CN112451488A - 一种植物来源类外泌体制品的制备方法和应用 - Google Patents
一种植物来源类外泌体制品的制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种植物来源类外泌体制品的制备方法及其应用,所述方法包括将植物适宜部位匀浆后差速离心,然后超滤获得植物来源类外泌体的浓缩液。本发明方法适用范围广,成本和动力消耗明显降低,并且制备的产品具有更好的标准化程度,产品质量高,并且更易应用。
Description
技术领域
本发明属于生物技术领域,涉及一种植物来源类外泌体制品的制备方法和应用,具体涉及从植物适宜部位提取具有生物活性的类外泌体制品的方法及其应用。
背景技术
外泌体是活生物体细胞分泌的一种具有脂质双层膜结构, 且富含蛋白质、脂质和核酸等内容物的微小膜泡。目前已发现的外泌体存在于多种生物体液中, 包括血液、乳汁、唾液、菌培养液以及植物体内。他们携带和传递重要的信号分子, 是细胞间通讯的重要载体。外泌体有多种分离和纯化方法,比较常用的有差速离心法、密度梯度离心法、超滤法、多聚物沉淀法和免疫磁珠等方式,其中超高速离心配合密度梯度离心是公认的分离方法“金标准”。
近年来,研究人员利用外泌体的分离方法从可食用植物中提取得到类外泌体结构,其形貌、组成和功能与外泌体有一定的相似性。相关实验证明,植物来源类外泌体进入哺乳动物体内后,参与相关信号通路并可完成跨界调控,发挥类似原植物的治疗保健功能。例如葡萄类外泌体能够被肠道巨噬细胞选择性吸收,从而改善葡聚糖硫酸钠(DSS)诱导的小鼠结肠炎;柠檬类外泌体通过特异性靶向肿瘤部位和激活TRAIL介导的细胞凋亡抑制慢性髓细胞白血病肿瘤的生长。可见,植物来源类外泌体具有广阔的开发应用价值,而建立一种适合于工业化生产的类外泌体制备方法是首要任务。
目前,植物类外泌体的提取方式主要通过超高速离心法。例如,在提取生姜类外泌体时,将粗滤过的生姜汁在4℃下以10000 g离心1 h,取上清液重复2-3次除去细胞碎片,再用100000 g离心80 min沉淀得到生姜类外泌体。但是,该法获得的类外泌体纯度低,结构易被破坏,且受样品粘度的影响以及超高速离心机仪器的限制。因此,有必要开发一种简洁、经济,容易保存的植物类外泌体制备技术。
发明内容
有鉴于现有技术的问题,本发明开发了一种新的植物来源类外泌体制品的制备方法。本发明方法包括将植物适宜部位匀浆后差速离心,然后超滤获得植物来源类外泌体的浓缩液。实验结果表明,本发明的方法适用范围广,成本和动力消耗明显降低,并且制备的产品具有更好的标准化程度,产品质量高,并且更易应用。
具体来说,本发明采用了以下技术方案:
一种植物来源类外泌体制品的制备方法,包括以下步骤:
(1)取植物适宜部位,冲洗表面污渍,超纯水润洗后加入适量生理盐水或PBS浸泡,匀浆,于4℃离心20~60 min,收集上清液,离心力为500~1000 g;
(2)取步骤(1)的上清液4℃离心30~120 min,收集上清液,离心力为3000~20000 g;
(3)取步骤(2)的上清液超滤取滤液,滤膜截留分子量10~100kDa;
(4)取步骤(3)的滤液,超滤浓缩,滤膜截留分子量3~10kDa,获得植物来源类外泌体的浓缩液。
优选地,该方法进一步包括以下步骤:
(5)在步骤(4)所得类外泌体浓缩液中加入冻干保护剂和适量水溶解,制得冻干用溶液;
(6)将步骤(5)得到的冻干用溶液分装后进行冻干,得到植物类外泌体冻干粉。
