發(fā)布時(shí)間：2018-06-23 16:50

  本文選題：腦源性神經(jīng)營(yíng)養(yǎng)因子融合蛋白(BDNF-PTD) + 大腦中動(dòng)脈栓塞模型��； 參考：《南方醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 研究背景 血腦屏障主要由血管內(nèi)皮細(xì)胞和腦組織的星狀膠質(zhì)細(xì)胞的足突構(gòu)成,具有內(nèi)皮細(xì)胞之間緊密連接并且缺乏內(nèi)吞囊的特點(diǎn),可限制血液和腦組織之間物質(zhì)的自由交換,因此大多數(shù)藥物很難通過血腦屏障進(jìn)入大腦發(fā)揮治療疾病作用。腦源性神經(jīng)營(yíng)養(yǎng)因子(Brain-derived neurotrophic factor, BDNF)為生物體內(nèi)天然的蛋白分子,在神經(jīng)系統(tǒng)的生長(zhǎng)發(fā)育中,對(duì)神經(jīng)元的生存,分化起重要作用；具有增強(qiáng)學(xué)習(xí)記憶等功能,但同樣難以通過血腦屏障進(jìn)入中樞神經(jīng)系統(tǒng)治療神經(jīng)退行性疾病。目前臨床上缺少一種有效的,非侵入性的方法使得BDNF通過血腦屏障而發(fā)揮生物學(xué)效應(yīng),因而限制了BDNF在臨床上的應(yīng)用。所以,以何種給藥系統(tǒng)使BDNF進(jìn)入大腦是發(fā)揮BDNF臨床治療作用應(yīng)用的關(guān)鍵。目前在實(shí)驗(yàn)室階段研究的腦靶向給藥系統(tǒng)主要包括： ①腦血管內(nèi)灌注或腦內(nèi)直接注射給藥。 ②病毒載體介導(dǎo)基因后的腦內(nèi)直接注射給藥。 ③脂質(zhì)體包裹藥物后系統(tǒng)途徑給藥。 ④腦靶向單克隆抗體偶聯(lián)藥物后系統(tǒng)途徑給藥。 但這些方法有很多的不足： ①腦血管內(nèi)灌注或腦內(nèi)注射給藥有極大的不順應(yīng)性,且給藥后,藥物分布在很小的腦部位。 ②病毒基因介導(dǎo)給藥雖然是很好的入腦載體,但并不適合臨床上的急性缺血的腦損傷的治療,因?yàn)槟X損傷后在很短的時(shí)間內(nèi)給藥才能對(duì)神經(jīng)元細(xì)胞有保護(hù)和修復(fù)作用。而且病毒載體對(duì)人的免疫系統(tǒng),及細(xì)胞染色體組有副作用。 ③用脂質(zhì)體包裹藥物能增加藥物的脂溶性,但腦對(duì)脂質(zhì)體的攝入是絕對(duì)的和非特異性的,增加了藥物因全身分布而帶來的副作用和成本。 ④單克隆抗體偶聯(lián)藥物進(jìn)行靜脈給藥能夠使得BDNF通過血腦屏障到達(dá)大腦而發(fā)揮生物學(xué)效應(yīng),但這種方法得到的藥物,制備過程繁瑣,成本高；腦靶向用單克隆抗體需先進(jìn)行人源化改造。 如何尋找種有效的,非侵入性的給藥系統(tǒng)方法,使得BDNF進(jìn)入大腦發(fā)揮生物學(xué)效應(yīng)? 來源于人免疫缺陷病毒的TAT蛋白能夠跨膜導(dǎo)入細(xì)胞內(nèi)部的發(fā)現(xiàn),給我們解決這個(gè)問題提供了很好的思路。蛋白轉(zhuǎn)導(dǎo)域(PTD)是指那些小于20個(gè)氨基酸、帶正電荷,可以穿過大多數(shù)細(xì)胞膜的穿膜肽的一個(gè)富含堿性氨基酸區(qū)域,帶正電荷的多肽片段與蛋白轉(zhuǎn)導(dǎo)功能相關(guān)聯(lián)�？梢詫⒋蠓肿舆\(yùn)輸入幾乎所有的哺乳動(dòng)物細(xì)胞內(nèi),無(wú)需特殊的環(huán)境。其對(duì)給合物的大小沒有嚴(yán)格的限制,已經(jīng)在腫瘤,免疫治療等臨床前研究中取得了很大的進(jìn)展。 本實(shí)驗(yàn)室前期通過基因工程方法制備了一種能通過血腦屏障的腦源性神經(jīng)營(yíng)養(yǎng)因子-PTD融合蛋白(BDNF-PTD),實(shí)驗(yàn)證明其能通過血腦屏障。