CN115305258B - 过表达shp2的基因工程化间充质干细胞外囊泡的制备方法和应用 - Google Patents
过表达shp2的基因工程化间充质干细胞外囊泡的制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种过表达SHP2的基因工程化间充质干细胞外囊泡的制备方法和应用，将MSC在减血清低糖DMEM培养基中培养，采用编码PTPN11基因的慢病毒转染MSC，得到MSC‑SHP2，转入无外泌体的血清低糖DMEM培养基中培养，收集上清，通过超滤法进行粗提，再通过亲和层析法进一步提取，能够收集到稳定表达SHP2的间充质干细胞外囊泡。本发明的工程化间充质干细胞外囊泡制备工艺简单成熟，生物相容性好，可作为一种新型治疗制剂，具有重要的研究价值和广阔的应用前景。
Description
技术领域
本发明涉及生物技术领域，尤其涉及一种过表达SHP2的基因工程化间充质干细胞外囊泡的制备方法和应用。
背景技术
随着全球老龄化的加剧，阿尔茨海默病(Alzheimer’s disease,AD)的患病率在全球范围内持续上升。目前65岁以上人群中约有11％的人患有AD，85岁以上人群的比例高达42％。AD是一种进行性地神经退行性疾病，目前尚无有效的治疗方法。尽管存在争议，但β-淀粉蛋白(Aβ)和微管相关蛋白tau的积累被认为是AD的主要病理机制之一。然而，针对Aβ和tau的药物开发面临巨大的困难和令人失望的结果，如Aducanumab、Crenezumab和Semorinemab对AD患者认知和功能下降的缓解作用不明显。此外，一些用于胆碱功能恢复的抗AD药物存在严重的副作用，如他克林的肝毒性和利伐他明的胃肠道不良反应。当务之急是发现和开发更有效的治疗AD的药物。
自噬作为一种清除异常蛋白积聚的重要机制，在清除Aβ、磷酸化tau蛋白和受损细胞器的积累方面发挥关键作用。在AD的发病机制中，自噬功能障碍是导致AD相关蛋白沉积和缺陷线粒体积累的潜在细胞机制。最近的一项研究表明，自噬失调发生在Aβ形成和神经退行性疾病之前，这一发现说明，在AD治疗中，恢复脑内自噬水平可能比清除Aβ或tau更重要，而且线粒体自噬在神经元保护和记忆改善中的关键作用已被证实。然而，现有介导线粒体自噬的诱导物或化合物在大脑富集少，并且毒性大，限制了其对AD病理的探索和研究以及未来的临床转化应用。有研究表明，雷帕霉素、UrolithinA、烟酰胺核苷(NR)及烟酰胺单核苷酸(NMN)等可通过增强PINK1/Parkin依赖性线粒体自噬逆转AD小鼠的记忆缺陷，然而，在针对轻度至中度AD患者的临床试验中，未能改善认知功能。因此，需要进一步开发依赖于线粒体自噬的AD治疗药物。
近年来，细胞外囊泡(extracellular vesicles,EVs)作为一种极具潜力的治疗平台受到了广泛关注。这些EVs是细胞主动释放的纳米级膜囊泡，对特定疾病具有敏感性和特异性，是一类新的生物标志物，能够有效地递送治疗性药物，可用作新型生物治疗剂。间充质干细胞(Mesenchymal stemcells，MSCs)外囊泡不仅可以行使MSCs的组织修复和免疫调控功能外，而且具备诸多独特优点，比如低免疫原性、无成瘤风险、体内稳定性高等，最重要的是可穿透血脑屏障，在治疗神经退行性疾病方面展现出了在临床治疗和药物负载方面的巨大应用潜力。如Morris Losurdo等(Intranasal delivery of mesenchymal stem cell-derived extracellular vesicles exerts immunomodulatory and neuroprotectiveeffects in a 3xTg model of Alzheimer's disease)通过鼻腔通道输送间充质干细胞外囊泡(MSC-EVs)可以抑制小鼠大脑中小胶质细胞的激活，为阿尔茨海默病的治疗提供了一个疗法，但该间充质干细胞外囊泡的治疗效果十分有限。
