ScAAV9-hIGF1逆轴突靶向运动神经元干预ALS小鼠模型作用及机制研究

发布时间：2018-05-16 23:10

本文选题：肌萎缩侧索硬化 + scAAV9　；参考：《河北医科大学》2016年博士论文

【摘要】：第一部分Sc AAV9-h IGF1逆轴突靶向转染运动神经元的理论和实践目的:研究发现,AAV9可以通过外周给药逆轴突靶向转染脑和脊髓的神经元,而且自身互补双链AAV(sc AAV)比传统单链AAV转染效率高,蛋白表达速度快。本部分首先来验证1×1012vg/ml的AAV9-GFP和s c AAV9-h IGF1病毒载体是否能通过肌肉注射逆轴突靶向转染SOD1G93A A LS成年小鼠的运动神经元,并实现蛋白的持续表达,建立sc AAV9为载体、营养因子为基础的基因治疗,为进一步治疗肌萎缩侧索硬化提供可行的实验依据。方法:1随机选取60天龄成年SOD1G93A ALS小鼠,给予双侧腓肠肌一次性注射AAV9-GFP病毒载体,10ul/肌,即1×1010vg/肌。三周后,麻醉处死动物,冰冻切片,在荧光显微镜下直接观察肌肉和脊髓的绿色荧光蛋白(GFP)的表达。2选取60天龄成年SOD1G93AALS小鼠,随机分为空白对照组(control组,n=15)和,AAV9-GFP组给予一次性肌肉注射AAV9-GFP 10ul/肌,即1×1010vg/肌,注射肌肉包括双侧的咬肌、肱二头肌、肱三头肌、肋间肌、腹肌、股二头肌、股四头肌、腓肠肌,n=15,每天观察两组动物发病时间和生存期。用蛋白印迹技术方法检测终末期两组小鼠GFP蛋白的表达。3随机选取60天龄成年SOD1G93A ALS小鼠,给予双侧腓肠肌一次性注射sc AAV9-h IGF1病毒载体,10ul/肌,即1×1010vg/肌,n=3。三周后,麻醉,多聚甲醛灌注,取小鼠脊髓腰膨大组织,运用免疫荧光技术在荧光显微镜下观察腰髓前角运动神经元sc AAV9-h IGF1逆轴突靶向转染神经元及h IGF1蛋白的表达情况。4随机选取60天龄成年SOD1G93A ALS小鼠,给予双侧腓肠肌一次性注射sc AAV9-h IGF1病毒载体,10ul/肌,即1×1010vg/肌。三周后,麻醉处死动物,迅速取脊髓腰膨大组织,用ELISA技术检测h IGF1蛋白在小鼠腓肠肌和对应腰髓的相对表达量。结果:1一次性双侧腓肠肌注射AAV9-GFP,10μl/肌,三周后,冰冻切片,荧光显微镜下直接观察,可见腓肠肌组织、神经轴突和运动神经元都有绿色荧光蛋白(GFP)的表达;2 AAV9-GFP小鼠与空白对照组小鼠的发病时间和生存期无明显差异,两组发病时间中位数分别为96d、97d;生存期中位数均为120d,说明AAV9-GFP干预不影响小鼠的发病时间和生存期,整个实验过程未发现AAV9对小鼠有明显不良反应。终末期小鼠麻醉后取腰髓组织,运用蛋白印迹方法检测GFP蛋白的表达,发现终末期仍有GFP蛋白的表达,说明AAV9能够使基因在体内持续表达蛋白;3免疫荧光显示sc AAV9-h IGF1能逆轴突靶向转染ALS成年小鼠的腰髓运动神经元,并表达h IGF1蛋白;4 ELISA检测h IGF1蛋白在注射的腓肠肌及对应腰段脊髓的表达量,腰段脊髓h IGF1蛋白表达量为注射肌肉的35%,转染效率约为26%。第二部分肌肉注射sc AAV9-h IGF1干预ALS小鼠模型的疗效观察目的:我们用60d龄和90d龄的同窝拷贝数相同的肌萎缩侧索硬化SOD1G93A转基因小鼠为动物模型,随机分为sc AAV9-h IGF1干预组和AAV9-GFP对照组,给予一次性多部位肌肉注射,给药剂量为每块肌肉1×1010vg(10μl),观察sc AAV9-h IGF1是否对运动功能有改善作用,是否对肌肉组织、腰髓运动神经元有保护作用,能否减少星形胶质细胞和小胶质细胞的增生,最重要的是否能够推迟小鼠发病及延长生存期。方法:1动物分组:60天和90天龄的同窝SOD1G93A转基因ALS小鼠,随机一次性肌肉注射sc AAV9-h IGF1和AAV9-GFP,60天和90天龄小鼠雌雄各15对。2肌肉注射部位包括双侧的咬肌、肱二头肌、肱三头肌、肋间肌、腹肌、股二头肌、股四头肌、腓肠肌,sc AAV9-h IGF1和AAV9-GFP的用药量为10ul/肌。3每周观察并记录小鼠的评分,运动功能(转轮时间),体重等变化,每天观察并记录小鼠的发病及终末时间。小鼠的发病时间定为当天三次转轮时间(恒速15转/分)最长时间不能达到180s,记为发病;小鼠终末时间为背卧位30s不能自行翻身。ALS小鼠110d龄时记录小鼠的步长和足迹。4两组ALS小鼠110d龄时,麻醉后迅速取腓肠肌组织,冰冻切片,用HE,NADH,MGT等染色方法观察ALS小鼠腓肠肌组织的病理变化。5两组ALS小鼠110d龄时,麻醉后灌注取材,取腰髓组织,震荡切片用SMI32,GFAP,Iba1免疫组化染色观察小鼠腰髓运动神经元、星形胶质细胞和小胶质细胞数量的变化。