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  本文關(guān)鍵詞：AAV9-IGF1對SOD1G93A小鼠運動神經(jīng)元閾電位的影響　出處：《河北醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 肌萎縮側(cè)索硬化 SOD1G93A轉(zhuǎn)基因鼠 動作電位 AAV9-IGF1 興奮性

【摘要】：目前已識別了超氧化物歧化酶1(superoxide dismutase 1,SOD1)基因100多種突變,占家族型ALS患者的~20%。人SOD1鼠模型的臨床表型及病理基礎(chǔ)與ALS散發(fā)性及家族性患者最為相近。其中,有三種SOD1G85R、SOD1G37R及SOD1G93A已被廣泛的應(yīng)用于ALS的轉(zhuǎn)基因鼠模型。SOD1是表達(dá)153個氨基酸多肽的同型二聚體酶,每個亞單元結(jié)合一個銅及一個鋅離子。銅負(fù)責(zé)酶的催化活性,鋅負(fù)責(zé)穩(wěn)定蛋白的結(jié)構(gòu)。SOD1的主要功能包括自由基的清除,通過催化超氧化物陰離子轉(zhuǎn)化成分子氧和過氧化氫,通過谷胱甘肽過氧化物酶及過氧化氫酶分解成水。SOD1分布廣泛,主要分布于細(xì)胞質(zhì)中,線粒體及其他亞細(xì)胞器中也能被檢測到。腺相關(guān)病毒(adeno-associated virus,AAV)是一種小的,無包膜病毒,已經(jīng)成為基因治療領(lǐng)域研究的主題。它介導(dǎo)的基因轉(zhuǎn)導(dǎo)是安全有效的,也是作為臨床研究的一種治療工具。大量的動物模型顯著的證據(jù)表明了重組腺相關(guān)病毒(recombinant adeno-associated virus,r AAVs)的有效性及安全性。其中,將攜帶編碼人胰島素生長因子1基因重組腺相關(guān)病毒(insulin like growth factor 1 delivery by recombinant adeno-associated virus,r AAV-IGF1)注射到SOD1G93A模型小鼠的呼吸及后肢肌肉中,這延長了生存期及延緩了運動的削減。AAV帶菌者具有逆向傳遞的特性,如脊髓神經(jīng)元是選擇性的靶組織,允許局部分泌的人胰島素生長因子1(insulin like growth factor 1,IGF1)對周圍細(xì)胞有廣泛的作用,并不僅僅局限于傳遞病毒的運動神經(jīng)元細(xì)胞。中樞神經(jīng)系統(tǒng)中,AAV研究最多的血清型是1,2,5,8,9,及重組。血清型的效果取決于腦分布、物種及靶細(xì)胞類型。這些血清型有效地轉(zhuǎn)導(dǎo)給神經(jīng)元,星形膠質(zhì)細(xì)胞、少突膠質(zhì)細(xì)胞及小膠質(zhì)細(xì)胞轉(zhuǎn)導(dǎo)受限,使用特定細(xì)胞激活子能夠得到改善。研究證實,腺相關(guān)病毒血清型9(adeno-associated virus 9,AAV9)能通過血腦屏障且有效靶向腦及脊髓中的神經(jīng)元、星形膠質(zhì)細(xì)胞。小鼠中,AAV9介導(dǎo)的中樞神經(jīng)系統(tǒng)通過小腦延髓池途徑的靶向基因傳遞,與其它AAV血清型相比,AAV9是在中樞神經(jīng)系統(tǒng)中傳遞更好的載體,能更好的透過血腦屏障。為了探究肌萎縮側(cè)索硬化(amyotrophic lateral sclerosis,ALS)癥狀前期運動神經(jīng)元電生理的改變及IGF1對電生理的影響,本實驗采用側(cè)腦室及腦實質(zhì)兩種方法注射AAV9-IGF1及AAV9-GFP(green fluorescent protein,GFP)于SOD1G93A新生鼠中,觀察運動皮層M2區(qū)的表達(dá)情況,選用IGF1表達(dá)量多的方法注射AAV9-IGF/AAV-GFP,并記錄20-40天SOD1G93A小鼠運動皮層2區(qū)(motor cortex 2 area,M2)的動作電位,分析AAV9-IGF1對SOD1G93A小鼠癥狀前期運動神經(jīng)元動作電位的影響。目的:1記錄SOD1G93A轉(zhuǎn)基因及非轉(zhuǎn)基因小鼠的動作電位,并比較動作電位的閾電位、頻率、動作電位幅度、潛伏期及上升速率是否存在差別;2通過側(cè)腦室及腦實質(zhì)注射AAV9-IGF1/AAV-GFP于SOD1G93A新生鼠(出生24h內(nèi))中,分別觀察其在運動皮層M2區(qū)的表達(dá)效果;3分別記錄腦實質(zhì)注射AAV9-IGF1/AAV-GFP的20-40天SOD1G93A轉(zhuǎn)基因小鼠M2區(qū)的動作電位,并比較閾電位、頻率、動作電位幅度、潛伏期及上升速率是否存在差別。方法:本實驗應(yīng)用C57/6小鼠及SOD1G93A轉(zhuǎn)基因及非轉(zhuǎn)基因同窩對照小鼠,SOD1G93A轉(zhuǎn)基因小鼠的鑒定通過PCR擴增的方法。采用腦組織及側(cè)腦室兩種方法注射AAV9-IGF1/AAV-GFP,選用20-40天C57/6、SOD1G93A轉(zhuǎn)基因和非轉(zhuǎn)基因小鼠的腦片,用免疫組化及免疫熒光方法觀察AAV9-IGF1及AAV-GFP在運動皮層M2區(qū)的表達(dá),選用表達(dá)量多的方法注射AAV9-IGF1/AAV-GFP于SOD1G93A轉(zhuǎn)基因新生鼠中,采用全細(xì)胞膜片鉗技術(shù)記錄20-40天小鼠腦片M2的動作電位,并統(tǒng)計分析。結(jié)果:1應(yīng)用全細(xì)胞膜片鉗技術(shù),記錄20-40天SOD1G93A轉(zhuǎn)基因(h SOD1G93A)組及非轉(zhuǎn)基因(m SOD1WT)組小鼠腦片運動皮層M2區(qū)動作電位:兩組閾電位有區(qū)別,但差異無統(tǒng)計學(xué)意義;兩組頻率中,SOD1G93A轉(zhuǎn)基因組高于同窩對照組,150p A電流刺激時差異有統(tǒng)計學(xué)意義(150p A,P=0.040.05),200-500p A電流刺激時,差異無統(tǒng)計學(xué)意義;動作電位幅度,兩組之間差異無統(tǒng)計學(xué)意義;兩組潛伏期比較,差異無統(tǒng)計學(xué)意義;兩組上升速率,SOD1G93A轉(zhuǎn)基因組高于同窩對照組(450p A除外),250p A電流刺激時差異有統(tǒng)計學(xué)意義(250p A,P=0.0320.05)。