CN112029802A - 一种富含人角质细胞生长因子-2的外泌体制备方法及其应用 - Google Patents
一种富含人角质细胞生长因子-2的外泌体制备方法及其应用 Download PDFInfo
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Abstract
本发明涉及一种富含人角质细胞生长因子‑2的外泌体制备方法及其应用，属于生物医药技术领域。本发明通过利用KGF‑2过表达载体，在干细胞中过表达KGF‑2，获得富含KGF‑2的外泌体。富含KGF‑2的外泌体具有极好的稳定性，外泌体脂质双层膜结构可以有效保护KGF‑2，富含KGF‑2的外泌体可以以冻干粉的形式存在，方便使用，可以通过雾化等方式直接达到肺部迅速发挥药效，局部停留时间长、浓度高，黏膜停留时间短、全身停留时间短，降低全身毒性，能提高生物利用度，具有较高的经济效益。
Description
技术领域
本发明涉及一种富含人角质细胞生长因子-2的外泌体制备方法及其应用，属于生物医药技术领域。
背景技术
人角质细胞生长因子(Keratinocyte growth factor-2，KGF-2)又叫做成纤维细胞生长因子-10(Fibroblast Growth Factor-10，FGF-10)，是1996年发现的一种促进上皮细胞生长的细胞因子。研究表明，KGF-2基因主要表达在胚胎的发育过程之中，以及在特定的成年组织如肺、前列腺。在胚胎发育过程中KGF-2是起始肢芽形成并促进其生长的关键性细胞因子。KGF-2由208个氨基酸组成，N端有39个氨基酸的疏水信号肽，成熟蛋白有169个氨基酸(40-208aa)，理论分子量为19.3kDa，其特异性地结合于靶细胞表面的酪氨酸激酶受体FGFR2IIIb(KGFR)、FGFR1而触发信号传导，从而发挥生理作用，刺激上皮细胞再生、分化和迁移。体外活性研究表明，KGF-2在0.1-5nM(2-100ng/mL)浓度时角质上皮细胞Bal/MK具有显著的促分裂作用，对成纤维细胞NIH3T3无作用。
KGF-2用于治疗和预防由任何因素造成的急性肺损伤/呼吸窘迫综合征，包括特种医学，如高原性肺损伤、军事医学、运动医学和其他疾病致肺损伤/呼吸窘迫综合征。以上疾病可以是亚临床、轻度、中度、重度和危及生命的肺损伤。研究发现KGF-2通过保持肺泡表面活性物质动态平衡，促进细胞增生和迁移修复肺损伤，它在保护气血屏障完整，维持细胞骨架和细胞间连接稳态，促进肺水清除，降低或避免肺泡-毛细血管应激衰竭中有重要作用。但是，KGF-2对酸和热不稳定，阻碍其在临床运用。
外泌体(exosome)，是一种直径大约30-100nm的细胞分泌的微小膜泡，具有脂质双层膜结构，能很好地保护其包被的物质核酸或蛋白。因此，可以利用过表达KGF-2的间充质干细胞，获取大批量富含KGF-2的外泌体，用于肺损伤等治疗。
发明内容
本发明的目的是为解决如何获得KGF-2的外泌体的技术问题。
为达到解决上述问题的目的，本发明所采取的技术方案是提供一种富含人角质细胞生长因子-2的外泌体制备方法；包括以下步骤：
步骤1：构建EGFP和KGF-2的融合表达载体；
步骤2：包装过表达慢病毒；
步骤3：将干细胞培养于干细胞培养基，并用慢病毒转染干细胞；
步骤4：过表达KGF-2的干细胞，置于无血清培养基中培养，收集细胞上清，利用高速离心或者外泌体提取试剂盒的方式，提取外泌体。
优选地，上述步骤3中的干细胞为间充质干细胞。
优选地，上述步骤3中的干细胞包括人绒毛膜间充质干细胞、脐带间充质干细胞和脂肪干细胞。
本发明提供的一种富含人角质细胞生长因子-2的外泌体在治疗和预防急性肺损伤/呼吸窘迫综合征中的应用。
优选地，所述肺损伤包括发生在任何年龄段的亚临床、轻度、中度、重度和危及生命的肺部损伤，急性、亚急性和慢性的肺部损伤，以及呼吸道损伤。
本发明提供的一种富含人角质细胞生长因子-2的外泌体在制备治疗和预防急性肺损伤/呼吸窘迫综合征制剂中的应用。