阿尔茨海默病是一种多因素病因的复杂疾病，虽然通过对遗传物质研究和借助生物信息学方法确定了很多影响因子，并开发了多种临床研究药物，但很多药物治疗测试效果均不是很理想，因此，提供一种用于阿尔茨海默病治疗的新型药物迫在眉睫。目前关于基因工程化间充质干细胞外囊泡在阿尔茨海默病治疗的研究还未见报道。
发明内容
为解决上述技术问题，本发明公开了一种基因工程化的间充质干细胞外囊泡的制备方法，并使其高表达SHP2(EVs-SHP2)，具有显著的线粒体自噬诱导功能和改善认知障碍，该细胞外囊泡生物相容性好，制备工艺简单成熟，在阿尔茨海默病治疗中呈现显著的疗效。
本发明的第一个目的是提供一种过表达SHP2的基因工程化间充质干细胞外囊泡的制备方法，包括以下步骤：
S1、将间充质干细胞在减血清低糖DMEM培养基培养纯化，得到间充质干细胞系；
S2、采用编码PTPN11基因的慢病毒转染步骤S1得到的间充质干细胞系，得到过表达SHP2的间充质干细胞(MSC-SHP2)；
S3、提取步骤S2中过表达SHP2的间充质干细胞的细胞外囊泡，得到所述过表达SHP2的基因工程化间充质干细胞外囊泡(EVs-SHP2)；所述的提取为将所述过表达SHP2的间充质干细胞在无外泌体的血清低糖DMEM培养基中培养，收集上清，通过超滤法进行粗提，再通过亲和层析法进一步提取。
进一步地，本发明中的低糖DMEM培养基指培养基中葡萄糖含量≤1100mg/L，减血清低糖DMEM培养基指葡萄糖含量≤1100mg/L，且相对于血清低糖DMEM培养基配方中减少50-90％的血清用量。
进一步地，在步骤S1中，所述间充质干细胞的来源包括但不限于骨髓、脂肪、肌肉等。
进一步地，在步骤S2中，Src同源性结构域蛋白酪氨酸磷酸酶2(Protein tyrosinephosphatase,SHP2)由PTPN11基因编码，SHP2在间充质干细胞的过表达优选为转染效率高的慢病毒介导。与内源表达的方法相比，细胞外囊泡通过机械或化学技术实现蛋白的外源装载，不仅方法复杂，而且载药效率不可控，破坏细胞外囊泡完整性。SHP2是一种胞内蛋白，本领域技术人员公知，胞内蛋白作为胞浆成分，在细胞外囊泡形成的过程中会包裹部分胞内蛋白，而本发明提供了一种制备方法，利用慢病毒在间充质干细胞高表达SHP2蛋白，使分泌的细胞外囊泡稳定表达胞内蛋白SHP2。
进一步地，在步骤S2中，通过嘌呤霉素筛选得到过表达蛋白质酪氨酸磷酸酶2的间充质干细胞。
进一步地，所述过表达SHP2的基因工程化间充质干细胞外囊泡的粒径为30～150nm左右，电位为0～-20mV左右。
进一步地，在步骤S3中，所述的超滤法为将上清用0.22～0.45μm滤膜过滤，收集过滤后的液体。
进一步地，在步骤S3中，所述的亲和层析法为采用含CD63抗体的亲和层析介质进行亲和层析。
进一步地，在步骤S3中，与以往单一的细胞外囊泡提纯方法相比，本发明通过组合型提取工艺平台进行提纯，首先通过超滤法进行粗提，所获得产物再次通过亲和层析方法进一步提取，获得较高纯度和活性的细胞外囊泡。
本发明的第二个目的是提供上述制备方法制备得到的过表达SHP2的基因工程化间充质干细胞外囊泡。
本发明的第三个目的是提供上述过表达SHP2的基因工程化间充质干细胞外囊泡在制药中的应用。
进一步地，上述基因工程化间充质干细胞可用于预防或治疗神经退行性疾病，如阿尔茨海默病、帕金森病、亨廷顿病等。
进一步地，过表达SHP2的间充质干细胞外囊泡在制备阿尔茨海默病预防或治疗药物中的应用。
进一步地，所述过表达SHP2的基因工程化间充质干细胞外囊泡可靶向神经细胞的线粒体，诱导线粒体自噬。
进一步地，所述过表达SHP2的基因工程化间充质干细胞外囊泡可在脑部富集。
进一步地，所述过表达SHP2的基因工程化间充质干细胞外囊泡可提高阿尔茨海默病个体大脑的线粒体自噬，改善脑部神经炎性反应、神经细胞损伤和β-淀粉蛋白的聚集。
进一步地，所述过表达SHP2的间充质干细胞外囊泡的给药方式为静脉注射。
本发明的第四个目的是提供一种抗阿尔茨海默病制剂，所述抗阿尔茨海默病制剂包括上述过表达SHP2的基因工程化间充质干细胞外囊泡。