2通過側(cè)腦室及腦實質(zhì)兩種方法注射AAV9-IGF1/AAV-GFP,應(yīng)用免疫組化及免疫熒光方法觀察20-40天小鼠腦片M2區(qū)IGF1的表達(dá)情況。在小鼠腦片運動皮層M2區(qū)中,與側(cè)腦室注射相比,腦實質(zhì)注射的方法IGF1表達(dá)量更高,且差異有統(tǒng)計學(xué)意義。3應(yīng)用全細(xì)胞膜片鉗技術(shù),記錄腦實質(zhì)注射AAV9-IGF1/AAV-GFP20-40天SOD1G93A轉(zhuǎn)基因組小鼠腦片M2區(qū)的動作電位:與AAV9-GFP組相比,AAV9-IGF1組閾電位明顯減小,且差異有統(tǒng)計學(xué)意義;兩組頻率,差異無統(tǒng)計學(xué)意義;與AAV9-GFP組相比,AAV9-IGF1組動作電位幅度降低,但差異無統(tǒng)計學(xué)意義;與AAV9-GFP組相比,AAV9-IGF1組動作電位的潛伏期縮短,且在150p A、500p A電流刺激時,差異有統(tǒng)計學(xué)意義(P0.05);兩組上升速率比較,差異無統(tǒng)計學(xué)意義。結(jié)論:1與同窩對照組相比,SOD1G93A轉(zhuǎn)基因鼠癥狀前期運動神經(jīng)元有興奮性增高的趨勢。2與側(cè)腦室注射相比,腦實質(zhì)注射在20-40天小鼠M2區(qū)的IGF1表達(dá)量更高。3 IGF1降低了SOD1G93A轉(zhuǎn)基因鼠癥狀前期運動神經(jīng)元的閾電位,發(fā)揮了神經(jīng)保護(hù)作用。
[Abstract]:Has identified the superoxide dismutase 1 (superoxide dismutase 1, SOD1) 100 kinds of gene mutations, ~20%. patients accounted for SOD1 rat model of familial ALS clinical phenotypes and pathologic basis and ALS in sporadic and familial patients were mostly similar. Among them, there are three kinds of SOD1G85R, SOD1G37R and.SOD1 transgenic mouse model SOD1G93A has been widely used in ALS is the same two dimeric enzyme expression polypeptide of 153 amino acids, each sub unit with a copper and a zinc ion. The catalytic activity of the enzyme responsible for the removal of copper, the main function structure of.SOD1 is responsible for the zinc stable proteins including free radicals, superoxide anion catalyzed by into molecular oxygen and hydrogen peroxide, decomposed into water.SOD1 widely distributed through glutathione peroxidase and catalase, mainly distributed in the cytoplasm, mitochondria and other subcellular organelles can also be detected by adenovirus. Close the virus (adeno-associated virus, AAV) is a small, non enveloped virus, has become the field of gene therapy. Gene transduction mediated by theme it is safe and effective, but also as a therapeutic tool for clinical research. A large number of animal models significant evidences to prove that the recombinant adeno-associated virus (recombinant adeno-associated virus, R, AAVs) efficacy and safety. Among them, the encoding human insulin-like growth factor 1 gene recombinant adeno-associated virus (insulin like growth factor 1 delivery by recombinant adeno-associated virus, R AAV-IGF1) was injected into the SOD1G93A mouse model of respiratory and hindlimb muscles, the prolonged survival and slow motion cut the.AAV carrier has the characteristics of reverse transfer, such as spinal cord neurons are the target tissue selective, allowing local secretion of human insulin-like growth factor 1 (insulin like Growth factor 1, IGF1) has a wide effect on the surrounding cells, motor neurons is not limited