CN108853501A - 磁感蛋白在改善神经退行性疾病中的应用 - Google Patents
磁感蛋白在改善神经退行性疾病中的应用 Download PDFInfo
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Abstract
本发明涉及磁遗传学领域，尤其涉及磁感蛋白在改善神经退行性疾病中的应用。本发明通过实验证明，将磁感应蛋白导入PD细胞结果表明，在磁场作用下，PD细胞存活率得到提高，PD细胞中α‑synuclein、GSK‑3β水平被降低。将磁感应蛋白导入AD大鼠模型，在磁场作用下，AD模型大鼠海马CA1区Bcl‑2表达升高、Bax蛋白表达降低，大鼠的学习、记忆能力得到提高。由此证明磁感应蛋白能够具有改善神经退行性疾病的作用。本发明还提供了改善神经退行性疾病的药物，该药物包括磁感应蛋白或表达磁感应蛋白的基因工程载体。
Description
技术领域
本发明涉及磁遗传学领域，尤其涉及磁感蛋白在改善神经退行性疾病中的应用。
背景技术
神经退行性疾病(Neurodegenerative disease，ND)是一类慢性、进行性神经疾病。不同类型神经退行性疾病的病变部位和病因虽然各不相同，但大脑特定区域的迟发性神经细胞退行性病变、细胞丢失是他们的共同特征，因此统称为神经退行性疾病。
神经退行性疾病主要包括：老年性痴呆(Alzheimer’s disease，AD，阿尔茨海默病)；帕金森病(Parkinson＇s disease，PD)，亨廷顿病(Huntington’s disease,HD)，脊髓小脑共济失调(spinal cerbellar ataxias)，齿状核红核苍白球丘脑下核萎缩(denatatorubropallidoluydian atrophy)，肌萎缩侧索硬化症(Amyotrophic LateralSclerosis,ALS)，脊髓肌萎缩症(Spinal Musclar Atrophy)等。
目前，对神经退行性疾病病因及发病机制的研究一直在继续，而随着对神经退行性疾病研究的不断深入，发现越来越多的因素与神经退行性疾病发生和发展有关。1、氧化应激：研究表明，由于抗氧化防御系统功能缺陷所致的自由基产生增加，在PD发病中起主要作用。例如谷胱甘肽(GSH)是体内的一种重要的自由基清除剂，在PD病人黑质部位，GSH大量减少，且减少量与PD的严重程度呈正相关；2、线粒体功能障碍：AD患者脑内存在mtDNA缺陷和氧化磷酸化异常。另外，AD患者神经元线粒体功能发生障碍后，将导致神经元能量供给不足同时释放大量ROS，诱发氧化应激损伤，钙调节失衡，最终触发神经元凋亡；3、兴奋性毒素：在AD患者脑组织中谷氨酸能系统正常结构发生改变，谷氨酸转运体及谷氨酸重摄取的功能下降。另外，β淀粉样前体蛋白和Tau蛋白可以抑制细胞外谷氨酸的摄入，这种抑制作用将导致细胞外谷氨酸水平增高，从而产生兴奋性毒性作用。在PD患者和PD实验动物模型中发现，由背侧丘脑下核投射到黑质纹状体的谷氨酸能神经元大量增加，这些研究证实了多巴胺(DA)能神经元上谷氨酸受体的过度激活是诱发兴奋性毒性细胞死亡的原因之一。同时，谷氨酸摄取障碍亦加重了谷氨酸受体的过度激活；4、免疫炎症：PD患者脑中，伴随着脑黑质致密部DA能神经元选择性丧失的是大量表达组织相容性白细胞抗原DR的小胶质细胞，这被认为是小胶质细胞激活的重要标志。5、Ca2+失衡:AD患者脑中Na+/Ca2+交换较正常人明显提高，患者血清中Ca2+浓度降低，可以使甲状旁腺激素分泌增加，通过蛋白途径激活腺苷酸环化酶使细胞内环磷酸腺苷浓度升高，从而促进钙内流，诱导了脑细胞内Ca2+的增高，进而通过破坏线粒体功能等途径降低细胞能量代谢而发生痴呆。6、细胞凋亡：研究发现，AD患者神经元减少，认知功能降低，是由于细胞凋亡导致的，AD患者脑中细胞凋亡速率是正常对照组的50多倍。
