BDNF-PTD融合蛋白逆转大脑缺血导致神经死亡的药效学和机制研究

发布时间：2018-06-23 16:50

本文选题：脑源性神经营养因子融合蛋白(BDNF-PTD) + 大脑中动脉栓塞模型　；参考：《南方医科大学》2010年硕士论文

【摘要】： 研究背景血脑屏障主要由血管内皮细胞和脑组织的星状胶质细胞的足突构成,具有内皮细胞之间紧密连接并且缺乏内吞囊的特点,可限制血液和脑组织之间物质的自由交换,因此大多数药物很难通过血脑屏障进入大脑发挥治疗疾病作用。脑源性神经营养因子(Brain-derived neurotrophic factor, BDNF)为生物体内天然的蛋白分子,在神经系统的生长发育中,对神经元的生存,分化起重要作用；具有增强学习记忆等功能,但同样难以通过血脑屏障进入中枢神经系统治疗神经退行性疾病。目前临床上缺少一种有效的,非侵入性的方法使得BDNF通过血脑屏障而发挥生物学效应,因而限制了BDNF在临床上的应用。所以,以何种给药系统使BDNF进入大脑是发挥BDNF临床治疗作用应用的关键。目前在实验室阶段研究的脑靶向给药系统主要包括： ①脑血管内灌注或脑内直接注射给药。 ②病毒载体介导基因后的脑内直接注射给药。 ③脂质体包裹药物后系统途径给药。 ④脑靶向单克隆抗体偶联药物后系统途径给药。但这些方法有很多的不足： ①脑血管内灌注或脑内注射给药有极大的不顺应性,且给药后,药物分布在很小的脑部位。 ②病毒基因介导给药虽然是很好的入脑载体,但并不适合临床上的急性缺血的脑损伤的治疗,因为脑损伤后在很短的时间内给药才能对神经元细胞有保护和修复作用。而且病毒载体对人的免疫系统,及细胞染色体组有副作用。 ③用脂质体包裹药物能增加药物的脂溶性,但脑对脂质体的摄入是绝对的和非特异性的,增加了药物因全身分布而带来的副作用和成本。 ④单克隆抗体偶联药物进行静脉给药能够使得BDNF通过血脑屏障到达大脑而发挥生物学效应,但这种方法得到的药物,制备过程繁琐,成本高；脑靶向用单克隆抗体需先进行人源化改造。如何寻找种有效的,非侵入性的给药系统方法,使得BDNF进入大脑发挥生物学效应? 来源于人免疫缺陷病毒的TAT蛋白能够跨膜导入细胞内部的发现,给我们解决这个问题提供了很好的思路。蛋白转导域(PTD)是指那些小于20个氨基酸、带正电荷,可以穿过大多数细胞膜的穿膜肽的一个富含碱性氨基酸区域,带正电荷的多肽片段与蛋白转导功能相关联。可以将大分子运输入几乎所有的哺乳动物细胞内,无需特殊的环境。其对给合物的大小没有严格的限制,已经在肿瘤,免疫治疗等临床前研究中取得了很大的进展。本实验室前期通过基因工程方法制备了一种能通过血脑屏障的脑源性神经营养因子-PTD融合蛋白(BDNF-PTD),实验证明其能通过血脑屏障。在此基础上,本文研究了BDNF-PTD在动物脑缺血模型中保护神经元功能的药效学及其作用机制。目的研究本实验中心制备的BDNF-PTD在动物脑缺血模型中保护神经元功能的初步药效学功能；通过神经功能缺陷评分对非给药组和给药组进行神经功能缺陷评价；研究本实验中心制备的BDNF-PTD在动物脑缺血模型中保护神经元功能的作用机制。方法一、建立SD大鼠脑局部缺血模型大鼠用10%水合氯醛(3ml／100g)腹腔注射麻醉,仰卧固定在手术台上,在颈部正中切开,剪开前筋膜,钝性分离胸锁乳突肌和胸骨舌骨肌之间的间隙,暴露左侧颈总动脉(CCA)和迷走神经,并分离。往远心段找出颈外动脉(ECA)和颈内动脉(ICA),干净分离ECA、ICA。结扎ECA近分叉端和在距分叉处10mm结扎CCA,并在CCA近分叉处套线待用,用动脉夹夹住ICA。在距分叉处5mm的CCA上用眼科剪45°剪一小斜口,插入栓线,通过分叉处进入ICA,提起在CCA近分叉处准备好的线,打好结但不能太紧,栓线能通过为合适,松开ICA处的动脉夹,继续使栓线顺ICA入颅方向推进,直到推进至分叉处17mm为止。将线栓和CCA固定,剪掉多余的栓线,缝合筋膜和皮肤。根据Bederson评分法进行神经功能缺陷程度的评分。