优选地，所述制剂包括喷雾剂、滴剂和注射剂。
相比现有技术，本发明具有如下有益效果：
本发明提供了一种富含人角质细胞生长因子-2的外泌体制备方法；富含KGF-2的外泌体具有极好的稳定性，外泌体脂质双层膜结构可以有效保护KGF-2，富含KGF-2的外泌体可以以冻干粉的形式存在，方便使用，可以通过雾化等方式直接达到肺部迅速发挥药效，局部停留时间长、浓度高，黏膜停留时间短、全身停留时间短，降低全身毒性，能提高生物利用度，具有高经济效益。
附图说明
图1为PCR获取目的基因片段时，融合PCR方法原理示意图；
图2为融合表达EGFP和KGF-2的外泌体的全内反射荧光显微镜照片，每一个绿色荧光点代表融合表达EGFP和KGF-2的外泌体。
图3为小鼠肺部组织经苏木素伊红HE染色照片，其中A图为对照组肺组织染色照片，表现为正常肺组织；B图为博来霉素损伤组的肺组织染色照片，表现为肺组织明显损伤；C图为外泌体治疗组的肺组织染色照片，表现为肺组织的肺损伤得到显著改善。
具体实施方式
为使本发明更明显易懂，兹以优选实施例，并配合附图作详细说明如下：
本发明提供一种富含人角质细胞生长因子-2的外泌体制备方法；包括以下步骤：
步骤1：构建EGFP和KGF-2的融合表达载体；
步骤2：包装过表达慢病毒；
步骤3：将干细胞培养于干细胞培养基，并用慢病毒转染干细胞；
步骤4：过表达KGF-2的干细胞，置于无血清培养基中培养，收集细胞上清，利用高速离心或者外泌体提取试剂盒的方式，提取外泌体。
上述步骤3中的干细胞为间充质干细胞。干细胞包括人绒毛膜间充质干细胞、脐带间充质干细胞和脂肪干细胞。
本发明提供的一种富含人角质细胞生长因子-2的外泌体在治疗和预防急性肺损伤/呼吸窘迫综合征中的应用。其中肺损伤包括发生在任何年龄段的亚临床、轻度、中度、重度和危及生命的肺部损伤，急性、亚急性和慢性的肺部损伤，以及呼吸道损伤。
本发明提供一种富含人角质细胞生长因子-2的外泌体在制备治疗和预防急性肺损伤/呼吸窘迫综合征制剂中的应用；其中制剂包括喷雾剂、滴剂和注射剂。
本发明的目的在于提供一种富含人角质细胞生长因子-2的外泌体的生产制备方法。为了达到这一目的，本发明提供的技术方案是通过一种过表达KGF-2的间充质干细胞以获取富含KGF-2的外泌体。KGF-2具有生物活性，对来源于内胚层、中胚层和外胚层的细胞，如上皮细胞、真皮细胞、成纤维细胞、内皮细胞，干细胞等具有调节、修复和再生作用。富含KGF-2的外泌体主要用于任何因素造成的肺损伤和/或肺水肿，包括发生在任何年龄段，如成人、儿童等的亚临床、轻度、中度、重度和危及生命的肺部损伤，急性、亚急性和慢性的肺部损伤，特种医学疾病致肺部损伤，如航空、航天、航海、潜水等，特殊地理气候致肺部损伤，如极地、冷环境、热环境等，军事医学疾病致肺部损伤，如核辐射、爆炸与投射物及其他(激光、振动、电磁辐射、声损伤等)和宇宙保障等，运动医学疾病致肺部损伤，如跑步、游泳等，化学中毒、生物危害、人兽接触、特殊作业、灾难意外等致肺部损伤，误吸(如腐蚀性液体或气体)、溺水、感染(如细菌、病毒、真菌、分支杆菌、立克次体、卡氏肺孢子虫等)、炎症、药物和化学品致肺部损伤，创伤，败血症、脏器出血、反复输血、心肺分流、血栓栓塞、全身各器官病变致肺部损伤，弥漫性血管内凝血、血栓性血小板减少性紫癜、溶血尿毒综合症、热射病和其他血管炎症性综合症致肺部损伤，代谢性疾病、酸碱平衡紊乱、尿毒症、脏器移植、肿瘤及并发症致肺部损伤，尤其是肺损伤/急性呼吸窘迫综合征、高原性肺水肿、高氧致肺损伤、化疗药物引起的肺损伤和肺移植后原发性肺功能不全，以及呼吸道损伤。
实施例1
本发明提供一种过表达KGF-2干细胞外泌体的制备方法，包括：干细胞过表达KGF-2、提取细胞培养液上清中的外泌体。所述干细胞包括人绒毛膜间充质干细胞、脐带间充质干细胞及脂肪干细胞。具体包括：
1.干细胞过表达KGF-2。首先构建EGFP和KGF-2(NM_004465.2)的融合表达载体。
