CN116790490A - 一种胶原结合型的外泌体、外泌体冻干粉及制备方法 - Google Patents
一种胶原结合型的外泌体、外泌体冻干粉及制备方法 Download PDFInfo
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Abstract
本发明涉及外泌体制备技术领域，具体公开了一种胶原结合型的外泌体，外泌体的制备方法包括：构建重组表达载体pHBLV‑CMV‑CD63‑EF1‑ZsGreen‑T2A‑puro，重组表达载体的核苷酸序列包括如SEQ ID NO:1所示；将重组表达载体转染干细胞，使干细胞分泌出具有胶原结合特性的外泌体。本发明制备得到的外泌体能很好地靶向损伤处的胶原，从而能更稳定的发挥作用。本发明还公开了外泌体冻干粉，可常温长期保存和运输，且可有效保留活性，能广泛方便使用于多种损伤修复，使用方便，用途广泛。
Description
技术领域
本发明涉及外泌体制备制备技术领域，具体涉及一种胶原结合型的外泌体、外泌体冻干粉及制备方法。
背景技术
外泌体是一类由细胞主动分泌的脂质双分子层结构囊泡，外泌体中包含大量因子、RNA和DNA等物质，参与细胞间的信息交流，对细胞间的相互作用起到重要桥梁作用，因而应用较为广泛。然而现有外泌体在应用仍然存在靶向性不佳的问题，因此如何使干细胞分泌的外泌体在局部靶向稳定发挥作用至关重要，近年来，随着生物技术的发展，通过基因编辑方式修饰干细胞，并使分泌的外泌体包含目的分子是研究的热点，如公开号为CN115093459A的中国专利公开了一种可在外泌体表面进行轴突靶向修饰的递送复合物及其对外泌体的修饰方法，其靶向轴突的外泌体具有良好的血液稳定性、隐身性、靶向性，可有效识别中枢神经***轴突组织。公开号为CN115177742A的中国专利公开了一种载药脑靶向外泌体的制备方法及其应用，其外泌体来源于小鼠单核巨噬细胞白血病细胞的细胞上清，所述的脑靶向外泌体载药体系能够将药物负载至外泌体内，且无明显生物毒性作用，尾静脉少量给药即可透过血脑屏障进入中枢实现抑制血管紧张素(RAS)的表达。
外泌体在促进伤口愈合方面具有较大潜力，可应用于损伤，在动物模型中已被证明可以促进胶原蛋白的合成以及成纤维细胞和角质形成细胞的增殖和迁移。现有新技术中，外泌体在损伤上的应用不断在研究和改进，如将外泌体掺入凝胶后应用于损伤，由于外泌体递送速率缓慢而稳定，从而比外泌体单次直接给药更为有益。然而外泌体在损伤应用上，同样存在缺乏靶向性、外泌体局部逸散而导致疗效降低的问题，因此，开发一种可靶向结合损伤部位的外泌体对提高外泌体疗效、使外泌体更好的应用于损伤治疗具有很好的研究价值和应用前景。
此外，现有的外泌体在保存与使用过程中，常用的方式为将流动的液态外泌体在-80℃的条件下保存，冷冻保存液中采用二甲基亚砜/甘油进行保护，储存成本较高且不便。并且使用二甲基亚砜/甘油与外泌体制备的混合物容易对机体细胞造成刺激损伤，降低外泌体的生物疗效。因此，本发明旨在开发一种胶原结合型的外泌体及胶原结合型外泌体冻干粉的制备方法，以制备得到一种能在常温下保存、运输且有效保留活性的胶原结合型外泌体冻干粉。
发明内容
本发明所解决的技术问题在于提供一种外泌体，以解决现有技术中干细胞来源的外泌体缺乏靶向性，局部损伤应用过程中容易逸散、疗效不佳的问题。
本发明所解决的技术问题之二在于提供一种外泌体冻干粉，解决现有技术中外泌体缺乏靶向性、疗效不佳，且在保存过程中容易损坏和降解失活的问题。