细胞外囊泡可以被设计成表达各种蛋白具有靶向组织或提供信号调节功能失调的细胞，而本发明通过对EVs产生细胞MSC进行基因修饰，使其高表达SHP2，制备得到过表达SHP2的基因工程化间充质干细胞外囊泡，在发挥间充质干细胞组织修复能力的基础上，通过SHP2诱导神经细胞线粒体自噬，从而恢复脑部线粒体自噬，清除受损线粒体以及Aβ聚集，缓解阿尔茨海默病患者脑部神经元损伤和神经炎性状态，逆转认知缺陷，进而显著改善阿尔茨海默病的病理进程。
借由上述方案，本发明至少具有以下优点：
本发明的间充质干细胞来源广泛，易获取、分离，可大规模扩增。基因工程化的细胞外囊泡制备工艺简单成熟，生物安全性高，细胞外囊泡作为递送载体治疗属于无细胞疗法，排斥几率很低。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合详细附图说明如后。
附图说明
为了使本发明的内容更容易被清楚的理解，下面根据本发明的具体实施例并结合附图，对本发明作进一步详细的说明。
图1为本发明流式细胞术检测间充质干细胞的纯度结果分析图；
图2为本发明中间充质干细胞在编码ptpn11基因的慢病毒转染后，SHP2高表达的免疫荧光和流式结果分析图；
图3为本发明中透射电镜显示工程化细胞外囊泡的粒径大小及zeta电位检测结果图；
图4为本发明中工程化细胞外囊泡的SHP2表达水平的western blot结果图；
图5为本发明中工程化细胞外囊泡进入SH-SY5Y细胞线粒体的免疫荧光结果图；
图6为本发明中工程化细胞外囊泡诱导SH-SY5Y细胞的线粒体自噬免疫荧光结果图；
图7为本发明中工程化细胞外囊泡诱导SH-SY5Y细胞的自噬电镜结果图；
图8为本发明中工程化细胞外囊泡诱导SH-SY5Y细胞的自噬的信号通路检测western blot结果图；
图9为本发明工程化细胞外囊泡对SH-SY5Y细胞线粒体电位影响的流式分析图；
图10为本发明工程化细胞外囊泡对SH-SY5Y细胞凋亡影响的MTT和LDH释放分析结果图；
图11为本发明工程化细胞外囊泡对SH-SY5Y细胞凋亡影响的AnnexinⅤ/PI流式分析图；
图12为本发明中工程化细胞外囊泡对SH-SY5Y细胞的凋亡信号通路影响的western blot结果图；
图13为本发明工程化细胞外囊泡对SH-SY5Y细胞ROS产生的影响；
图14为本发明中工程化细胞外囊泡对巨噬细胞NLRP3炎症小体影响的免疫荧光结果分析图；
图15为本发明中工程化细胞外囊泡对巨噬细胞炎症因子分泌的影响结果图；
图16为本发明中工程化细胞外囊泡对巨噬细胞炎症信号通路影响的westernblot结果分析图；
图17为本发明中工程化细胞外囊泡对巨噬细胞内P65的胞浆核定位影响的免疫荧光分析结果图；
图18为本发明中工程化细胞外囊泡在Aβ1-42诱导的阿尔茨海默病模型小鼠的生物分布及SHP2的表达分析；
图19为本发明中工程化细胞外囊泡对阿尔茨海默病小鼠脑部的线粒体自噬影响的结果分析图；
图20为本发明中工程化细胞外囊泡对阿尔茨海默病小鼠脑部Aβ1-42的清除影响结果图；
图21为本发明中工程化细胞外囊泡对阿尔茨海默病小鼠脑部ROS产生和细胞凋亡的影响；
图22为本发明中工程化细胞外囊泡对阿尔茨海默病小鼠脑部NLRP3炎症小体形成的影响；
图23为本发明中工程化细胞外囊泡对阿尔茨海默病小鼠脑部炎症信号通路表达和细胞因子产生的影响；
图24为本发明中工程化细胞外囊泡对阿尔茨海默病小鼠脑部小胶质细胞浸润的影响；
图25为本发明中工程化细胞外囊泡对阿尔茨海默病小鼠脑部Aβ1-42含量的影响；
图26为本发明中工程化细胞外囊泡对阿尔茨海默病小鼠脑部细胞损伤的影响。
图27为本发明中工程化细胞外囊泡对阿尔茨海默病小鼠脑部突出重塑性和乙酰胆碱产生的影响；
图28为本发明中工程化细胞外囊泡对阿尔茨海默病小鼠脑部MDA、SOD和GSH产生的影响；
图29为本发明中水迷宫实验评估工程化细胞外囊泡对阿尔茨海默病小鼠记忆力的影响；
