本文選題：肌萎縮側(cè)索硬化 + scAAV9�。� 參考：《河北醫(yī)科大學(xué)》2016年博士論文

【摘要】：第一部分Sc AAV9-h IGF1逆軸突靶向轉(zhuǎn)染運(yùn)動(dòng)神經(jīng)元的理論和實(shí)踐目的:研究發(fā)現(xiàn),AAV9可以通過(guò)外周給藥逆軸突靶向轉(zhuǎn)染腦和脊髓的神經(jīng)元,而且自身互補(bǔ)雙鏈AAV(sc AAV)比傳統(tǒng)單鏈AAV轉(zhuǎn)染效率高,蛋白表達(dá)速度快。本部分首先來(lái)驗(yàn)證1×1012vg/ml的AAV9-GFP和s c AAV9-h IGF1病毒載體是否能通過(guò)肌肉注射逆軸突靶向轉(zhuǎn)染SOD1G93A A LS成年小鼠的運(yùn)動(dòng)神經(jīng)元,并實(shí)現(xiàn)蛋白的持續(xù)表達(dá),建立sc AAV9為載體、營(yíng)養(yǎng)因子為基礎(chǔ)的基因治療,為進(jìn)一步治療肌萎縮側(cè)索硬化提供可行的實(shí)驗(yàn)依據(jù)。方法:1隨機(jī)選取60天齡成年SOD1G93A ALS小鼠,給予雙側(cè)腓腸肌一次性注射AAV9-GFP病毒載體,10ul/肌,即1×1010vg/肌。三周后,麻醉處死動(dòng)物,冰凍切片,在熒光顯微鏡下直接觀察肌肉和脊髓的綠色熒光蛋白(GFP)的表達(dá)。2選取60天齡成年SOD1G93AALS小鼠,隨機(jī)分為空白對(duì)照組(control組,n=15)和,AAV9-GFP組給予一次性肌肉注射AAV9-GFP 10ul/肌,即1×1010vg/肌,注射肌肉包括雙側(cè)的咬肌、肱二頭肌、肱三頭肌、肋間肌、腹肌、股二頭肌、股四頭肌、腓腸肌,n=15,每天觀察兩組動(dòng)物發(fā)病時(shí)間和生存期。用蛋白印跡技術(shù)方法檢測(cè)終末期兩組小鼠GFP蛋白的表達(dá)。3隨機(jī)選取60天齡成年SOD1G93A ALS小鼠,給予雙側(cè)腓腸肌一次性注射sc AAV9-h IGF1病毒載體,10ul/肌,即1×1010vg/肌,n=3。三周后,麻醉,多聚甲醛灌注,取小鼠脊髓腰膨大組織,運(yùn)用免疫熒光技術(shù)在熒光顯微鏡下觀察腰髓前角運(yùn)動(dòng)神經(jīng)元sc AAV9-h IGF1逆軸突靶向轉(zhuǎn)染神經(jīng)元及h IGF1蛋白的表達(dá)情況。4隨機(jī)選取60天齡成年SOD1G93A ALS小鼠,給予雙側(cè)腓腸肌一次性注射sc AAV9-h IGF1病毒載體,10ul/肌,即1×1010vg/肌。三周后,麻醉處死動(dòng)物,迅速取脊髓腰膨大組織,用ELISA技術(shù)檢測(cè)h IGF1蛋白在小鼠腓腸肌和對(duì)應(yīng)腰髓的相對(duì)表達(dá)量。結(jié)果:1一次性雙側(cè)腓腸肌注射AAV9-GFP,10μl/肌,三周后,冰凍切片,熒光顯微鏡下直接觀察,可見(jiàn)腓腸肌組織、神經(jīng)軸突和運(yùn)動(dòng)神經(jīng)元都有綠色熒光蛋白(GFP)的表達(dá);2 AAV9-GFP小鼠與空白對(duì)照組小鼠的發(fā)病時(shí)間和生存期無(wú)明顯差異,兩組發(fā)病時(shí)間中位數(shù)分別為96d、97d;生存期中位數(shù)均為120d,說(shuō)明AAV9-GFP干預(yù)不影響小鼠的發(fā)病時(shí)間和生存期,整個(gè)實(shí)驗(yàn)過(guò)程未發(fā)現(xiàn)AAV9對(duì)小鼠有明顯不良反應(yīng)。終末期小鼠麻醉后取腰髓組織,運(yùn)用蛋白印跡方法檢測(cè)GFP蛋白的表達(dá),發(fā)現(xiàn)終末期仍有GFP蛋白的表達(dá),說(shuō)明AAV9能夠使基因在體內(nèi)持續(xù)表達(dá)蛋白;3免疫熒光顯示sc AAV9-h IGF1能逆軸突靶向轉(zhuǎn)染ALS成年小鼠的腰髓運(yùn)動(dòng)神經(jīng)元,并表達(dá)h IGF1蛋白;4 ELISA檢測(cè)h IGF1蛋白在注射的腓腸肌及對(duì)應(yīng)腰段脊髓的表達(dá)量,腰段脊髓h IGF1蛋白表達(dá)量為注射肌肉的35%,轉(zhuǎn)染效率約為26%。第二部分肌肉注射sc AAV9-h IGF1干預(yù)ALS小鼠模型的療效觀察目的:我們用60d齡和90d齡的同窩拷貝數(shù)相同的肌萎縮側(cè)索硬化SOD1G93A轉(zhuǎn)基因小鼠為動(dòng)物模型,隨機(jī)分為sc AAV9-h IGF1干預(yù)組和AAV9-GFP對(duì)照組,給予一次性多部位肌肉注射,給藥劑量為每塊肌肉1×1010vg(10μl),觀察sc AAV9-h IGF1是否對(duì)運(yùn)動(dòng)功能有改善作用,是否對(duì)肌肉組織、腰髓運(yùn)動(dòng)神經(jīng)元有保護(hù)作用,能否減少星形膠質(zhì)細(xì)胞和小膠質(zhì)細(xì)胞的增生,最重要的是否能夠推遲小鼠發(fā)病及延長(zhǎng)生存期。方法:1動(dòng)物分組:60天和90天齡的同窩SOD1G93A轉(zhuǎn)基因ALS小鼠,隨機(jī)一次性肌肉注射sc AAV9-h IGF1和AAV9-GFP,60天和90天齡小鼠雌雄各15對(duì)。2肌肉注射部位包括雙側(cè)的咬肌、肱二頭肌、肱三頭肌、肋間肌、腹肌、股二頭肌、股四頭肌、腓腸肌,sc AAV9-h IGF1和AAV9-GFP的用藥量為10ul/肌。3每周觀察并記錄小鼠的評(píng)分,運(yùn)動(dòng)功能(轉(zhuǎn)輪時(shí)間),體重等變化,每天觀察并記錄小鼠的發(fā)病及終末時(shí)間。小鼠的發(fā)病時(shí)間定為當(dāng)天三次轉(zhuǎn)輪時(shí)間(恒速15轉(zhuǎn)/分)最長(zhǎng)時(shí)間不能達(dá)到180s,記為發(fā)病;小鼠終末時(shí)間為背臥位30s不能自行翻身。