preliminary discussion on the effectiveness in brain iron metabolism of Hephaestin.
Method
Here to selective 40 clearing Wistar rat, and the embolism reperfusion model was divided randomly into 8 groups, control group(5), sham operation group(5), ischemic reperfusion group(30), and the last groups were divided randomly into 6 subsections,every subsections has 5 rats,that is 4h,8h,16h,32h, 64h,128h after ischemic reperfusion.The ischemic refusion model was established, immunohistochemistry staining, and the Leica Q550IW system was used to analyze the picture,the mean optical density values in cerebral cortex, corpus striatum was worked out in different reperfusion time.
Result
Hephaestin was expressed in cerebral cortex,corpus striatum in normal rat.After 8 hours of cerebral ischemia reperfusion,the expression of Hephaestin was increasing obviously,and it could last out 64 hours after reperfusion.At 16 to 32 hours after reperfusion it get to peak(P<0.05),and at 128 hours it was diminished greatly (P<0.05).
Conclusion
The change of expression of hephaestin in the cortex and striatum during acute cerebral ischemia and reperfusion was greatly.From the study we can see the expression of Hephaestin was most obviously after reperfusion 16hour to 32hour,and after reperfusion 128hour the expression appeared the lowest leval. and the change may play an important role in the pathphysiology after acute cerebral ischemic.It is possible that the expression of Hephaestin is closely related to the degree of injury of brain tissue and the level of iron in the cerebral tissue after cerebral injury.
引文
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[3]谢俊霞,姜宏,王俊.参与脑铁代谢的两种新蛋白[J].神经科学进展(三),北京：高等教育出版社,2004,619-625.
[4]常彦忠,乔亚超.Hephaestin在脑铁代谢中的作用[J].生物物理学报,2009,25：119-120.
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[7]Minnerup J, Heidrich J,Wellmann J, et al. Meta analysis of the efficacy of granulocyte colony stimulating factor in animal models of focal cerebral ischemia[J]. Stroke,2008,39(6):1855-1861.
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[11]龚彪,李长清,黄剑.SD大鼠线栓法局灶性脑缺血/再灌注模型的改进[J].重庆医学,2006,35(4)：313-315.
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[3]Vulpe C D,Kuo Y M,Murphy T L,et al.Hephaestin,a ceruloplasmin homologue implicated in intestinal iron transport,is defective in the sla mouse.Nat Genet.1999,21(2):195-199.
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[5]Chen H J,Attieh Z K,Su T,et al. Hephaestin is a ferroxidase that maintains partial activity in sex linked anemia mice. Blood,2004,103:3933-3939.
[6]Anderson( J,Frazer D M.McKie A T,ct al.The ceruloplasmin homolog hephaestin and the control of intestinal iron absorption. Blood Cells Mol Dis,2002,29(3):367-375.
[7]David M,Hudson,Susan B,Curtis,et al. Human hephaestin expression is not limited to enterocytes of the gastrointestinal tract but is also found in the antrum, the enteric nervous system, and pancreatic β-cells. Am J Physiol Gastrointest Liver Physiol,2010,298:G425-G432.
[8]Kuo YM,Su T,Chen H,et al.Mislocalisation of hephaestin,a muhicopper ferroxidase involved in basolateral intestinal iron transport,in the sex linked anaemia mouse. Gut,2004,53:201～206.
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[13]Gregory J. Anderson, David M.et al. The expression and regulation of the iron transport molecules Hephaestin and Iregl.Cell Biochemistry and Biophysics,2002,36:137-146.
[14]Syed BA, Beaumont NJ, Patel A, Naylor CE, Bayele HK, Joannou CL, Rowe PS, Evans RW, Srai SK. Analysis of the human hephaestin gene and protein:comparative modeling of the N-terminus ecto-domain based upon ceruloplasmin. Protein Eng,2002,15(3):14-205.
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[2]龚彪,李长清,黄剑.SD大鼠线栓法局灶性脑缺血/再灌注模型的改进[J].重庆医学,2006,35(4)：313 315.
[3]谢俊霞,姜宏,王俊.参与脑铁代谢的两种新蛋白[J].神经科学进展(三),北京：高等教育出版社,2004,619-625.
[4]常彦忠,乔亚超.Hephaestin在脑铁代谢中的作用[J].生物物理学报,2009,25：119-120.
[5]David M,Hudson,Susan B,Curtis,et al. Human hephaestin expression is not limited to enterocytes of the gastrointestinal tract but is also found in the antrum, the enteric nervous system, and pancreatic β-cells. Am J Physiol Gastrointest Liver Physiol,2010,298:G425-G432.