结果:1 Sc AAV9-h IGF1干预组小鼠肌肉萎缩明显减轻,后肢的外展较有力,步长较AAV-GFP组小鼠明显改善;2 Sc AAV9-h IGF1干预小鼠(60天龄给药)发病时间中位数雄雌分别延长了24天(121d vs.97d),18天(118d vs.100d);生存期中位数雌雄分别延长了29天(149d vs.120d),24天(147d vs.123d)。Sc AAV9-h IGF1干预小鼠(90天龄给药)生存期中位数雌雄分别延长了15天(138d vs.123d),14天(137d vs.123d);3无论是60天龄还是90天龄sc AAV9-h IGF1干预的ALS小鼠在神经功能评分,体重,运动功能方面都比AAV9-GFP对照组小鼠有明显改善;4肌肉病理提示AAV9-GFP对照组小鼠的肌纤维可见明显的萎缩(HE染色),呈神经源性损伤(NADH染色),线粒体损伤较重(MGT染色);Sc AAV9-h IGF1治疗组小鼠肌肉萎缩改善,也有神经源性损伤的表现,线粒体损伤较轻;5免疫组织化学显示sc AAV9-h IGF1干预小鼠与AAV9-GFP对照组小鼠相比,腰髓前角运动神经元(SMI32阳性)数量减少明显改善(P0.05);星形胶质细胞(GFAP阳性)和小胶质细胞(Iba1阳性)的免疫组织化学结果显示sc AAV9-h IGF1能明显减轻转基因小鼠腰髓星形胶质细胞和小胶质细胞增生的程度(P0.05)。第三部分Sc AAV9-h IGF1对ALS小鼠模型保护机制研究目的:IGF1作为神经生长因子中的一员,对中枢及外周神经系统都具有保护作用,大量实验表明IGF1能够促进运动神经元存活,并证实在临床实验中有很高的安全性。很多实验,包括我们前部分的实验,都证实了IGF1能够延缓SOD1G93A ALS小鼠模型疾病的进展,改善运动功能和延迟肌肉萎缩,减轻胶质细胞反应,保护运动神经元,延长生存期。那么IGF1是通过什么机制对SOD1G93AALS小鼠模型起作用的呢?目前尚没有一致的答案。据报道,IGF1能够通过抑制凋亡保护运动神经元和减少神经胶质细胞释放肿瘤坏死因子-α(TNF-α)和一氧化氮(NO)。另有实验表明鞘内注射IGF1能激活PI3K/Akt和ERK信号通路,促进运动神经元的存活,我们本部分实验的目的是进一步研究IGF1对SOD1G93AALS小鼠模型的保护机制,为IGF1能够真正用于患者提供可靠实验依据。方法:1同窝拷贝数相同的80天SOD1G93A雄性小鼠随机分为sc AAV9-h IGF1干预组和AAV9-GFP对照组,肌肉注射部位包括;双侧咬肌、肱二头肌、肱三头肌、肋间肌、腹肌、股二头肌、股四头肌、腓肠肌;10ul/肌,干预14d后麻醉,新鲜取材,迅速提取腰髓RNA,利用基因芯片筛选有统计学差异的基因。2 90天和60天龄同窝拷贝数相同的SOD1G93A ALS小鼠随机分为sc AAV9-h IGF1干预组和AAV9-GFP对照组,肌肉注射双侧的咬肌、肱二头肌、肱三头肌、肋间肌、腹肌、股二头肌、股四头肌、腓肠肌,10ul/肌。干预后110天取材,利用实时定量PCR验证选出的基因,用蛋白印迹和免疫荧光定量和定位蛋白的表达。3运用免疫荧光及蛋白印迹方法证实IGF1的抗凋亡机制,凋亡指标包括TUNEL,cleaved-caspase 3和9,Bax及Bcl-2。4利用CRISPR-Cas9技术,30天龄鞘内注射干预,靶向敲除中枢神经系统运动神经元的IGF1基因,90天龄取材,利用实时定量PCR,蛋白印迹及免疫组化、免疫荧光等技术进一步验证选出的基因。结果:1基因芯片筛选结果发现sc AAV9-h IGF1干预小鼠腰髓的DAO基因水平明显高于AAV9-GFP对照组(P0.05);2实时定量PCR验证sc AAV9-h IGF1干预小鼠腰髓的DAO基因水平明显高于AAV9-GFP对照组(P0.05)。免疫荧光和蛋白印迹检查显示DAO主要定位于运动神经元,sc AAV9-h IGF1治疗组小鼠腰髓的DAO蛋白水平明显高于AAV9-GFP对照组(P0.05);3 Sc AAV9-h IGF1治疗组小鼠腰髓TUNEL,cleaved-caspase 3和9,Bax等促凋亡指标下调,抗凋亡Bcl-2上调;4利用CRISPR-Cas9系统,靶向敲降中枢神经系统的IGF1基因,小鼠的症状明显加重,DAO蛋白水平出现明显下调,D-serine水平随之上升,同时cleaved-caspase 3和9促凋亡指标上调。结论:1 Sc AAV9能够携带目的基因通过肌肉注射靶向转染成年SOD1G93AALS小鼠的运动神经元。2 IGF1对SOD1G93A ALS小鼠的运动神经元和肌纤维有保护作用,能够推迟小鼠的发病时间和延长生存期。3 IGF1对SOD1G93A ALS小鼠的保护机制是通过上调DAO,降解D-serine,减轻兴奋性中毒,抑制凋亡,保护运动神经元。
[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.
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R-332;R744.8

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