ALS小鼠110d齡時(shí)記錄小鼠的步長(zhǎng)和足跡。4兩組ALS小鼠110d齡時(shí),麻醉后迅速取腓腸肌組織,冰凍切片,用HE,NADH,MGT等染色方法觀察ALS小鼠腓腸肌組織的病理變化。5兩組ALS小鼠110d齡時(shí),麻醉后灌注取材,取腰髓組織,震蕩切片用SMI32,GFAP,Iba1免疫組化染色觀察小鼠腰髓運(yùn)動(dòng)神經(jīng)元、星形膠質(zhì)細(xì)胞和小膠質(zhì)細(xì)胞數(shù)量的變化。結(jié)果:1 Sc AAV9-h IGF1干預(yù)組小鼠肌肉萎縮明顯減輕,后肢的外展較有力,步長(zhǎng)較AAV-GFP組小鼠明顯改善;2 Sc AAV9-h IGF1干預(yù)小鼠(60天齡給藥)發(fā)病時(shí)間中位數(shù)雄雌分別延長(zhǎng)了24天(121d vs.97d),18天(118d vs.100d);生存期中位數(shù)雌雄分別延長(zhǎng)了29天(149d vs.120d),24天(147d vs.123d)。Sc AAV9-h IGF1干預(yù)小鼠(90天齡給藥)生存期中位數(shù)雌雄分別延長(zhǎng)了15天(138d vs.123d),14天(137d vs.123d);3無(wú)論是60天齡還是90天齡sc AAV9-h IGF1干預(yù)的ALS小鼠在神經(jīng)功能評(píng)分,體重,運(yùn)動(dòng)功能方面都比AAV9-GFP對(duì)照組小鼠有明顯改善;4肌肉病理提示AAV9-GFP對(duì)照組小鼠的肌纖維可見(jiàn)明顯的萎縮(HE染色),呈神經(jīng)源性損傷(NADH染色),線粒體損傷較重(MGT染色);Sc AAV9-h IGF1治療組小鼠肌肉萎縮改善,也有神經(jīng)源性損傷的表現(xiàn),線粒體損傷較輕;5免疫組織化學(xué)顯示sc AAV9-h IGF1干預(yù)小鼠與AAV9-GFP對(duì)照組小鼠相比,腰髓前角運(yùn)動(dòng)神經(jīng)元(SMI32陽(yáng)性)數(shù)量減少明顯改善(P0.05);星形膠質(zhì)細(xì)胞(GFAP陽(yáng)性)和小膠質(zhì)細(xì)胞(Iba1陽(yáng)性)的免疫組織化學(xué)結(jié)果顯示sc AAV9-h IGF1能明顯減輕轉(zhuǎn)基因小鼠腰髓星形膠質(zhì)細(xì)胞和小膠質(zhì)細(xì)胞增生的程度(P0.05)。第三部分Sc AAV9-h IGF1對(duì)ALS小鼠模型保護(hù)機(jī)制研究目的:IGF1作為神經(jīng)生長(zhǎng)因子中的一員,對(duì)中樞及外周神經(jīng)系統(tǒng)都具有保護(hù)作用,大量實(shí)驗(yàn)表明IGF1能夠促進(jìn)運(yùn)動(dòng)神經(jīng)元存活,并證實(shí)在臨床實(shí)驗(yàn)中有很高的安全性。很多實(shí)驗(yàn),包括我們前部分的實(shí)驗(yàn),都證實(shí)了IGF1能夠延緩SOD1G93A ALS小鼠模型疾病的進(jìn)展,改善運(yùn)動(dòng)功能和延遲肌肉萎縮,減輕膠質(zhì)細(xì)胞反應(yīng),保護(hù)運(yùn)動(dòng)神經(jīng)元,延長(zhǎng)生存期。那么IGF1是通過(guò)什么機(jī)制對(duì)SOD1G93AALS小鼠模型起作用的呢?目前尚沒(méi)有一致的答案。據(jù)報(bào)道,IGF1能夠通過(guò)抑制凋亡保護(hù)運(yùn)動(dòng)神經(jīng)元和減少神經(jīng)膠質(zhì)細(xì)胞釋放腫瘤壞死因子-α(TNF-α)和一氧化氮(NO)。另有實(shí)驗(yàn)表明鞘內(nèi)注射IGF1能激活PI3K/Akt和ERK信號(hào)通路,促進(jìn)運(yùn)動(dòng)神經(jīng)元的存活,我們本部分實(shí)驗(yàn)的目的是進(jìn)一步研究IGF1對(duì)SOD1G93AALS小鼠模型的保護(hù)機(jī)制,為IGF1能夠真正用于患者提供可靠實(shí)驗(yàn)依據(jù)。方法:1同窩拷貝數(shù)相同的80天SOD1G93A雄性小鼠隨機(jī)分為sc AAV9-h IGF1干預(yù)組和AAV9-GFP對(duì)照組,肌肉注射部位包括;雙側(cè)咬肌、肱二頭肌、肱三頭肌、肋間肌、腹肌、股二頭肌、股四頭肌、腓腸肌;10ul/肌,干預(yù)14d后麻醉,新鮮取材,迅速提取腰髓RNA,利用基因芯片篩選有統(tǒng)計(jì)學(xué)差異的基因。2 90天和60天齡同窩拷貝數(shù)相同的SOD1G93A ALS小鼠隨機(jī)分為sc AAV9-h IGF1干預(yù)組和AAV9-GFP對(duì)照組,肌肉注射雙側(cè)的咬肌、肱二頭肌、肱三頭肌、肋間肌、腹肌、股二頭肌、股四頭肌、腓腸肌,10ul/肌。干預(yù)后110天取材,利用實(shí)時(shí)定量PCR驗(yàn)證選出的基因,用蛋白印跡和免疫熒光定量和定位蛋白的表達(dá)。3運(yùn)用免疫熒光及蛋白印跡方法證實(shí)IGF1的抗凋亡機(jī)制,凋亡指標(biāo)包括TUNEL,cleaved-caspase 3和9,Bax及Bcl-2。4利用CRISPR-Cas9技術(shù),30天齡鞘內(nèi)注射干預(yù),靶向敲除中樞神經(jīng)系統(tǒng)運(yùn)動(dòng)神經(jīng)元的IGF1基因,90天齡取材,利用實(shí)時(shí)定量PCR,蛋白印跡及免疫組化、免疫熒光等技術(shù)進(jìn)一步驗(yàn)證選出的基因。結(jié)果:1基因芯片篩選結(jié)果發(fā)現(xiàn)sc AAV9-h IGF1干預(yù)小鼠腰髓的DAO基因水平明顯高于AAV9-GFP對(duì)照組(P0.05);2實(shí)時(shí)定量PCR驗(yàn)證sc AAV9-h IGF1干預(yù)小鼠腰髓的DAO基因水平明顯高于AAV9-GFP對(duì)照組(P0.05)。