其原则如下：0分：无神经功能缺损症状,行为正常；1分：前肢屈曲；2分：中度神经功能缺损,抵抗对侧推力下降伴前肢屈曲,无转圈行为；3分：重度神经功能缺损,抵抗对侧推力下降伴前肢屈曲,有转圈行为。1、2、3级为模型成功的标准。二、脑源性神经营养因子融合蛋白(BDNF-PTD)对大鼠局灶性脑缺血治疗作用的初步药效学研究通过线栓法制作大鼠大脑中动脉栓塞(MACO)模型。永久性栓塞1个小时后腹腔给每只动物模型注射50ug (50ug/ml)的BDNF-PTD,24小时后断头取脑,大脑切片TTC染色,比较栓塞1小时后给药模型组与未给药模型组的脑梗死面积,观察BDNF-PTD保护大脑缺血神经元死亡的情况。三、脑源性神经营养因子融合蛋白(BDNF-PTD)对大鼠局灶性脑缺血治疗作用的机制研究 BDNF能提高神经元抵抗缺血的能力,具有促进受损神经元的修复,再生,调节神经结构的重建,促进脑损伤后认知功能恢复等作用。其作用的机制可能与两个主要的信号途径有关：磷脂酰肌醇-3-激酶(PI3K)途径和细胞分裂素(丝裂原)活化蛋白激酶(MAPK/ERK)途径。它们通过激活转录蛋白(如cAMP效应元件结合蛋白)而影响基因的表达来促进神经元的生长和存活。而促分裂原活化蛋白激酶和磷脂酰肌醇-3-激酶途径都与BDNF-TrkB受体信号调节有关。我们研制的BDNF-PTD具有逆转神经元死亡的功能,是否也是通过这些通路来发挥作用,这将在本课题研究中进行验证。通过线栓法制作大鼠大脑中动脉栓塞(MCAO)模型。永久性栓塞1个小时后腹腔给每只动物模型注射50ug (50ug/ml)的BDNF-PTD,24小时后断头取脑蛋白,通过免疫印迹法(WB),验证BDNF-PTD是否可激活ERK1／2细胞外信号调节激酶和磷脂酰肌醇3激酶(PI3K)。结果一、成功建立SD大鼠脑局部缺血模型大鼠大脑缺血模型制作后,栓塞24小时后,发现给药组和未给药模型组、阴性对照组,在神经功能活动上有不同的表现。根据Bederson评分法对每组模型进行神经功能缺陷程度的评分,给药组大部分处于0到1分。而未给药模型组及阴性对照组有抵抗对侧推力下降伴前肢屈曲,和转圈追尾运动,大部分处于2到3分。二、脑源性神经营养因子融合蛋白(BDNF-PTD)对大鼠局灶性脑缺血神经保护治疗作用的初步研究大鼠大脑中动脉栓塞模型在栓塞1小时给药,随后24内观察大鼠偏瘫症状,与未给药模型组相比,给药组大鼠偏瘫症状得到明显的改善。永久性栓塞24小时后,大脑冠状切片TTC染色。梗死面积用Mean±SD表示,BDNF-PTD给药组与未给药模型组、阴性对照组,比较结果有显著性差别(F=26.791,P=0.0000.01)。发现1小时给药模型组大鼠脑坏死区域面积与未给药模型组相比明显减少75%～80%。三、脑源性神经营养因子融合蛋白(BDNF-PTD)对大鼠局灶性脑缺血治疗作用的机制研究研究脑源性神经营养因子融合蛋白(BDNF-PTD)对大鼠局灶性脑缺血治疗作用的机制。在本课题中,实验结果证明,BDNF-PTD激活ERK1／2,使其磷酸化,给药模型组中pERK1/2蛋白表达水平较正常组均有显著性升高(P0.01)；与未给药模型组中pERK1/2蛋白表达水平比较也均有显著性升高(P0.05)。另外,实验结果也证明,BDNF-PTD能上调PI3K,给药模型组中PI3K蛋白表达水平较正常组均有显著性升高(P=0.0000.01)；与未给药模型组中PI3K蛋白表达水平比较也均有显著性升高(P=0.0000.01)；而未给药模型组与正常组比较差异没有统计学意义(P0.05)。结论一、在大鼠脑缺血模型中,脑源性神经营养因子融合蛋白(BDNF-PTD)能通过血脑屏障并且能发挥生物学效应。跟未给药模型组对比,显示BDNF-PTD能很好的裸护脑缺血后的神经元。二、在大鼠脑缺血模型中,证实了脑源性神经营养因子融合蛋白(BDNF-PTD)通过激活ERK细胞外信号调节激酶的磷酸化和上调磷脂酰肌醇3激酶(P3K),从而促进神经元细胞的存活。
[Abstract]:Background of the study