[6]Hu Q, Chen C, Yan J, et al. Therapeutic application of genel silencing MMP-9 in a middle cerebral artery occlusion induced focal ischemia rat model[J]. Exp Neurol,2009,216(1):35-46.
[7]Minnerup J, Heidrich J,Wellmann J, et al. Meta analysis of the efficacy of granulocyte colony stimulating factor in animal models of focal cerebral ischemia[J]. Stroke,2008,39(6):1855-1861.
[8]刘运权,戴颖.线栓法大鼠局灶性脑缺血模型的改进[J].中国临床解剖学杂志,2005,23(2)：222-223.
[9]高永强,杨巧莲,王伟.大鼠局造型脑缺血模型制备及取脑方法的研究[J].包头医学院报,2009,25(4)：6-9.
[10]厉建元,张亚卓,姚维成等.两种方法制作大鼠大脑中动脉栓塞再灌注模型比较[J].青岛大学医学院报,2008,44(5)：382-384.
[11]龚彪,李长清,黄剑.SD大鼠线栓法局灶性脑缺血/再灌注模型的改进[J].重庆医学,2006,35(4)：313-315.
[12]曹霞等,鼠须替代尼龙线栓塞大鼠大脑中动脉造成局灶性脑缺血模型研究.中国病理生理杂志2001,17(4)：381-382.
[13]王维治,罗祖明.神经病学,第5版,北京：人民卫生出版社,2005,134-141.
[14]董瑞剑,赵仁亮,沈若武,等.大鼠实验性脑缺血耐受模型的建立与评价[J]. 齐鲁医学杂志,2007,8,22(4)：320-322.
[15]马长升,马文领,戴维国,等.插线法制作大鼠局灶性脑栓塞再灌注模型的研究[J].解剖学杂志,1999,22(3)：209-213.
[16]宋宁,王俊,姜宏,等.亚铁氧化酶Hephaestin的研究进展[J].生理科学进展,2007,38(4)：336-338.
[17]Vulpe C D.Kuo Y M,Murphy T L,et al.Hephaestin,a ceruloplasmin homologue implicated in intestinal iron transport,is defective in the sla mouse. Nat Genet.1999,21(2):195-199.
[18]David M,Hudson,Susan B,Curtis,et al. Human hephaestin expression is not limited to enterocytes of the gastrointestinal tract but is also found in the antrum, the enteric nervous system, and pancreatic β-cells. Am J Physiol Gastrointest Liver Physiol,2010,298:G425-G432.
[19]温仲民,包仕尧,王晓云等.大鼠急性脑缺血后Hephaestine表达变化的研究[J].苏州大学学报(医学版)2004,24(2)：147-150.
[20]Memezawa H,Minamisawa H,Smith ML,et al. Ischemic penumbra in a model of reversible middle cerebral artery occlusion in the rat.Exp Brain Res 1992,89(6):72-78.
[21]吕金荣.微量元素铁与人体健康[J].微量元素与健康研究,2006,23(3)：63-64.
[22]Chen H J,Su T,Attieh Z K,et al.Systemic regulation of Hephaestin and Iregl revealed in studies of genetic and nutritional iron deficiency,Blood,2003, 102(5):1893-1899.
[23]Syed BA,Beaumont NJ,Patel A,et al. Analysis of the human hephaestin gene and protein:comparative modeling of the N-terminus ecto-domain based upon eeruloplasmin. Protein Eng,2002,15:205～214.
[24]Chen H J,Attieh Z K,Su T,et al. Hephaestin is a ferroxidase that maintains partial activity in sex linked anemia mice. Blood,2004,lO3:3933-3939.
[25]Gregory J. Anderson, David M.et al. The expression and regulation of the iron transport molecules Hephaestin and Iregl.Cell Biochemistry and Biophysics,2002,36:137-146.
[26]关鹏,王娜,石欢欢等.DFO对心肌细胞铁代谢的影响.河北师范大学学报,2007,31(3)：392-395.
[27]李艳伟,钱忠明,李琳.急性脑缺血与铁代谢失衡[J].中华神经医学杂志,2007,6(4)：431-432.
[28]Millerot E, Prigent-Tessier AS, Bertrand NM,et al.Serum ferritin in stroke:amarkerofincreasedbodyiron storesor stroke severity [J].J Cereb Blood Flow Metab,2005,25(10):1386-1393.
[29]Anderson( J,Frazer D M.McKie A T,ct al.The ceruloplasmin homolog hephaestin and the control of intestinal iron absorption.Blood Cells Mol Dis,2002,29(3):367-375.
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