本发明所解决的技术问题之三在于提供所述的外泌体冻干粉的制备方法。
本发明所解决的技术问题采用以下技术方案来实现：
一种胶原结合型的外泌体，外泌体的制备方法包括：
构建重组表达载体pHBLV-CMV-CD63-EF1-ZsGreen-T2A-puro，重组表达载体的核苷酸序列包括如SEQ ID NO:1所示；
将重组表达载体转染干细胞，使干细胞分泌出具有胶原结合特性的外泌体。
进一步地，所述干细胞优选为人脂肪间充质干细胞或人骨髓间充质干细胞或人脐带间充质干细胞或人脲原性干细胞。本发明的技术方案中，包括但不限于上述优选的干细胞，也可以采用其它的人体干细胞。
进一步地，所述重组表达载体采用慢病毒转染的方式进行转染，慢病毒转染的实施步骤包括：
将不含内毒素的所述重组表达载体转染至淋巴细胞，培养后收集细胞液得到病毒液；
将病毒液与培养后的干细胞混合，将感染成功的干细胞培养至60～70％用于外泌体的提取。
一种外泌体冻干粉，采用如上所述的外泌体为原料、冻干保护剂为辅料制备而成，所述冻干保护剂包括30-40g/L海藻糖、2.0-3.0mM硫酸锰、0.01-0.015mol/L磷酸盐缓冲液，冻干保护剂的pH值为7.35～7.45。
进一步地，所述外泌体与冻干保护剂的质量比为1:1。
进一步地，所述外泌体为外泌体浓缩液，外泌体浓缩液的浓度为400～600ug/ml。
进一步地，所述冻干保护剂包括35g/L海藻糖、2.5mM硫酸锰、0.012mol/L磷酸盐缓冲液，冻干保护剂的pH值为7.4。
所述的外泌体冻干粉的制备方法，将外泌体与干细胞混合，过滤后置于液氮中速冻，速冻5～10min后转移至真空冷冻干燥机中冷冻干燥得到外泌体冻干粉。
有益效果：本发明所述的外泌体，其采用基因编辑技术构建独特的慢病毒载体，并转染干细胞，从而使干细胞分泌出具有胶原结合特性的外泌体，所述的外泌体能靶向地与损伤处的胶原结合，从而更为稳定的发挥作用。
本发明所述的外泌体冻干粉，有效保留活性，可靶向结合于损伤部位，有效促进局部细胞增殖和血管生成，且可常温长期保存和运输，可广泛方便使用于多种损伤修复，使用方便，用途广泛。
本发明所述的外泌体冻干粉制备方法，其采用的冻干保护剂可为外泌体提供稳定存在的微环境，又可以在使用过程中辅助外泌体功能，有效促进局部损伤的血管再生，同时外泌体冻干保护剂呈现弱碱性，可中和损伤局部的弱酸性环境，使损伤局部PH趋向于贴合机体细胞适宜生长增殖的PH值；采用独特的冻干保护剂结合液氮速冻预处理技术，替代了传统外泌体保存液中常用的甘油和二甲亚砜防冰晶形成作用，以及胎牛血清白蛋白对外泌体的蛋白保护作用，避免外泌体冻干粉在使用过程中对机体的刺激和异种蛋白引起的免疫反应，并能通过冻干保护剂形成独特的保护膜，在冻干保护剂各成分的协同作用下减少低温和干燥过程中外泌体的损伤，使外泌体生物膜保持完整，很好的维持外泌体中生物分子的活性。
附图说明
图1是pHBLV-CMV-MCS-EF1-ZsGreen-T2A-puro载体结构示意图；
图2是pHBLV-CMV-CD63-EF1-ZsGreen-T2A-puro载体结构示意图；
图3为CD63-BSP外泌体对比于未转染外泌体(EXO)在体外鼠尾胶原结合缓释效果图；
图4为CD63-BSP外泌体冻干前(CD63-BSP EXO)后(F-CD63-BSP EXO)外泌体电镜对照图；
图5为CD63-BSP外泌体冻干前(CD63-BSP EXO)后(F-CD63-BSP EXO)外泌体电镜粒径对照分析图；
图6为CD63-BSP外泌体冻干粉常温保存与-80℃保存两个月后对脐静脉内皮细胞增殖影响；