免疫熒光和蛋白印跡檢查顯示DAO主要定位于運(yùn)動(dòng)神經(jīng)元,sc AAV9-h IGF1治療組小鼠腰髓的DAO蛋白水平明顯高于AAV9-GFP對(duì)照組(P0.05);3 Sc AAV9-h IGF1治療組小鼠腰髓TUNEL,cleaved-caspase 3和9,Bax等促凋亡指標(biāo)下調(diào),抗凋亡Bcl-2上調(diào);4利用CRISPR-Cas9系統(tǒng),靶向敲降中樞神經(jīng)系統(tǒng)的IGF1基因,小鼠的癥狀明顯加重,DAO蛋白水平出現(xiàn)明顯下調(diào),D-serine水平隨之上升,同時(shí)cleaved-caspase 3和9促凋亡指標(biāo)上調(diào)。結(jié)論:1 Sc AAV9能夠攜帶目的基因通過(guò)肌肉注射靶向轉(zhuǎn)染成年SOD1G93AALS小鼠的運(yùn)動(dòng)神經(jīng)元。2 IGF1對(duì)SOD1G93A ALS小鼠的運(yùn)動(dòng)神經(jīng)元和肌纖維有保護(hù)作用,能夠推遲小鼠的發(fā)病時(shí)間和延長(zhǎng)生存期。3 IGF1對(duì)SOD1G93A ALS小鼠的保護(hù)機(jī)制是通過(guò)上調(diào)DAO,降解D-serine,減輕興奮性中毒,抑制凋亡,保護(hù)運(yùn)動(dòng)神經(jīng)元。
[Abstract]:The theoretical and practical purposes of the first part of the Sc AAV9-h IGF1 reverse axonal targeting of the transfected motoneurons: the study found that AAV9 can transfect neurons in the brain and spinal cord through the reverse axon targeting of the peripheral drug, and the complementary double chain AAV (SC AAV) is more efficient and faster than the traditional single strand AAV (SC AAV). The first part of this part is to verify 1 x 1012vg/ Whether the AAV9-GFP and S C AAV9-h IGF1 virus vector of ML can be transfected into the motor neuron of SOD1G93A A LS adult mice by intramuscular injection of the reverse axon target, and realize the continuous expression of the protein, establish the SC AAV9 as the carrier and the gene therapy based on the nutrition factor, and provide the feasible experimental basis for the further treatment of amyotrophic lateral sclerosis. 1 SOD1G93A ALS mice of 60 days of age were randomly selected to give AAV9-GFP viral vector of bilateral gastrocnemius and 10ul/ muscle, that is, 1 x 1010vg/ muscle. After three weeks, the animals were killed and frozen, and the expression of green fluorescent protein (GFP) in muscle and spinal cord was observed directly under the fluorescence microscope, and 60 days of age SOD1G93AALS mice were selected with 60 days of age. The machine was divided into the blank control group (group control, n=15) and the group AAV9-GFP was given a one-time muscle injection of AAV9-GFP 10ul/ muscle, that is, 1 x 1010vg/ muscle. The injection muscles included bilateral masseter, biceps brachii, brachial triceps, intercostal muscle, abdominal muscle, two head of femoral head, four muscles of the femoris, gastrocnemius, n=15, and daily observation of the time and life period of two groups of animals. The trace technique was used to detect the expression of GFP protein of the end stage two groups of mice.3 randomly selected adult SOD1G93A ALS mice at 60 days of age, giving bilateral gastrocnemius injection of SC AAV9-h