由于神经退行性疾病的发病机制非常复杂，寻找对神经退行性疾病的治疗方法仍是目前亟待解决的难题，而随着对这类疾病研究的不断深入，针对不同发病机制的治疗方法和药物大量的涌现。主要药物种类包括：脑细胞活化剂、神经保护剂(例如：钙离子拮抗剂、抗氧化药物、NMDA受体拮抗剂、抗炎药物、抗凋亡药物等)、雌激素替代物、神经干细胞移植、神经营养因子、具有营养作用的小分子药物或中医药。
但是，目前，对神经退行性疾病的治疗主要仍是延缓病情的发展，无法逆转其病情，且疗效十分的有限。且鉴于神经退行性疾病致病因素的多样性，阻断一个或两个途径不能明显减少神经元的全面的功能障碍和损失。因此，对于神经退行性疾病的治疗仍有待开发更加有效的治疗方法。
发明内容
有鉴于此，本发明要解决的技术问题在于提供磁感蛋白在改善神经退行性疾病中的应用，本发明通过促进磁感蛋白与铁离子的结合，提高ND模型细胞的存活率、抑制ND相关蛋白标志物的表达，改善ND症状。
本发明提供了磁感蛋白在制备降低帕金森病相关蛋白水平的药物中的应用。
本发明实施例中，所述帕金森病相关蛋白为α-突触核蛋白和/或糖原合成酶激酶-3β。
一些实施例中，所述磁感蛋白选自铁硫蛋白、铁蛋白、胶原蛋白、热休克蛋白、角蛋白、神经营养因子、钙调蛋白、GP120蛋白、血红蛋白或胰岛素。
一些具体实施例中，所述磁感蛋白为磁感应蛋白Isca。
本发明以多巴胺能神经细胞MES23.5细胞为研究对象，用1-甲基-4-苯基吡啶离子(1-methyl-4-phenyl-pyridinium，MPP+)诱导建立PD的体外细胞损伤模型。以rAAV病毒为载体，将编码磁感应蛋白Isca的基因转染上述模型细胞，然后将实验组细胞培养于磁场环境中培养24h，经检测发现，实验组细胞中的α-synuclein、GSK-3β的磷酸化蛋白蛋白表达量均低于模型组，且实验组的细胞存活率高于模型组。说明磁感蛋白的导入以及磁场的作用能够起到神经保护的作用。
本发明还提供了磁感蛋白在制备抑制凋亡基因表达和/或促进凋亡抑制基因表达的药物中的应用。
本发明中，凋亡基因为Bax基因；所述凋亡抑制基因为Bcl-2基因
一些实施例中，所述磁感蛋白选自铁硫蛋白、铁蛋白、胶原蛋白、热休克蛋白、角蛋白、神经营养因子、钙调蛋白、GP120蛋白、血红蛋白或胰岛素。
一些具体实施例中，所述磁感蛋白为磁感应蛋白Isca。
本发明将水迷宫训练后符合标准的SD大鼠以Aβ构建AD大鼠模型，之后向模型大鼠侧脑室注射rAAV2/1-Isca-EGFP，实验组大鼠于磁场环境中培养24h，经检测，实验组大鼠与模型组相比，磁感蛋白组和磁场组中Bcl-2蛋白表达上调，Bax蛋白表达下调，说明磁感蛋白及其与磁场的相互作用能够促进抗凋亡的发生，进而改善AD模型大鼠的记忆、认知及情感状态。且证明导入ISCA基因的大鼠在磁场作用下可提高AD模型大鼠的学习、记忆能力。
本发明还提供了磁感蛋白在制备改善神经退行性疾病中的应用。
本发明中，所述神经退行性疾病为老年性痴呆、帕金森病、亨廷顿病、脊髓小脑共济失调、齿状核红核苍白球丘脑下核萎缩、肌萎缩侧索硬化症或脊髓肌萎缩症。
一些实施例中，磁感蛋白选自铁硫蛋白、铁蛋白、胶原蛋白、热休克蛋白、角蛋白、神经营养因子、钙调蛋白、GP120蛋白、血红蛋白或胰岛素。
一些具体实施例中，所述磁感蛋白为磁感应蛋白Isca。
本发明还提供了一种改善神经退行性疾病的药物，包括磁感应蛋白或能够表达磁感应蛋白的基因工程载体。
本发明中，所述神经退行性疾病为老年性痴呆、帕金森病、亨廷顿病、脊髓小脑共济失调、齿状核红核苍白球丘脑下核萎缩、肌萎缩侧索硬化症或脊髓肌萎缩症。