图7为本发明所述的外泌体冻干粉与常规方法得到的外泌体冻干粉的对比效果图。
具体实施方式
为了使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解，下面结合具体实施例进一步阐述本发明。
实施例1
本实施例所述的外泌体，其制备方法包括：
(一)pHBLV-CMV-CD63-EF1-ZsGreen-T2A-puro载体构建
1)根据GeneBank中人CD63的mRNA上CDS序列、BSP序列的基因信息，设计合成XbaI-CD63-EF1-ZsGreen-T2A-puro-NotI基因DNA片段(汉恒生物科技(上海)有限公司)。分别用限制性内切酶XbaI和NotI酶切合成的XbaI-CD63-EF1-ZsGreen-T2A-puro-NotI基因的双链DNA分子，酶切体系：Xba I:1μL，Not I:1μL，缓冲液:3μL，合成的DNA:1ug，补充水至30ul，37℃酶切4h，回收酶切产物。
2)用限制性内切酶XbaI和NotI酶对如图1所示
pHBLV-CMV-MCS-EF1-ZsGreen-T2A-puro(汉恒生物科技(上海)有限公司)进行切割，酶切体系：Xba I:1μL，Not I:1μL，缓冲液:3μL，pHBLV-CMV-MCS-EF1-ZsGreen-T2A-puro质粒:1μg，补充水至30μL。37℃酶切4小时，回收载体骨架。
3)将步骤1)中的酶切产物与步骤2)中的载体骨架连接，连接体系为：T4DNA连接酶:1μL，缓冲液:1μL，回收的合成DNA:20ng，回收的质粒:10ng。16℃连接过夜后，转化进大肠杆菌，筛选阳性菌并提取其质粒，得到如图2所示的重组质粒pHBLV-CMV-CD63-EF1-ZsGreen-T2A-puro。重组质粒的测序结果如SEQ ID NO:1所示。
(二)瞬时转染
从液氮中取出293T细胞，放在37℃水浴锅内解冻，离心后用培养基稀释后培养在多聚赖氨酸包被的T25培养皿中，当密度达到60％-70％时，可用于后续的转染实验。
将不含有内毒素的pHBLV-CMV-CD63-EF1-ZsGreen-T2A-puro载体15μg，pLP-110μg，pLP-210μg，pLP-VSVG 5μg转染到293T细胞内，加入DNA-钙磷酸盐混合物，培养10h左右更换培养基，继续培养60h之后，收集细胞培养液，离心后收集上清，用0.45-micro的滤膜过滤，继续离心，弃去上清，用PBS重悬沉淀；将病毒液保存于-80℃。
病毒滴度检测：将HEK 293T细胞接种于24孔板中，转染时，将保存于-80℃冰箱中病毒液37℃水浴融解，用含有体积分数5％胎牛血清FBS的细胞培养基进行10倍梯度稀释，从10-1稀释到10-10。从对应的培养孔吸取培养液并加入经倍数稀释的病毒液,于CO2培养箱孵育48小时，加入完全培养基。4天后，根据Invitrogen公司的TRIZOL操作说明书进行RNA抽提，RNA逆转录后获得cDNA，最后在Bio-Rad的iQ5上进行Real-time定量PCR法检测，获得较高滴度的病毒为1x108TU/ml。
(三)pHBLV-CMV-CD63-EF1-ZsGreen-T2A-puro载体转染干细胞
将保存于-80℃的人骨髓间充质干细胞hBMSC于37℃水浴解冻，培养至60～70％融合度时用于后续转染。除了本实施例采用的hBMSC，还可以采用干细胞(人脂肪间充质干细胞(hADSC)、人脐带间充质干细胞(hUCSC)、人脲原干细胞(hUSC)等。