IGF1 virus, 10ul/ muscle, that is, 1 x 1010vg/ muscle, n=3. three weeks later, anesthesia, and polyoxymethylene perfusion, taking the spinal cord swelling tissue of the mice, using immunofluorescence technique in fluorescosis Under light microscope, the expression of SC AAV9-h IGF1 reverse axon transfected neurons and H IGF1 protein in the anterior lumbar spinal cord motor neurons were observed randomly and.4 randomly selected 60 days old adult SOD1G93A ALS mice, giving the bilateral gastrocnemius injection SC AAV9-h IGF1 viral vector, the 10ul/ muscle, that is, the 1 x muscle. After three weeks, the animals were killed and the ridges were quickly removed. ELISA technique was used to detect the relative expression of H IGF1 protein in the gastrocnemius and the corresponding lumbar pulp in mice. Results: 1 a one-time bilateral gastrocnemius muscle was injected with AAV9-GFP, 10 mu l/ muscle, and three weeks later, frozen section and direct observation under fluorescence microscope showed that the gastrocnemius muscle tissue, the axon process and the motor neuron all have green fluorescent protein (GFP). There was no obvious difference between the onset time and the survival time of 2 AAV9-GFP mice and the blank control group, the median of the two groups was 96d, 97d, and the median of the survival period was 120d, indicating that the AAV9-GFP intervention did not affect the time and the survival time of the mice. The whole experimental course did not find the obvious adverse reaction to the mice. After anaesthesia, the GFP protein expression was detected by Western blot, and the expression of GFP protein was detected by Western blot. It was found that the end stage still had the expression of GFP protein, indicating that AAV9 could express the protein in the body continuously; 3 the immunofluorescence showed that SC AAV9-h IGF1 could transfect the ALS adult mice with the lumbar medullary motor neuron, and expressed the H IGF1 protein; 4 ELISA. The expression of H IGF1 protein in the gastrocnemius and the lumbar spinal cord was detected. The expression of H IGF1 protein in the lumbar spinal cord was 35% of the injected muscle, and the transfection efficiency was about 26%. second part of the muscle injection of SC AAV9-h IGF1 to interfere with the ALS mouse model: We used the same amyotrophic lateral cord with the same copy number of the same fossa in 60d age and 90d age. The sclerosing SOD1G93A transgenic mice were randomly divided into the SC AAV9-h IGF1 intervention group and the AAV9-GFP control group. The mice were injected with one time multi site muscle and the dosage was 1 x 1010vg (10 L) per muscle. It