1.1.所用材料：(1)pCDH表达载体pCDH-CMV-MCS-EF1-Puro购自上海元敏生物科技有限公司。(2)蛋白纯化仪购自GE公司。(3)实时定量PCR仪购自BioRad公司。(4)质粒抽提试剂盒购自Qiagen公司。(5)BCA蛋白定量试剂盒(PierceTM BCA Protein Assay kit，Pierce#23253)。(6)BamHI和EcoRI限制性内切酶购自NEB公司。(7)荧光显微镜购自Leica公司。
1.2.PCR获取目的基因片段。融合PCR方法说明：(1)引物设计：需要将A片段的3’端引物含有B片段的一部分，B片段的5’端引物含有A片段的一部分，这样分别扩增获得A和B就含有了互补片段。如附图1中第一步中的P2含有一部分B片段序列，P3含有A片段一部分序列；(2)分别PCR扩增A和B片段，25μL体系一次扩增8管含有一管阴性对照，切胶回收这两个片段。(3)以A和B两个片段为模板，进行融合PCR实验。融合PCR分为两步，操作步骤如下：
第一步：(不加引物)
循环参数：95℃预变性5min，94℃变性20s、57℃退火20s、72℃延伸40s进行10个循环，72℃延伸5min。如附图1第二步：(第一步不需要添加引物，因为让A和B片段具有互补片段，可以互为引物扩增)
第二步：10个循环后，取出反应管加入引物。
循环参数：95℃预变性5min，94℃变性20s、60℃退火20s、72℃延伸40s进行10个循环，72℃延伸5min。待反应完毕后，PCR产物测序。
1.3.通过酶切连接入目的载体。分析酶切位点，选择合适的酶切位点进行酶切链接目的序列与载体。
基因名称:人KGF-2 基因长度:627bp
克隆载体:pCDH-CMV-MCS-EF1-Puro 克隆位点:EcoRI/BamHI
克隆载体抗性:Amp 测序反应:2
1.4.测序鉴定。
1.5.包装过表达慢病毒。
1.6.干细胞转染过表达慢病毒。干细胞培养于干细胞培养基，置于37℃、5％CO2培养箱中培养。细胞生长至70％左右，慢病毒转染干细胞，慢病毒感染复数(multiplicity ofinfection，MOI)值为10左右，同时还加入了polybrene(4ug/ml)。转染3天后进行荧光显微镜下观察EGFP表达，提取RNA进行polymerase chain reaction(PCR)验证KGF-2基因过表达，提取蛋白进行western blot验证KGF-2蛋白过表达。
2.提取细胞培养液上清中的外泌体
过表达KGF-2的干细胞，置于无血清培养基中培养24小时，收集细胞上清，利用高速离心或者外泌体提取试剂盒的方式，提取外泌体。
实施例2
本发明使用一种检测外泌体过表达KGF-2的方法。通过本实验室建立的高通量纳米生物芯片液体活检系统(High-throughput Nano-bio Chip Integrated System forLiquid Biopsy,HNCIB)，实现同时一步捕获和识别外泌体表面的膜蛋白，其原理是以包被有外泌体特异性捕获抗体的芯片为载体，原位捕获外泌体，利用全内反射荧光显微镜(Total Internal Reflective Fluorescence Microscopy,TIRFM)观察融合表达EGFP和KGF-2的外泌体的绿色荧光信号。如附图2为融合表达EGFP和KGF-2的外泌体的全内反射荧光显微镜照片，每一个绿色荧光点代表融合表达EGFP和KGF-2的外泌体。
实施例3