本发明实施例中，所述能够表达磁感应蛋白的基因工程载体为表达磁感应蛋白的rAAV病毒载体。
一些实施例中，磁感蛋白选自铁硫蛋白、铁蛋白、胶原蛋白、热休克蛋白、角蛋白、神经营养因子、钙调蛋白、GP120蛋白、血红蛋白或胰岛素。
一些具体实施例中，所述磁感蛋白为磁感应蛋白Isca。
本发明提供药物的剂型为注射剂；其中，所述表达磁感应蛋白的rAAV病毒载体的滴度为5×1011cfu/mL。
所述注射剂给予的位置为侧脑室。给予注射剂的位置包含神经细胞或神经元。
药物给予的剂量为10μl/只大鼠，注射时间20min，注射速度0.5μl/min，注射完毕后留针10min。
本发明还提供了一种改善神经退行性疾病的方法，将磁感蛋白导入神经细胞，在磁场作用下促进磁感蛋白与铁离子的结合。
一些实施例中，所述磁感蛋白选自铁硫蛋白、铁蛋白、胶原蛋白、热休克蛋白、角蛋白、神经营养因子、钙调蛋白、GP120蛋白、血红蛋白或胰岛素。
一些具体实施例中，所述磁感蛋白为磁感应蛋白Isca。
本发明中，所述神经细胞为神经元或神经胶质细胞。
一些实施例中，所述神经细胞为黑质神经细胞、少突神经胶质细胞或黑质多巴胺能神经元细胞。
本发明中，神经退行性疾病为老年性痴呆、帕金森病、亨廷顿病、脊髓小脑共济失调、齿状核红核苍白球丘脑下核萎缩、肌萎缩侧索硬化症或脊髓肌萎缩症。
本发明通过实验证明，将磁感应蛋白导入PD细胞结果表明，在磁场作用下，PD细胞存活率得到提高，PD细胞中α-synuclein、GSK-3β水平被降低。将磁感应蛋白导入AD大鼠模型，在磁场作用下，AD模型大鼠海马CA1区Bcl-2表达升高、Bax蛋白表达降低，大鼠的学习、记忆能力得到提高。由此证明磁感应蛋白能够具有改善神经退行性疾病的作用。本发明还提供了改善神经退行性疾病的药物，该药物包括磁感应蛋白或表达磁感应蛋白的基因工程载体。
附图说明
图1示各组细胞OD值与存活率；
图2示α-synuclein、GSK-3β表达情况；其中，各泳道依次为：1对照组；2模型组；3磁感蛋白组；4磁场组。
具体实施方式
本发明提供了磁感蛋白在改善神经退行性疾病中的应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
下面结合实施例，进一步阐述本发明：
实施例1
帕金森病(Parkinson＇s disease，PD)是严重影响中老年人群生活质量和身心健康的神经系统退行性疾病之一，是以黑质－纹状体多巴胺能神经元进行性变性坏死、多巴胺递质缺乏为主要病理基础的常见老年中枢神经系统退行性疾病，具有发病率高、起病隐袭等特点。近年来研究发现帕金森相关蛋白α-突触核蛋白(α-synuclein)、糖原合成酶激酶-3β(GSK-3β)、Tau蛋白对于PD的发病和发展以及多巴胺神经元的保护具有重要的调节作用。
1、实验步骤
(1)MPP+造模
以多巴胺能神经细胞MES23.5细胞为研究对象，用1-甲基-4-苯基吡啶离子(1-methyl-4-phenyl-pyridinium，MPP+)诱导建立PD的体外细胞损伤模型。
MES23.5细胞接种于含5％(体积分数)胎牛血清、1％(质量浓度)谷氨酰胺、2％(质量浓度)50×Sato＇s溶液和2％(体积分数)青/链霉素的DMEM/F12培养基，置于37℃、5％(体积分数)CO2、饱和湿度的细胞培养箱中培养，用0.25％(质量浓度)胰酶消化传代，收集对数生长期细胞制成细胞悬液。将细胞以细胞密度1×105接种到96孔板中，加入终浓度为100μ的MPP+培养液培养24h，建立体外PD模型细胞,即模型组。
(2)导入ISCA基因
以rAAV病毒为载体，将编码磁感应蛋白Isca的基因转染上述模型细胞，培养条件如上，形成磁感蛋白组。
(3)磁场培养