was observed whether SC AAV9-h IGF1 could improve the exercise function, and it had protective effect on the muscle tissue and the lumbar medullary motor neurons. Whether or not the proliferation of astrocytes and microglia can be reduced, the most important thing is to postpone the onset of mice and prolong the survival time. Methods: 1 animals were divided into groups: 60 days and 90 days old SOD1G93A transgenic ALS mice, random injection of SC AAV9-h IGF1 and AAV9-GFP at random, and 15 pairs of.2 muscle injection parts of male and female male and male mice in 90 days of age. The masseter muscle, biceps brachii, biceps brachii, brachial triceps, intercostal muscle, abdominal muscle, two biceps femoris, four biceps femoris, gastrocnemius, SC AAV9-h IGF1 and AAV9-GFP were used to observe and record the score of 10ul/ muscle.3 weekly, exercise function (wheel time), weight change, observe and record the onset and end time of mice every day. Mice hair The time of the disease was determined as the three wheel time of the same day (constant speed, 15 turn / score), the longest time could not reach 180s, which was recorded as the disease. The end time of the mouse was the back position, 30s could not turn over the.ALS mice and recorded the step length of the mice and the footprints of the.4 two groups of ALS mice 110D age. After anesthesia, the gastrocnemius muscle tissue, frozen section, HE, NADH, MGT, etc. Methods the pathological changes of the gastrocnemius tissue of ALS mice were observed in.5 two group ALS mice at 110D age. After anesthesia, the lumber tissue was taken after anesthesia and the spinal cord tissue was taken. The concussion section was stained with SMI32, GFAP and Iba1 immunohistochemical staining to observe the changes in the number of mouse lumbar medullary motor neurons, astrocytes and microglia. Results: the muscle atrophy of mice in the 1 Sc AAV9-h IGF1 intervention group. The contraction was obviously reduced, the abduction of the hind limbs was stronger, and the step length was obviously better than that of the AAV-GFP group, and the median of the onset time of the 2 Sc AAV9-h IGF1 intervention mice was 24 days (121d vs.97d), 18 days (118d vs.100d), and the median male and male division of the survival period was 29 days (149d vs.120d), and 24 days (147d vs.123d). The median survival period of the intervention mice (90 days of age) was prolonged for 15 days (138D vs.123d) and 14 days (137d vs.123d); 3 ALS mice with SC AAV9-h IGF1 intervention at 60 days of age or 90 days of age were significantly improved in the neurological score, weight, and exercise function than in the AAV9-GFP pairs of mice; 4 muscle pathology hints AAV9-GFP control group. The muscle fibers of the mice were obviously atrophied (HE staining), showed neurogenic injury (NADH staining), and the mitochondrial damage was heavier (MGT staining); the muscle atrophy improved in the Sc AAV9-h IGF1 treatment group and the expression of neurogenic injury, and the mitochondrial damage was lighter. 