在此基礎(chǔ)上,本文研究了BDNF-PTD在動(dòng)物腦缺血模型中保護(hù)神經(jīng)元功能的藥效學(xué)及其作用機(jī)制。 目的 研究本實(shí)驗(yàn)中心制備的BDNF-PTD在動(dòng)物腦缺血模型中保護(hù)神經(jīng)元功能的初步藥效學(xué)功能；通過神經(jīng)功能缺陷評(píng)分對(duì)非給藥組和給藥組進(jìn)行神經(jīng)功能缺陷評(píng)價(jià)；研究本實(shí)驗(yàn)中心制備的BDNF-PTD在動(dòng)物腦缺血模型中保護(hù)神經(jīng)元功能的作用機(jī)制。 方法 一、建立SD大鼠腦局部缺血模型 大鼠用10%水合氯醛(3ml／100g)腹腔注射麻醉,仰臥固定在手術(shù)臺(tái)上,在頸部正中切開,剪開前筋膜,鈍性分離胸鎖乳突肌和胸骨舌骨肌之間的間隙,暴露左側(cè)頸總動(dòng)脈(CCA)和迷走神經(jīng),并分離。往遠(yuǎn)心段找出頸外動(dòng)脈(ECA)和頸內(nèi)動(dòng)脈(ICA),干凈分離ECA、ICA。結(jié)扎ECA近分叉端和在距分叉處10mm結(jié)扎CCA,并在CCA近分叉處套線待用,用動(dòng)脈夾夾住ICA。在距分叉處5mm的CCA上用眼科剪45°剪一小斜口,插入栓線,通過分叉處進(jìn)入ICA,提起在CCA近分叉處準(zhǔn)備好的線,打好結(jié)但不能太緊,栓線能通過為合適,松開ICA處的動(dòng)脈夾,繼續(xù)使栓線順I(yè)CA入顱方向推進(jìn),直到推進(jìn)至分叉處17mm為止。將線栓和CCA固定,剪掉多余的栓線,縫合筋膜和皮膚。 根據(jù)Bederson評(píng)分法進(jìn)行神經(jīng)功能缺陷程度的評(píng)分。其原則如下：0分：無(wú)神經(jīng)功能缺損癥狀,行為正常；1分：前肢屈曲；2分：中度神經(jīng)功能缺損,抵抗對(duì)側(cè)推力下降伴前肢屈曲,無(wú)轉(zhuǎn)圈行為；3分：重度神經(jīng)功能缺損,抵抗對(duì)側(cè)推力下降伴前肢屈曲,有轉(zhuǎn)圈行為。1、2、3級(jí)為模型成功的標(biāo)準(zhǔn)。 二、腦源性神經(jīng)營(yíng)養(yǎng)因子融合蛋白(BDNF-PTD)對(duì)大鼠局灶性腦缺血治療作用的初步藥效學(xué)研究 通過線栓法制作大鼠大腦中動(dòng)脈栓塞(MACO)模型。永久性栓塞1個(gè)小時(shí)后腹腔給每只動(dòng)物模型注射50ug (50ug/ml)的BDNF-PTD,24小時(shí)后斷頭取腦,大腦切片TTC染色,比較栓塞1小時(shí)后給藥模型組與未給藥模型組的腦梗死面積,觀察BDNF-PTD保護(hù)大腦缺血神經(jīng)元死亡的情況。 三、腦源性神經(jīng)營(yíng)養(yǎng)因子融合蛋白(BDNF-PTD)對(duì)大鼠局灶性腦缺血治療作用的機(jī)制研究 BDNF能提高神經(jīng)元抵抗缺血的能力,具有促進(jìn)受損神經(jīng)元的修復(fù),再生,調(diào)節(jié)神經(jīng)結(jié)構(gòu)的重建,促進(jìn)腦損傷后認(rèn)知功能恢復(fù)等作用。其作用的機(jī)制可能與兩個(gè)主要的信號(hào)途徑有關(guān)：磷脂酰肌醇-3-激酶(PI3K)途徑和細(xì)胞分裂素(絲裂原)活化蛋白激酶(MAPK/ERK)途徑。它們通過激活轉(zhuǎn)錄蛋白(如cAMP效應(yīng)元件結(jié)合蛋白)而影響基因的表達(dá)來促進(jìn)神經(jīng)元的生長(zhǎng)和存活。而促分裂原活化蛋白激酶和磷脂酰肌醇-3-激酶途徑都與BDNF-TrkB受體信號(hào)調(diào)節(jié)有關(guān)。我們研制的BDNF-PTD具有逆轉(zhuǎn)神經(jīng)元死亡的功能,是否也是通過這些通路來發(fā)揮作用,這將在本課題研究中進(jìn)行驗(yàn)證。 