将实验组细胞培养于磁场环境中培养24h，其他条件同上，形成磁场组。
(4)结果检测
由此，实验形成模型组、磁感蛋白组和磁场组三组实验组，同时，以正常培养的MES23.5细胞为对照组，采用MTT检测各组细胞的增殖情况，采用Western blot检测帕金森相关蛋白α-synuclein、GSK-3β的表达情况。
2、实验结果
(1)MTT检测细胞增殖情况
向96孔板中加入5mg/ml的MTT，37℃培养4小时后，加入150μl的DMSO，震荡10min，在酶标仪上选择490nm的波长，检测各个孔的吸光度，计算细胞存活率。结果如表1和图1：
细胞存活率(％)＝实验组吸光度均值/空白对照组×100％
表1各组细胞存活率
| 组别 | OD值 | 存活率/％ |
| 对照组 | 0.852±0.012 | 100 |
| 模型组 | 0.498±0.013 | 58.5 |
| 磁感蛋白组 | 0.604±0.033 | 70.9 |
| 磁场组 | 0.764±0.014 | 89.7 |
从表中可以看出，模型组相对于对照组来说，细胞存活率降低，进行磁感蛋白的导入后，细胞存活率提高，进行磁场刺激后，细胞存活率进一步提高。
(2)α-synuclein、GSK-3β表达情况
收集上述四组细胞，分别加入含有RIPA、蛋白酶抑制剂、磷酸酶抑制剂的混合裂解液冰上裂解30min，收集细胞碎片和裂解液，4℃、12000r/min离心5min，取上清，提取蛋白，按照同样的方法重复提取蛋白3次。
BCA蛋白定量；SDS-PAGE电泳，转膜，5％(质量浓度)脱脂奶粉室温封闭2h；加入α-synuclein单克隆抗体(1:1000)、GSK-3β单克隆抗体(1:1000)、Phospho-GSK3β(1:800)，4℃过夜。加入辣根过氧化物酶标记的二抗(1:5000)反应1h；膜上加ECL显影剂，反应2min，常规显影，用Image Lab软件对蛋白的表达进行半定量分析，分别计算各蛋白的灰度值比。结果如图2
从图2可以看出，空白对照组比较，模型组α-synuclein、GSK-3β的磷酸化蛋白表达量水平显著升高，说明造模成功。磁感蛋白组和磁场组的α-synuclein、GSK-3β的磷酸化蛋白蛋白表达量均低于模型组，说明磁感蛋白的导入以及磁场的作用能够起到神经保护的作用。
实施例2：
阿尔兹海默症(Alzheimer＇s disease，AD)是一种进行性的神经退行性疾病，临床上表现为记忆和认知功能障碍，语言和行为能力缺失，患者后期表现出严重的失忆症和运动功能减弱，最终导致死亡。现在的研究发现，β淀粉样蛋白(Aβ)在大脑内的沉积是AD的主要病理病因，海马区细胞凋亡与Aβ神经毒性作用密切相关，而Bcl-2和Bax在细胞凋亡的基因调控过程中起着至关重要的作用。
1、实验步骤
(1)实验动物筛选
采用Morris水迷宫对50只SD大鼠进行训练，每日上午及下午相同时间各训练1次，连续4天。Morris水迷宫第Ⅲ象限设置没入水中平台(水面下1cm)，将大鼠于4个象限边缘1/2弧度处头朝池壁入水，记录大鼠入水后站上平台时间(逃避潜伏期)，经60s未找到平台的大鼠，将其引领至平台，放置30s，引导学习记忆。第5天把大鼠Ⅰ象限中点面朝水池壁放入水池中，记录大鼠找到平台的时间(逃避潜伏期)及游泳速度，连测4次，计算每只大鼠的平均逃避潜伏期及平均游泳速度，以这两项指标反映大鼠的学习能力，计算每只大鼠的平均逃避潜伏期、平均游泳速度与参考值之差所占该鼠的平均值的比值，保留介于该值±20％的大鼠，将学习记忆能力及游泳速度异较大者剔除。
(2)动物模型的建立