Brain - derived neurotrophic factor ( BDNF ) is a natural protein molecule in the organism . Brain - derived neurotrophic factor ( BDNF ) plays an important role in the survival and differentiation of neurons in the development of nervous system .
It is difficult to enter the central nervous system through the blood - brain barrier to treat neurodegenerative diseases , but it is difficult to enter the central nervous system through the blood - brain barrier to treat neurodegenerative diseases . At present , there is a lack of effective and non - invasive method to make BDNF exert biological effect through the blood brain barrier , thus limiting BDNF ' s application in clinic .

( 1 ) Intracerebral perfusion or direct intracerebroventricular injection .

( 2 ) Direct injection administration in the brain after the viral vector - mediated gene .

( 3 ) Administration of liposome - encapsulated drug .

( 4 ) the brain targeting monoclonal antibody is coupled with the drug and then is administered by a systemic route .

However , there are many deficiencies in these methods :

( 1 ) There is a great irregularity in cerebral vascular infusion or intracerebroventricular injection , and after administration , the drug is distributed in very small brain regions .

( 2 ) the viral gene - mediated administration is a good entry - brain carrier , but is not suitable for the treatment of brain injury of acute ischemia in clinic , because the drug can be administered in a very short time after brain injury to protect and repair the neuron cells , and the virus vector has side effects on the human immune system and the cell chromosome group .

( 3 ) The liposome - encapsulated drug can increase the fat solubility of the drug , but the uptake of the brain to the liposome is absolute and nonspecific , and the side effect and cost caused by the whole body distribution of the drug are increased .

4 ) the monoclonal antibody - coupled drug is administered intravenously to enable BDNF to reach the brain through the blood brain barrier to exert biological effect , but the medicine prepared by the method has the advantages of complicated preparation process and high cost ;
Monoclonal antibody for brain targeting needs advanced pedestrian source transformation .

How to find effective and non - invasive methods of administration to BDNF to the brain to exert a biological effect ?

TAT protein derived from human immunodeficiency virus ( TAT ) can be introduced into cells from human immunodeficiency virus . It provides a good idea to solve this problem . The protein transduction domain is a rich basic amino acid region with positive charge and can pass through most cell membranes . It can transport macromolecule into almost all mammalian cells without special environment . It has no strict limitation on the size of the donor , and has made great progress in preclinical studies such as tumor and immunotherapy .

In the early stage of the lab , a brain - derived neurotrophic factor - fusion protein ( BDNF - ptd ) , which can pass through the blood - brain barrier , was prepared by genetic engineering . It was proved that it could pass through the blood - brain barrier . On the basis of this , the pharmacodynamics and mechanism of BDNF - protein in the protection of neuron function in animal cerebral ischemia model were studied .

Purpose

In order to study the effect of BDNF - pD prepared by the experimental center on the function of neuron in animal model of cerebral ischemia ;
performing neurological deficit evaluation on the non - administration group and the administration group through a neurological deficit score ;
To study the mechanism of the protective neuron function in the model of animal cerebral ischemia , the BDNF - derived neurotrophic factor ( BDNF ) prepared by the experimental center was studied .

method

1 . Establishment of a model of ischemic brain ischemia in SD rats

The external carotid artery ( ECA ) and the internal carotid artery ( ICA ) were cut off at the proximal bifurcation of CCA .