5 immune group chemistry showed that SC AAV9-h IGF1 intervened mice and AAV9-GFP control mice. In comparison, the number of SMI32 positive neurons in the anterior lumbar spinal cord decreased significantly (P0.05); the immunohistochemical results of astrocytes (GFAP positive) and microglia (Iba1 positive) showed that SC AAV9-h IGF1 could significantly reduce the degree of proliferation of astrocytes and microglia in the lumbar spinal cord of transgenic mice (P0.05). Third part Sc AAV. 9-h IGF1 protection mechanism of ALS mouse model: IGF1 as a member of the nerve growth factor, the protection of the central and peripheral nervous system, a large number of experiments show that IGF1 can promote the survival of motor neurons, and prove that there is a high safety in practical clinical experiments. Many experiments, including our previous experiments, have proved that IGF1 can delay the progression of SOD1G93A ALS mouse model disease, improve exercise function and delay muscle atrophy, reduce glial reaction, protect motor neurons and prolong life. Then what mechanism does IGF1 work on SOD1G93AALS mouse model? There is no consistent answer. It is reported that IGF1 can be suppressed by inhibition. Apoptosis protects motoneurons and reduces the release of tumor necrosis factor - alpha (TNF- alpha) and nitric oxide (NO) in glial cells. Other experiments have shown that intrathecal IGF1 can activate PI3K/Akt and ERK signaling pathways to promote the survival of motor neurons. The purpose of this part of the experiment is to further study the protection of IGF1 on SOD1G93AALS mice models. The protective mechanism provides reliable experimental basis for IGF1 for patients. Methods: 1 the 80 day SOD1G93A male mice with the same copy number of the same litter were randomly divided into the SC AAV9-h IGF1 intervention group and the AAV9-GFP control group. The intramuscular injection sites included bilateral masseter, biceps brachii, brachial triceps, intercostal, abdominal, two head, femoral four head, gastrocnemius muscle, and 10u. L/ muscle, after the intervention of 14d, after anesthesia, fresh extraction and rapid extraction of the lumbar spinal cord RNA, the SOD1G93A ALS mice with the same copy number at 90 days and 60 days of age were randomly divided into the