图30为本发明中新物体识别实验评估工程化细胞外囊泡对阿尔茨海默病小鼠学习能力的影响；
图31为本发明中旷场实验工程化细胞外囊泡对阿尔茨海默病小鼠新环境中学习能力的影响。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
本发明各实施例中的材料来源为：
编码Ptpn11的慢病毒购自汉恒生物科技有限公司；人神经母细胞瘤细胞株SH-SY5Y(SCSP-5014)和小鼠巨噬细胞株RAW264.7(TCM13)购自中国科学院细胞库。SHP2抗体(3397)购自Cell signaling technology公司；Mito-trackerTM Green(M7514),Lyso-trackerTMdeep red(L12492)购自Invitrogen公司；人源淀粉蛋白Aβ1-42(A9810)购自sigma公司；DCFDA-Cellular ROS assay kit(ab113851)购自Abcam公司；Thioflavine S(HY-D0972)购自MCE公司；Annexin V-FITC/propidium iodide assay kit(C1062S)购自碧云天生物技术有限公司；Human amyloidβ-peptide(1-42)elisa kit(PA082)购自碧云天生物技术有限公司；Cy5.5荧光染料(HY-D0924)购自MedChemExpress公司。
间充质干细胞由小鼠骨髓中提取并利用其贴壁特性在低糖DMEM培养基中进行纯化和扩增。
6-8周龄雌性C57BL/6小鼠购自常州卡文斯实验动物有限公司。小鼠按照中国科学院生化与细胞所实验动物管理委员会(IACUC)的指导操作方法进行处理。
下述实施例提供了一种基因工程化的间充质干细胞外囊泡，所述的细胞外囊泡采用慢病毒介导干细胞高表达SHP2，然后分离纯化得到所述的工程化囊泡。具体地，包括如下步骤：
(1)分离小鼠骨髓来源间充质干细胞后，利用编码PTPN11基因的慢病毒转染使MSC高表达SHP2蛋白。
(2)根据细胞外囊泡提取纯化方法，收集基因改造后的MSC分泌的细胞外囊泡。
本发明提供的基因工程化的细胞外囊泡递送治疗分子SHP2穿过血脑屏障在脑中富集，增强治疗效率，为脑部疾病的治疗提供新策略。基于上述细胞外囊泡构建的基因工程化细胞外囊泡生物制剂，克服了传统的针对Aβ和tau蛋白的药物所面临的困境，通过增强线粒体自噬，清除阿尔茨海默病小鼠脑部受损的线粒体和Aβ聚集，从而高效缓解阿尔茨海默病。
实施例1编码Ptpn11的慢病毒转染间充质干细胞使其过表达SHP2分子
(1)从C57BL/6小鼠的胫骨和股骨骨髓提取骨髓细胞，然后根据间充质干细胞的贴壁特性，利用低糖DMEM培养基进行纯化和扩增。根据MSC的特定标志物(CD34-、CD45-、CD105+、CD73+)利用流式细胞术检测纯度，结果显示(图1)，经过一定时间培养，MSC的纯度可达70％左右(图1)。
(2)小鼠骨髓间充质干细胞分离培养后，利用编码Ptpn11
(NM_001109992.1)的慢病毒转染间充质干细胞，经过5μg/mL嘌呤霉素的筛选获得高表达SHP2的间充质干细胞。免疫荧光和流式细胞术(图2)结果显示，SHP2在间充质干细胞上高表达，说明成功建立高表达SHP2的间充质干细胞。
实施例2高表达SHP2的间充质干细胞外囊泡(EVs-SHP2)的提取及表征
(1)高表达SHP2的间充质干细胞更换为无外泌体的血清低糖DMEM培养基培养48h，收集上清；
(2)将上清在4℃下依次经过300g离心10min，去沉淀，2000g离心10min，去沉淀，10000g离心30min，去沉淀，100000g离心70min，弃上清，加PBS悬浮沉淀，再100000g离心70min，经过0.22μm滤膜过滤后对滤液进行超滤浓缩，最后在磁珠表面包被外泌体表面标记物CD63，利用免疫亲和法分离间充质干细胞外囊泡；