to transfer the virus. The central nervous system, AAV of serotype 1,2,5,8,9 is the most, and depends on the effect of serum type. The recombinant brain distribution, species and target cell types. These serotypes efficiently transduced for neurons, astrocytes, oligodendrocytes and microglial cell transduction is limited, the use of specific cell activator can be improved. The research confirmed that adeno-associated virus serotype 9 (adeno-associated virus 9, AAV9) can pass through the blood-brain barrier and effective targeting to the brain and spinal cord neurons, astrocytes in mice. In the central nervous system AAV9 mediated gene transfer to the cisterna magna pathway target, compared with other AAV serotypes, AAV9 is the better carrier in the central nervous system, through the blood better Brain barrier. In order to explore the amyotrophic lateral sclerosis (amyotrophic lateral sclerosis, ALS) changes of electrophysiological pre motor neuron symptoms and IGF1 on electrophysiology, the lateral ventricle and brain parenchyma in two methods of injection of AAV9-IGF1 and AAV9-GFP (green fluorescent protein, GFP SOD1G93A) in neonatal rats, observe the expression of the motor cortex area M2, the expression of IGF1 AAV9-IGF/AAV-GFP injection quantity, and record the 20-40 days SOD1G93A mice cortex area 2 (motor cortex 2 area, M2) of the action potential, analysis on the effect of AAV9-IGF1 on early symptoms of SOD1G93A mice motor neuron action potentials. Objective: the records of 1 SOD1G93A transgenic and non transgenic mouse action potential, threshold potential, and to compare the frequency of action potential, action potential amplitude, latency and the rising rate of whether there exists difference; 2 by intracerebroventricular injection of AA and brain parenchyma V9-IGF1/AAV-GFP SOD1G93A in neonatal rats (born in 24h), respectively, to observe the expression effect in motor cortex area M2; 3 of the action potential were recorded in brain parenchyma injection of AAV9-IGF1/AAV-GFP 20-40 day SOD1G93A M2 transgenic mice, and compare the threshold potential, action potential amplitude, frequency, latency and the rising rate of whether there is a difference. The experimental application of C57/6 mice and SOD1G93A transgenic and non transgenic littermate control mice, method of identification of SOD1G93A transgenic mice by PCR amplification. The brain tissue and the lateral ventricle injection of AAV9-IGF1/AAV-GFP with two methods, 20-40 day C57/6, SOD1G93A transgenic and non transgenic mice, to observe the expression of AAV9-IGF1 and AAV-GFP in motor cortex M2 the use of immunohistochemistry and immunofluorescence method, the expression method of quantity of injection of AAV9-IGF1/AAV-GFP in transgenic SOD1G93A in neonatal rats, the The action