将保存于-80℃的病毒液37℃水浴解冻。将上述干细胞每孔2×105cells/m L接种于24孔板中，每孔加500μL含10％FBS的对应干细胞专用培养液培养过夜。将5μL病毒液加到500μL含2％FBS的对应干细胞专用培养液中混合均匀，然后换掉原有的培养液，培养过夜后再换回10％FBS的对应干细胞专用培养液，72h观察绿色荧光蛋白表达情况。将成功转染的干细胞保存于-80℃中。
(四)外泌体提取
将转染成功的干细胞(T-hBMSC)培养至60-70％，用于外泌体提取，同批次对应未转染的干细胞作为对照组。干细胞经过无血清培养基处理24h，分离的上清4℃条件下3500×g离心30min，离心后上清经过0.22μm过滤器过滤。过滤上清置于100kd的超滤浓缩离心管中在4℃条件下3500×g离心30min获得初次外泌体浓缩液，填充PBS以200000×g进行1小时超速离心。获得沉淀以大量PBS重悬，通过0.22μm过滤器过滤后，200000×g进行2小时超速离心，获得的沉淀用PBS重悬即为外泌体，冻存于-80℃中。
(五)外泌体的胶原结合性检测
将提取的外泌体解冻融化后与鼠尾胶原混合置于24孔板中，待凝固后，添加PBS置于37℃条件下孵育。每24h吸取PBS进行蛋白含量检测，连续14天检测结果如图3所示，转染干细胞外泌体组PBS中蛋白含量呈现缓慢增加趋势，而对照组的未转染干细胞外泌体呈现爆发式增加，表明转染干细胞外泌体具有良好的胶原结合特性，可靶向结合于胶原并达到缓慢释放的效果。
实施例2
外泌体冻干粉，其制备方法包括：收集实施例1中转染成功干细胞的上清液，离心，离心后的上清液经过0.22μm过滤器过滤。过滤后的上清置于100kd的超滤浓缩离心管中继续离心获得初次外泌体浓缩液，将初次外泌体浓缩液填充PBS后超速离心，取沉淀用量PBS重悬，然后过滤、离心，获得的沉淀继续用PBS重悬得到浓缩外泌体。浓缩外泌体与冻干保护剂以1:1混合均匀，然后0.22μm过滤器过滤于速热导杯中，立即在无菌操作柜中置于液氮中速冻，冷冻5～10min冻结，迅速转移至真空冷冻干燥机中，冷冻干燥24h，获得胶原结合性的促血管生成外泌体冻干粉剂，于室温下避光阴凉处存放。
冻干保护剂优选的包括35g/L海藻糖、2.5mM硫酸锰和0.012mol/L磷酸盐缓冲液，冻干保护剂的PH值为7.4。其中添加的海藻糖，可在外泌体冷冻和干燥的过程中形成一层特殊的保护膜，保护并维持外泌体中生物分子的活性，添加磷酸盐缓冲液，调节混合体系PH维持在7.37～7.45，此PH贴合人体最适宜细胞特别是免疫细胞的生存环境，在此PH下细胞可发挥最大功能效应。且弱碱性的冻干粉剂在使用过程中可中和损伤局部的弱酸性微环境，调节为最适宜人体细胞生长的弱碱性环境，外泌体在使用过程中更快更有效地发挥其生物学效应，快速启动损伤处细胞的生物修复功能并对外泌体的信号分子做出反应至关重要。锰作为人体所需的关键微量元素之一，是含锰超氧化物歧化酶、精氨酸酶和丙酮酸梭化酶等的关键元素，参与机体的三大代谢过程，有效促进细胞增殖、血管再生等过程，在冻干粉制备过程中添加既定含量的硫酸锰，作为冻干过程中的离子平衡保护剂的同时，还有效协同外泌体的生物学效应、提高生物活性，促进细胞增殖、血管再生等。
(一)外泌体冻干前、后的结构变化
外泌体冻干前、后的微观结构如图4所示，外泌体冻干粉冻干前、后的粒径如图5所示。冻干前后微观结构和粒径分布差异不大，说明外泌体冻干前后结构变化不大，外泌体损伤较少。