(3)并对步骤(2)获得的细胞外囊泡进行透射电镜分析其粒径大小及分布和Zeta电位分析，结果显示，SHP2的高表达并没有改变细胞外囊泡的粒径大小和电位，粒径在100nm左右，电位在0～-20mV左右(图3)；通过免疫印迹分析细胞外囊泡上SHP2的表达，结果显示，SHP2蛋白在来源于工程化间充质干细胞的外囊泡上显著性高表达，说明本发明成功建立稳定表达SHP2蛋白的细胞外囊泡体系(图4)。
实施例3 EVs-SHP2对神经细胞线粒体自噬的调控作用
(1)利用人源神经母细胞瘤细胞SH-SY5Y进行体外实验。免疫荧光和Western blot结果显示，工程化外囊泡可以将SHP2带入胞内并进入神经细胞的线粒体(图5)。
(2)淀粉样蛋白可以导致脑内老年斑的形成和神经细胞的凋亡，是导致阿尔茨海默病的重要因素。本发明利用Aβ1-42与SH-SY5Y共同孵育，并加入非工程化的EVs(非工程化的EVs制备方法同工程化的EVs-SHP2，只是不进行慢病毒转染)和工程化的EVs-SHP2处理24h，通过mito-tracker和lyso-tracker以及TOM20和LC3B的荧光分析线粒体自噬情况，荧光结果显示，EVs-SHP2处理后显著性增强神经细胞的线粒体自噬水平(图6)。
(3)利用电镜观察自噬小体的数量，结果显示，EVs-SHP2处理后显著性增多神经细胞自噬小体的数量(图7)。而且Western blot结果显示LC3BII/I的比例明显增强(图8)。
实施例4 EVs-SHP2对神经细胞凋亡的调控作用
(1)Aβ1-42可以造成细胞凋亡和线粒体损伤，流式细胞术结果显示，SH-SY5Y细胞在Aβ1-42处理后，线粒体膜电位显著下降，而EVs-SHP2可以恢复线粒体膜电位(图9)。
(2)利用MTT和检测乳酸脱氢酶LDH释放评估EVs-SHP2对Aβ1-42造成的细胞凋亡的改善作用，结果显示，与EVs相比，EVs-SHP2能显著性改善细胞的凋亡(图10)，并且AnnexinⅤ/PI染色也证实这一点(图11)。另外，细胞凋亡信号通路的Western blot结果也显示，EVs-SHP2显著性抑制凋亡信号通路的激活(图12)。
实施例5 EVs-SHP2对巨噬细胞的炎性改善作用
(1)神经炎性反应在阿尔茨海默病的发病中也发挥重要作用，Aβ1-42造成的线粒体损伤会导致神经细胞释放大量的ROS，本发明通过免疫荧光实验证明EVs-SHP2抑制ROS产生(图13)。
(2)在transwell共培养体系中，在Aβ1-42处理后，将SH-SY5Y细胞与RAW264.7细胞共同培养24h，免疫荧光观察NLRP3的表达评估EVs-SHP2对炎症小体形成的抑制作用，结果显示，SH-SY5Y在Aβ1-42处理后显著增强巨噬细胞NLRP3的表达，而EVs-SHP2抑制炎症小体的形成(图14)；并且巨噬细胞释放的细胞因子IL-6、TNFα以及IL-1β被EVs-SHP2显著性的抑制(图15)。通过Western blot检测NLRP3活化的信号通路，结果显示，EVs-SHP2显著性抑制Aβ1-42处理造成的炎性小体信号通路激活(图16)。
(3)在巨噬细胞炎症发生的过程中，伴随着转录因子P65的磷酸化入核促进炎症性细胞因子的表达，于是本发明继续观察了P65的入核情况，免疫荧光结果显示，EVs-SHP2显著性抑制Aβ1-42处理后P65的入核(图17)。
实施例6 EVs-SHP2对阿尔茨海默病小鼠模型的脑部线粒体自噬诱导作用
(1)将5mg/mL的Aβ1-42注射到小鼠侧脑室构建阿尔茨海默病模型，将Cy5.5标记的EVs-SHP2尾静脉注射到小鼠体内6小时后，观察Cy5.5信号在各个器官的分布情况，结果显示，Cy5.5标记的EVs-SHP2可以靶向Aβ1-42的脑部，并增多脑部SHP2的蛋白表达(图18)。
(2)进一步观察SHP2随着EVs进入脑部后是否会恢复脑部的线粒体自噬。小鼠在5mg/mL的Aβ1-42处理14天，并每两天注射100μg的EVs-SHP2治疗14天，免疫荧光观察脑部自噬小体的标志物LC3B和线粒体标志物Tom20的相互作用，结果显示，经过EVs-SHP2的治疗后，显著性增强二者的共定位，说明线粒体自噬增强(图19)。并且LC3B与Aβ1-42的相互作用说明EVs-SHP2通过促进自噬增强对淀粉样蛋白的清除，有利于阿尔茨海默病的恢复(图20)。