potential of the whole cell patch clamp techniques 20-40 days M2 mouse brain slices, and statistical analysis. Results: 1 using whole cell patch clamp technique to record 20-40 days (H SOD1G93A) SOD1G93A transgenic and non transgenic group (m SOD1WT) mice brain slices of motor cortex area M2 action potential threshold potential: the two groups are different, but no statistically significant difference between the two groups; frequency in SOD1G93A group was higher than that of transgenic littermate control group, was statistically significant 150P A current stimulation (150P A, P=0.040.05) between 200-500p, A current stimulation, the difference was not statistically significant; the amplitude of action potential, no significant difference between the two groups were compared between the two groups; latency difference the two groups had no statistical significance; the rising rate of SOD1G93A group was higher than that of transgenic littermate control group (450p A), 250p was statistically significant difference (250p A stimulated A, P= 0.0320.05).2 by lateral ventricle and brain parenchyma in two ways Injection of AAV9-IGF1/AAV-GFP, immunohistochemistry and immunofluorescence method to observe the expression of 20-40 in mouse brain slices of M2 days IGF1. In the movement of mouse brain slices M2 cortex, compared with intracerebroventricular injection method, cerebral parenchyma injection the expression of IGF1 is higher, and the difference was statistically significant.3 using the whole cell patch clamp technique recording, action potential brain parenchyma injection AAV9-IGF1/AAV-GFP20-40 day SOD1G93A transgenic mice brain slices M2 region: compared with AAV9-GFP group, AAV9-IGF1 group significantly reduced the threshold potential, and the difference was statistically significant; two groups of frequencies, no significant difference; compared with group AAV9-GFP, group AAV9-IGF1 decreased the action potential amplitude, but without statistical significance. Differences; compared with AAV9-GFP group, AAV9-IGF1 group, action potential latency, and at 150P A, 500p A current stimulation, the difference was statistically significant (P0.05); group two increased rate comparison, difference There is no statistical significance. Conclusion: 1 littermates were compared with the control group, SOD1G93A transgenic mice compared with early symptoms of motor neuron excitability increased.2 and intracerebroventricular injection, cerebral parenchyma injection at 20-40 days of mouse M2 region of IGF1 is more highly expressed.3 IGF1 lowered the threshold potential of SOD1G93A transgenic mice early symptoms of motor neurons and play a neuroprotective effect.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R744.8

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