According to the Bederson ' s scoring method , the neurological deficit degree was scored . The following principles were as follows : 0 : No symptoms of neurological deficit and normal behavior ;
1 point : forelimb flexion ;
2 points : moderate neurological deficit , resistance to side thrust descending with forelimb flexion , no turning behavior ;
3 points : severe neurological deficit , resistance to side thrust decline with forelimb flexion , with swivel behavior . 1 , 2 , 3 are the criteria for the success of the model .

Study on the Effect of Brain - derived Neurotrophic Factor Fusion Protein ( BDNF ) on Focal Cerebral Ischemia in Rats

The rat brain middle cerebral artery embolism ( MACO ) model was made by wire embolism method . After permanent embolization for 1 hour , the brain was injected 50ug ( 50ug / ml ) in each animal model . After 24 hours , the brain was decapitated and the brain slices TTC stained . The cerebral infarction area of the model group and the untreated model group were compared after embolization for 1 hour .

Study on the mechanism of brain - derived neurotrophic factor fusion protein ( BDNF ) on focal cerebral ischemia in rats

BDNF - 3 - kinase pathway and mitogen - activated protein kinase ( MAPK / ERK ) pathway are involved in promoting the growth and survival of neurons by activating transcription proteins , such as cAMP effector binding proteins .

Rat models of middle cerebral artery occlusion were made by means of thread embolism . After 1 hour of permanent embolization , 50 ug ( 50 ug / ml ) of BDNF - DNA was injected intraperitoneally to each animal model . After 24 hours , the brain protein was decapitated and the brain protein was decapitated 24 hours later .

Results

First , we successfully established the brain ischemia model of SD rats .

After 24 hours of embolization in rats with cerebral ischemia model , it was found that the administration group and the untreated model group and the negative control group had different manifestations on the neurological activity . According to the Bederson scoring method , the scores of neurological deficits were scored , and the majority of the groups were 0 to 1 . The untreated model group and the negative control group had resistance to the reduction of the side thrust with the flexion of the forelimbs and the tail tracking movement , most of which were 2 to 3 points .

Preliminary study on the effect of brain - derived neurotrophic factor fusion protein ( BDNF ) on the protection and treatment of focal cerebral ischemia in rats

The cerebral middle cerebral artery occlusion model was administered in 1 hour after embolization , and the symptoms of hemiparalysis in rats were observed in 24 hours . After 24 hours of permanent embolism , the cerebral coronal slices were stained . The infarct size was expressed by Mean 卤 SD , and the results were significantly different from those in the untreated model group and the negative control group ( F = 26.791 , P = 0.0000 . 01 ) . The area area of brain necrosis was significantly reduced by 75 % ~ 80 % in 1 hour administration model group compared with the untreated model group .

Study on the mechanism of brain - derived neurotrophic factor fusion protein ( BDNF ) on focal cerebral ischemia in rats

Brain - derived neurotrophic factor fusion protein ( BDNF ) was used in the study of focal cerebral ischemia in rats . In this study , it was shown that the expression level of pERK 1 / 2 in the model group was significantly higher than that in the normal group ( P0.01 ) .
There was also a significant increase in the level of p / 2 protein expression in the untreated model group ( P0.05 ) .

In addition , the results of the experiment showed that the expression level of PI3 - 3 in the model group was significantly higher than that in the normal group ( P = 0.0000 . 01 ) .
Compared with the untreated model group , there was a significant increase ( P = 0.0000 . 01 ) .
There was no significant difference between the untreated model group and the normal group ( P0.05 ) .

Conclusion

1 . In the rat model of cerebral ischemia , the brain - derived neurotrophic factor fusion protein ( BDNF ) can exert a biological effect through the blood - brain barrier . Compared with the untreated model group , the brain - derived neurotrophic factor fusion protein ( BDNF - P ) can well protect the neurons after cerebral ischemia after cerebral ischemia .

Second , in the rat model of cerebral ischemia , it was confirmed that the brain - derived neurotrophic factor fusion protein ( BDNF - protein ) can regulate the phosphorylation of the kinase and up - regulate the phosphoinositide 3 kinase ( P3K ) by activating ERK extracellular signal , thereby promoting the survival of neuronal cells .
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R341

【参考文献】