(3)EVs-SHP2通过恢复线粒体自噬缓解了Aβ1-42诱导的脑部ROS的产生及细胞凋亡(图21)，并减少了炎性小体的数量(图22)。通过对脑组织炎性信号通路及炎性细胞因子产生的分析，结果显示，EVs-SHP2显著抑制炎性信号通路的激活及细胞因子表达，如IL-6、TNFα以及IL-1β(图23)。通过免疫组化实验观察EVs-SHP2对脑部小胶质细胞的影响，结果显示，EVs-SHP2改善Aβ1-42处理后导致的小胶质细胞的增多(图24)。
实施例7 EVs-SHP2对阿尔茨海默病小鼠模型的认知和记忆改善作用
(1)EVs-SHP2治疗阿尔茨海默病后，脑部自噬水平增高，加强对淀粉样蛋白的清除，然后用Elisa试剂盒检测脑部Aβ1-42水平，结果显示EVs-SHP2显著性减少Aβ1-42的聚集，ThioS染色结果也证明淀粉样蛋白斑块的减少(图25)。
(2)利用Nissl染色考察EVs-SHP2对损伤神经元细胞的修复作用，结果显示，Aβ1-42处理的小鼠脑部，Nissl染色变浅，说明神经元受损严重，当给与EVs-SHP2治疗后，改善了神经元损伤(图26)。
(3)利用SYP和PSD95抗体进行Western blot评估突触的可塑性，EVs-SHP2治疗后，改善Aβ1-42造成的突触损伤，并恢复乙酰胆碱的产生(图27)。并对脑部有关脂质氧化和抗氧化的指标进行检测，结果显示，EVs-SHP2抑制丙二醛MDA的产生以及促进抗氧化物质超氧化物歧化酶SOD和谷胱甘肽GSH的产生(图28)。
(4)经过EVs-SHP2治疗后，通过行为动物学实验评估对阿尔茨海默并小鼠的认知和记忆改善作用。水迷宫实验显示，EVs-SHP2显著缩短Aβ1-42造成的潜伏期时间的延长(图29)；新物体识别实验显示，EVs-SHP2显著增长小鼠对新物体的探索时间(图30)；以及旷场实验结果均显示，EVs-SHP2改善阿尔茨海默病小鼠的学习认知能力和记忆能力(图31)。
显然，上述实施例仅仅是为清楚地说明所作的举例，并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (6)
1.一种过表达SHP2的基因工程化间充质干细胞外囊泡在制备神经退行性疾病治疗药物中的应用，其特征在于，所述过表达SHP2的基因工程化间充质干细胞外囊泡的制备方法包括以下步骤：
S1、将间充质干细胞在减血清低糖DMEM培养基培养纯化，得到间充质干细胞系；
S2、采用编码PTPN11基因的慢病毒转染步骤S1得到的间充质干细胞系，得到过表达SHP2的间充质干细胞；
S3、提取步骤S2中过表达SHP2的间充质干细胞的细胞外囊泡，得到所述过表达SHP2的基因工程化间充质干细胞外囊泡；所述的提取为将所述过表达SHP2的间充质干细胞在无外泌体的血清低糖DMEM培养基中培养，收集上清，通过超滤法进行粗提，再通过亲和层析法进一步提取。
2.根据权利要求1所述的应用，其特征在于：所述减血清低糖DMEM培养基的葡萄糖含量≤ 1100 mg/L，且相对于血清低糖DMEM培养基配方中减少50-90%的血清用量。
3.根据权利要求1所述的应用，其特征在于：在步骤S3中，所述的超滤法为将上清用0.22 ~ 0.45 μm滤膜过滤，收集过滤后的液体。
4.根据权利要求1所述的应用，其特征在于：在步骤S3中，所述的亲和层析法为采用含CD63抗体的亲和层析介质进行亲和层析。
5.根据权利要求1所述的应用，其特征在于：所述过表达SHP2的基因工程化间充质干细胞外囊泡的粒径为30 ~ 150 nm，电位为0 ~ -20 mV。
6.根据权利要求1所述的应用，其特征在于：所述药物为阿尔茨海默病治疗药物。
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