Morris水迷宫训练后获得符合标准的40只健康SD大鼠，随机均分为对照组、模型组、磁感蛋白组和磁场组。模型组、磁感蛋白组和磁场组各组大鼠(共30只)腹腔注射1.25％D-半乳糖50mg/(kg·d)、连续6周致衰老。将Aβ25-35用生理盐水稀释为10g/L，37℃孵育1周，使其变为聚集状态(凝聚态)，侧脑室注射。
侧脑室注射：SD大鼠10％水合氯醛(300mg/kg)腹腔注射麻醉后固定于大鼠脑立体定位仪上，选择右侧侧脑室为注射靶区，钻开颅骨、暴露硬脑膜，微量进样器缓慢将Aβ25～35 10μg注入，注射时间为10min，留针10min，使Aβ25-35充分弥散，缝合皮肤，常规饲养。
模型成功标准：大鼠术后第7天，按(1)方法行Morris水迷宫实验，各组大鼠适应训练2天，至逃避潜伏期成绩基本稳定，于第3天测逃避潜伏期，造模大鼠逃避潜伏期大于参考值20％则认定为造模成功。
(3)导入ISCA基因
造模成功的大鼠20只，将大鼠麻醉，确定大鼠侧脑室注射靶点的三维坐标，在立体定向仪辅助下，用微量进样器向靶点注射rAAV2/1-Isca-EGFP，注射量10μl，滴度为5×1011/ml，注射时间20min，注射速度0.5μl/min，注射完毕后留针10min，用少量无菌骨蜡封闭骨孔，缝合头皮。
(4)磁场培养
将导入ISCA基因的10只大鼠培养于磁场环境中培养24h，其他条件同上，形成磁场组。
(5)结果检测
大鼠学习记忆能力对照组、模型组、磁感蛋白组和磁场组行Morris水迷宫实验，适应训练2天，第3天行定位航行及空间搜索实验。定位航行实验记录逃避潜伏期，空间搜索实验记录60s内大鼠穿越平台次数。
海马CA1区Bcl-2、Bax蛋白表达Bcl-2、Bax在细胞凋亡的基因调控过程中起着至关重要的作用。各组实验终点，处死大鼠，迅速取脑，置于4％多聚甲醛中固定，石蜡包埋、CA1区连续切片5片，常规HE染色及免疫组织化学染色。免疫组织化学染色采用链霉素－卵白素－生物素－过氧化物酶法(SABC)。每只大鼠选片1张，每张切片随机选取5个高倍视野进行显微镜下图象分析，各组所选取部位相同。Bcl-2、Bax阳性细胞为胞浆或胞核呈黄色，应用Image-proplus6.0图像分析软件计算平均吸光度。各实验组动物于相应时间点断头处死，取海马区脑组织，置冻存管中放入液氮保存。
2、实验结果
从学习记忆能力的实验结果可以看出，模型组大鼠的平均逃避潜伏期明显延长，穿越平台次数明显减少。导入ISCA基因后的磁感蛋白组的平均逃避潜伏期稍短于模型组，而在磁场组中该数值明显降低，提示，导入ISCA基因的大鼠在磁场作用下可提高AD模型大鼠的学习、记忆能力。
表2各组大鼠平均逃避潜伏期及穿越平台次数
| 组别 | 平均逃避潜伏期(s) | 穿越平台次数 |
| 对照组 | 14.86±3.12 | 7.95±0.86 |
| 模型组 | 57.25±6.13 | 2.86±0.72 |
| 磁感蛋白组 | 46.25±3.33 | 3.83±0.52 |
| 磁场组 | 24.76±4.18 | 6.86±0.96 |
(2)Bcl-2、Bax蛋白情况
Bcl-2是Bcl-2家族最主要的凋亡抑制基因，Bax是最主要的凋亡诱导基因。AD患者脑退化的神经细胞中，Bcl-2基因的含量明显减少。从表3可以看出，与对照组相比，模型组、磁感蛋白组和磁场组中Bcl-2蛋白和Bax蛋白的表达均有增加。与模型组相比，磁感蛋白组和磁场组中Bcl-2蛋白表达上调，Bax蛋白表达下调，说明磁感蛋白及其与磁场的相互作用能够促进抗凋亡的发生，进而改善AD模型大鼠的记忆、认知及情感状态。
表3各组大鼠CA1区Bcl-2、Bax蛋白表达情况
| 组别 | Bcl-2蛋白 | Bax蛋白 |
| 对照组 | 0.15±0.02 | 0.26±0.02 |
| 模型组 | 0.37±0.05 | 0.75±0.03 |