(二)外泌体冻干前、后对脐静脉内皮细胞(HUVECs)的生物学效应
将实施例1得到的的外泌体在-80℃保存两个月备用，将实施例2的制备方法得到的外泌体冻干粉在常温下保存两个月备用，均按照10ug/ml浓度添加于培养基与HUVECs培养，进行增殖实验，对照组为不添加外泌体培养基，结果如图6所示，表明相较于对照组而言，普通的-80℃保存的外泌体和室温保存外泌体冻干粉均明显促进HUVECs增殖，且CD63-BSP外泌体冻干粉相比普通-80℃保存的外泌体更能有效促进脐静脉内皮细胞增殖，说明本发明得到的外泌体冻干粉能在常温下保存，保存成本更低，更为方便的同时，本发明所述的外泌体冻干粉的生物活性能得到更好的保留，疗效更佳。
(三)采用本发明所述的冻干保护剂及方法制备得到的外泌体冻干粉与采用甘油、二甲亚砜为保护剂、不采用液氮速而在真空冷冻干燥机中直接干燥的外泌体冻干粉对脐静脉内皮细胞(HUVECs)的生物学效应。
分别取出本发明所述的外泌体冻干粉与采用甘油、二甲亚砜为保护剂，得到的外泌体冻干的外泌体冻干粉，按照10ug/ml浓度添加于培养基与HUVECs培养，进行增殖实验，空白对照组为不添加外泌体培养基。结果如图7所示，采用甘油、二甲亚砜为保护剂直接冻干的外泌体冻干粉组的HUVECs增殖甚至低于对照组，细胞增殖受到明显抑制，表明常规的采用甘油、二甲亚砜为保护剂制备的冻干粉剂直接与细胞培养过程中，对细胞有一定的毒性作用。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
Claims (8)
1.一种胶原结合型的外泌体，其特征在于，外泌体的制备方法包括：
构建重组表达载体pHBLV-CMV-CD63-EF1-ZsGreen-T2A-puro，重组表达载体的核苷酸序列包括如SEQ ID NO:1所示；
将重组表达载体转染干细胞，使干细胞分泌出具有胶原结合特性的外泌体。
2.根据权利要求1所述的胶原结合型的外泌体，其特征在于，所述干细胞包括人脂肪间充质干细胞或人骨髓间充质干细胞或人脐带间充质干细胞或人脲原性干细胞。
3.根据权利要求1所述的胶原结合型的外泌体，其特征在于，所述重组表达载体采用慢病毒转染的方式进行转染，慢病毒转染的实施步骤包括：
将不含内毒素的所述重组表达载体转染至淋巴细胞，培养后收集细胞液得到病毒液；
将病毒液与培养后的干细胞混合，将感染成功的干细胞培养至60～70％用于外泌体的提取。
4.一种外泌体冻干粉，其特征在于，采用如权利要求1～3任一所述的外泌体为原料、冻干保护剂为辅料制备而成，所述冻干保护剂包括30-40g/L海藻糖、2.0-3.0mM硫酸锰、0.01-0.015mol/L磷酸盐缓冲液，冻干保护剂的pH值为7.35～7.45。
5.根据权利要求4所述的外泌体冻干粉，其特征在于，所述外泌体与冻干保护剂的质量比为1:1。
6.根据权利要求4所述的外泌体冻干粉，其特征在于，所述外泌体为外泌体浓缩液，外泌体浓缩液的浓度为400～600ug/ml。
7.根据权利要求4所述的外泌体冻干粉，其特征在于，所述冻干保护剂包括35g/L海藻糖、2.5mM硫酸锰、0.012mol/L磷酸盐缓冲液，冻干保护剂的pH值为7.4。
8.如权利要求4所述的外泌体冻干粉的制备方法，其特征在于，将外泌体与干细胞混合，过滤后置于液氮中速冻，速冻5～10min后转移至真空冷冻干燥机中冷冻干燥得到外泌体冻干粉。
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