SC AAV9-h IGF1 intervention group and the AAV9-GFP control group with the same copy number of the same nest at 60 days of age, and the two side masseter muscles, the biceps, the biceps, the intercostal muscles and the abdominal muscles were intramuscularly injected. The two biceps femoris, the four head of the femoris, the gastrocnemius, and the 10ul/ muscle were harvested for 110 days, and the selected genes were tested by real-time quantitative PCR. The anti apoptotic mechanism of IGF1 was confirmed by immunofluorescence and Western blotting using Western blot and immunofluorescence quantitative and localizing protein expression.3, and the index of withering includes TUNEL, cleaved-caspase 3 and 9, Bax and Bcl-2.4. CRISPR-Cas9 technique and intrathecal injection of 30 days old were used to target the IGF1 gene of motor neurons in the central nervous system, 90 days of age, and the selected genes were further verified by real-time quantitative PCR, Western blot, immunohistochemistry and immunofluorescence. Results: 1 gene chip screening results showed that SC AAV9-h IGF1 interfered the lumbar spinal cord of mice. The level of DAO gene was significantly higher than that of the AAV9-GFP control group (P0.05); 2 real-time quantitative PCR verification that SC AAV9-h IGF1 intervention in the lumbar marrow of mice was significantly higher than that of the AAV9-GFP control group (P0.05). The immunofluorescence and Western blotting showed that DAO was mainly located in the motor neurons, and the level of the lumbar marrow of the mice was significantly higher. In the AAV9-GFP control group (P0.05), the waist marrow TUNEL, cleaved-caspase 3 and 9 in the 3 Sc AAV9-h IGF1 treatment group were down regulated and the anti apoptotic Bcl-2 up regulation. 4 using CRISPR-Cas9 system, the IGF1 gene of the central nervous system was targeted, the symptoms of mice were significantly increased, the level of DAO protein decreased obviously and the level increased accordingly. At the same time, cleaved-caspase 3 and 9 increase the apoptosis index. Conclusion: 1 Sc AAV9 can carry the target gene by intramuscular injection targeting the motoneuron.2 IGF1 transfected with adult SOD1G93AALS mice to protect the motoneurons and muscle fibers of SOD1G93A ALS mice. It can postpone the onset time of the mice and prolong the.3 IGF1 to SOD of the survival period. The protective mechanism of 1G93A ALS mice is through up regulating DAO, degrading D-serine, reducing excitotoxicity, inhibiting apoptosis, and protecting motor neurons.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R-332;R744.8

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