| 磁感蛋白组 | 0.42±0.03 | 0.64±0.05 |
| 磁场组 | 0.47±0.04 | 0.39±0.04 |
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (24)
1.磁感蛋白在制备降低帕金森病相关蛋白水平的药物中的应用。
2.根据权利要求1所述的应用，其特征在于，所述帕金森病相关蛋白为α-突触核蛋白和/或糖原合成酶激酶-3β。
3.根据权利要求1所述的应用，其特征在于，所述磁感蛋白选自铁硫蛋白、铁蛋白、胶原蛋白、热休克蛋白、角蛋白、神经营养因子、钙调蛋白、GP120蛋白、血红蛋白或胰岛素。
4.根据权利要求1所述的应用，其特征在于，所述磁感蛋白为磁感应蛋白Isca。
5.磁感蛋白在制备抑制凋亡基因表达和/或促进凋亡抑制基因表达的药物中的应用。
6.根据权利要求5所述的应用，其特征在于，所述凋亡基因为Bax基因；所述凋亡抑制基因为Bcl-2基因。
7.根据权利要求5所述的应用，其特征在于，所述磁感蛋白选自铁硫蛋白、铁蛋白、胶原蛋白、热休克蛋白、角蛋白、神经营养因子、钙调蛋白、GP120蛋白、血红蛋白或胰岛素。
8.根据权利要求5所述的应用，其特征在于，所述磁感蛋白为磁感应蛋白Isca。
9.磁感蛋白在制备改善神经退行性疾病中的应用。
10.根据权利要求9所述的应用，其特征在于，所述神经退行性疾病为老年性痴呆、帕金森病、亨廷顿病、脊髓小脑共济失调、齿状核红核苍白球丘脑下核萎缩、肌萎缩侧索硬化症或脊髓肌萎缩症。
11.根据权利要求9所述的应用，其特征在于，所述磁感蛋白选自铁硫蛋白、铁蛋白、胶原蛋白、热休克蛋白、角蛋白、神经营养因子、钙调蛋白、GP120蛋白、血红蛋白或胰岛素。
12.根据权利要求9所述的应用，其特征在于，所述磁感蛋白为磁感应蛋白Isca。
13.一种改善神经退行性疾病的药物，其特征在于，包括磁感应蛋白或能够表达磁感应蛋白的基因工程载体。
14.根据权利要求13所述的药物，其特征在于，所述神经退行性疾病为老年性痴呆、帕金森病、亨廷顿病、脊髓小脑共济失调、齿状核红核苍白球丘脑下核萎缩、肌萎缩侧索硬化症或脊髓肌萎缩症。
15.根据权利要求13所述的药物，其特征在于，所述能够表达磁感应蛋白的基因工程载体为表达磁感应蛋白的rAAV病毒载体。
16.根据权利要求13所述的药物，其特征在于，所述磁感蛋白选自铁硫蛋白、铁蛋白、胶原蛋白、热休克蛋白、角蛋白、神经营养因子、钙调蛋白、GP120蛋白、血红蛋白或胰岛素。
17.根据权利要求13所述的药物，其特征在于，所述磁感蛋白为磁感应蛋白Isca。
18.根据权利要求13～17任一项所述药物，其特征在于，其剂型为注射剂；其中，所述表达磁感应蛋白的rAAV病毒载体的滴度为5×1011cfu/mL。
19.一种改善神经退行性疾病的方法，其特征在于，将磁感蛋白导入神经细胞，在磁场作用下促进磁感蛋白与铁离子的结合。
20.根据权利要求19所述的方法，其特征在于，所述磁感蛋白选自铁硫蛋白、铁蛋白、胶原蛋白、热休克蛋白、角蛋白、神经营养因子、钙调蛋白、GP120蛋白、血红蛋白或胰岛素。
21.根据权利要求19所述的方法，其特征在于，所述磁感蛋白为磁感应蛋白Isca。
22.根据权利要求19所述的方法，其特征在于，所述神经细胞为神经元或神经胶质细胞。
23.根据权利要求19所述的方法，其特征在于，所述神经细胞为黑质神经细胞、少突神经胶质细胞或黑质多巴胺能神经元细胞。
24.如权利要求19所述的方法，其特征在于，所述神经退行性疾病为老年性痴呆、帕金森病、亨廷顿病、脊髓小脑共济失调、齿状核红核苍白球丘脑下核萎缩、肌萎缩侧索硬化症或脊髓肌萎缩症。
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