本发明提供一种利用过表达KGF-2干细胞外泌体治疗肺损伤的方法，包括：小鼠肺损伤模型制备、过表达KGF-2外泌体给药、肺损伤指标检测。肺损伤包括发生特种医学疾病致肺部损伤，如航空、航天、航海、潜水等，特殊地理气候致肺部损伤，如极地、冷环境、热环境等，军事医学疾病致肺部损伤，如核辐射、爆炸与投射物及其他(激光、振动、电磁辐射、声损伤等)和宇宙保障等，运动医学疾病致肺部损伤，如跑步、游泳等，化学中毒、生物危害、人兽接触、特殊作业、灾难意外等致肺部损伤，误吸(如腐蚀性液体或气体)、溺水、感染(如细菌、病毒、真菌、分支杆菌、立克次体、卡氏肺孢子虫等)、炎症、药物和化学品致肺部损伤，创伤，败血症、脏器出血、反复输血、心肺分流、血栓栓塞、全身各器官病变致肺部损伤，弥漫性血管内凝血、血栓性血小板减少性紫癜、溶血尿毒综合症、热射病和其他血管炎症性综合症致肺部损伤，代谢性疾病、酸碱平衡紊乱、尿毒症、脏器移植、肿瘤及并发症致肺部损伤。
具体包括：
1.小鼠肺损伤模型制备：
成年雄性C57BL/6野生型小鼠，饲养至8-10周，体重为23克左右进行气管内滴注博来霉素建立急性肺损伤小鼠模型。使用腹腔注射戊巴比妥钠麻醉小鼠，行气管插管，将博来霉素用PBS液配成1.25mg/ml溶液，按2.0ul/g分别气管内滴注PBS或博来霉素溶液，分别制作对照组和博来霉素肺损伤组。
2.过表达KGF-2外泌体给药：
小鼠肺损伤模型造模后24小时，进行气管内滴注过表达KGF-2外泌体治疗小鼠肺损伤。使用腹腔注射戊巴比妥钠麻醉小鼠，行气管插管，将外泌体用PBS液配成0.2mg/ml溶液，按2.0ul/g分别气管内滴注PBS或外泌体溶液。
3.肺损伤指标检测：
外泌体给药24小时后，小鼠腹腔内注射戊巴比妥钠麻醉后，放血致死，取部分肺组织，用4％福尔马林固定，行苏木素伊红HE染色，病理检查：局部的肺泡膜增厚、毛细血管内淤血、肺泡内出血、间质内中性粒细胞浸润、肺泡内中性粒细胞浸润。如附图3为小鼠肺部组织经苏木素伊红HE染色照片，其中A图为对照组肺组织染色照片，表现为正常肺组织；B图为博来霉素损伤组的肺组织染色照片，表现为肺组织明显损伤；C图为外泌体治疗组的肺组织染色照片，表现为肺组织的肺损伤得到显著改善。
以上所述，仅为本发明的较佳实施例，并非对本发明任何形式上和实质上的限制，应当指出，对于本技术领域的普通技术人员，在不脱离本发明的前提下，还将可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。凡熟悉本专业的技术人员，在不脱离本发明的精神和范围的情况下，当可利用以上所揭示的技术内容而做出的些许更动、修饰与演变的等同变化，均为本发明的等效实施例；同时，凡依据本发明的实质技术对上述实施例所作的任何等同变化的更动、修饰与演变，均仍属于本发明的技术方案的范围内。
Claims (7)
1.一种富含人角质细胞生长因子-2的外泌体制备方法；其特征在于：包括以下步骤：
步骤1：构建EGFP和KGF-2的融合表达载体；
步骤2：包装过表达慢病毒；
步骤3：将干细胞培养于干细胞培养基，并用慢病毒转染干细胞；
步骤4：过表达KGF-2的干细胞，置于无血清培养基中培养，收集细胞上清，利用高速离心或者外泌体提取试剂盒的方式，提取外泌体。
2.如权利要求1所述的一种富含人角质细胞生长因子-2的外泌体制备方法，其特征在于：所述步骤3中的干细胞为间充质干细胞。
3.如权利要求1所述的一种富含人角质细胞生长因子-2的外泌体制备方法，其特征在于：所述步骤3中的干细胞包括人绒毛膜间充质干细胞、脐带间充质干细胞和脂肪干细胞。
4.一种富含人角质细胞生长因子-2的外泌体在治疗和预防急性肺损伤/呼吸窘迫综合征中的应用。
5.如权利要求4所述的一种富含人角质细胞生长因子-2的外泌体在治疗和预防急性肺损伤/呼吸窘迫综合征中的应用，其特征在于：所述肺损伤包括发生在任何年龄段的亚临床、轻度、中度、重度和危及生命的肺部损伤，急性、亚急性和慢性的肺部损伤，以及呼吸道损伤。
6.一种富含人角质细胞生长因子-2的外泌体在制备治疗和预防急性肺损伤/呼吸窘迫综合征制剂中的应用。
7.如权利要求6所述的一种富含人角质细胞生长因子-2的外泌体在制备治疗和预防急性肺损伤/呼吸窘迫综合征制剂中的应用,其特征在于：所述制剂包括喷雾剂、滴剂和注射剂。
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