通過線栓法制作大鼠大腦中動(dòng)脈栓塞(MCAO)模型。永久性栓塞1個(gè)小時(shí)后腹腔給每只動(dòng)物模型注射50ug (50ug/ml)的BDNF-PTD,24小時(shí)后斷頭取腦蛋白,通過免疫印跡法(WB),驗(yàn)證BDNF-PTD是否可激活ERK1／2細(xì)胞外信號(hào)調(diào)節(jié)激酶和磷脂酰肌醇3激酶(PI3K)。 結(jié)果 一、成功建立SD大鼠腦局部缺血模型 大鼠大腦缺血模型制作后,栓塞24小時(shí)后,發(fā)現(xiàn)給藥組和未給藥模型組、陰性對(duì)照組,在神經(jīng)功能活動(dòng)上有不同的表現(xiàn)。根據(jù)Bederson評(píng)分法對(duì)每組模型進(jìn)行神經(jīng)功能缺陷程度的評(píng)分,給藥組大部分處于0到1分。而未給藥模型組及陰性對(duì)照組有抵抗對(duì)側(cè)推力下降伴前肢屈曲,和轉(zhuǎn)圈追尾運(yùn)動(dòng),大部分處于2到3分。 二、腦源性神經(jīng)營(yíng)養(yǎng)因子融合蛋白(BDNF-PTD)對(duì)大鼠局灶性腦缺血神經(jīng)保護(hù)治療作用的初步研究 大鼠大腦中動(dòng)脈栓塞模型在栓塞1小時(shí)給藥,隨后24內(nèi)觀察大鼠偏癱癥狀,與未給藥模型組相比,給藥組大鼠偏癱癥狀得到明顯的改善。永久性栓塞24小時(shí)后,大腦冠狀切片TTC染色。梗死面積用Mean±SD表示,BDNF-PTD給藥組與未給藥模型組、陰性對(duì)照組,比較結(jié)果有顯著性差別(F=26.791,P=0.0000.01)。發(fā)現(xiàn)1小時(shí)給藥模型組大鼠腦壞死區(qū)域面積與未給藥模型組相比明顯減少75%～80%。 三、腦源性神經(jīng)營(yíng)養(yǎng)因子融合蛋白(BDNF-PTD)對(duì)大鼠局灶性腦缺血治療作用的機(jī)制研究 研究腦源性神經(jīng)營(yíng)養(yǎng)因子融合蛋白(BDNF-PTD)對(duì)大鼠局灶性腦缺血治療作用的機(jī)制。在本課題中,實(shí)驗(yàn)結(jié)果證明,BDNF-PTD激活ERK1／2,使其磷酸化,給藥模型組中pERK1/2蛋白表達(dá)水平較正常組均有顯著性升高(P0.01)；與未給藥模型組中pERK1/2蛋白表達(dá)水平比較也均有顯著性升高(P0.05)。 另外,實(shí)驗(yàn)結(jié)果也證明,BDNF-PTD能上調(diào)PI3K,給藥模型組中PI3K蛋白表達(dá)水平較正常組均有顯著性升高(P=0.0000.01)；與未給藥模型組中PI3K蛋白表達(dá)水平比較也均有顯著性升高(P=0.0000.01)；而未給藥模型組與正常組比較差異沒有統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論 一、在大鼠腦缺血模型中,腦源性神經(jīng)營(yíng)養(yǎng)因子融合蛋白(BDNF-PTD)能通過血腦屏障并且能發(fā)揮生物學(xué)效應(yīng)。跟未給藥模型組對(duì)比,顯示BDNF-PTD能很好的裸護(hù)腦缺血后的神經(jīng)元。 二、在大鼠腦缺血模型中,證實(shí)了腦源性神經(jīng)營(yíng)養(yǎng)因子融合蛋白(BDNF-PTD)通過激活ERK細(xì)胞外信號(hào)調(diào)節(jié)激酶的磷酸化和上調(diào)磷脂酰肌醇3激酶(P3K),從而促進(jìn)神經(jīng)元細(xì)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 鹵 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 .
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R341

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