在如上方法中,所述冻干过程优选包括如下步骤:第一预冻过程,即降温至-40~-55℃,保持1~3 h;再进行第二预冻过程,即继续降温至-75~-85℃,保持3~6 h;然后开启真空泵,控制真空度为10~20 Pa,冷凝器温度控制在-50℃~-60℃,搁板温度逐步上升至25℃,冷冻干燥20~40 h。
在以上方法中,所述冻干保护剂包括乳糖、甘露醇、海藻糖、葡萄糖、蔗糖中的一种或几种。
在优选方案中,所述冻干保护剂按质量百分比计,冻干保护剂总量为5%~25%。
本发明还公开了上述植物来源类外泌体制品的应用,所述植物来源类外泌体制品单独或与其他成分联合用于护肤品、药品或药物递送***。
与现有技术相比,本发明的优点:
1、本发明的制备方法适用于新鲜和干燥的各类植物适宜部位类外泌体的制备,适用性广,技术迁移性强。采用低速离心与超滤相结合的制备工艺,仅需要常规离心机和超滤装置,市场供给充足,工业化程度高。与传统的超高速离心方法相比,无需特殊设备,成本及和运营后动力消耗有望显著降低。
2、本发明的制备方法可以克服传统超高速离心法制备类外泌体存在的分散难度大、类外泌体易变形、标准化程度低等问题。
3、通过冻干工艺制备植物来源类外泌体冻干粉,水复溶后还原率高,在保留植物原有活性的基础上,可以长期保存,便于质量控制和实际应用。
附图说明
图1为鱼腥草类外泌体透射电镜图。
图2为鱼腥草类外泌体直接冻干复溶液的透射电镜图。
图3为鱼腥草类外泌体加5%甘露醇冻干后复溶液的透射电镜图。
图4为鱼腥草类外泌体及其冻干粉对金黄色葡萄球菌的抑制结果。
图5为青梅果类外泌体对DSS诱导结肠炎小鼠结肠长度的影响。
图6为青梅果类外泌体治疗结肠炎小鼠的结肠组织学观察。
具体实施方式
发明的目的在于提供一种植物来源类外泌体的制备方法及其应用,发明人选取植物适宜部位匀浆,采用差速离心与超滤工艺结合的方法获得收率高、纯度好的类外泌体,并采用冻干技术制备冻干粉。活性实验表明,植物类外泌体及其冻干粉保留了植物原有的活性功能,且可长期保存。
一种植物来源类外泌体的制备方法,该方法包括如下步骤:
(1)取植物适宜部位,自来水冲洗表面污渍,超纯水润洗后加入适量生理盐水或PBS浸泡,匀浆,于4℃离心20~60 min,收集上清液,离心力为500~1000 g;
(2)取步骤(1)的上清液4℃离心30~120 min,收集上清液,所述离心力为3000-20000g;
(3)取步骤(2)的上清液超滤取滤液,滤膜截留分子量10~100kDa;
(4)取步骤(3)的滤液,超滤浓缩,滤膜截留分子量3~10kDa,所得浓缩液即为植物类外泌体。
进一步还包括以下步骤:
(5)在步骤(4)所得类外泌体中加入冻干保护剂和适量水溶解,制得冻干用溶液。冻干保护剂包括但不限于:乳糖、甘露醇、海藻糖、葡萄糖、蔗糖中的一种或几种。按质量百分比计,冻干保护剂总量为5%~25%。
(6)将步骤(5)得到的冻干用溶液分装后进行第一预冻过程,即降温至-40~-55°C,保持1~3 h;再进行第二预冻过程,即继续降温至-75~-85℃,保持3~6 h;然后开启真空泵,控制真空度为10~20 Pa,冷凝器温度控制在-50℃~-60℃,搁板温度逐步上升至25℃,冷冻干燥20-40 h,得到植物类外泌体冻干粉。
在一种实施方案中,本发明还公开了一种植物来源类外泌体的应用,植物来源类外泌体及其冻干粉的水复溶液可单独或与其他成分联合应用于护肤品。
在另一种实施方案中,本发明还公开了一种植物来源类外泌体的应用,植物来源类外泌体可应用于活性成分的递送,活性成分包括但不限于:疏水性化学药物、亲水性化学药物、生物药物等。
在再一种实施方案中,本发明还公开了一种植物来源类外泌体的应用,植物来源类外泌体及其冻干粉的水复溶液可单独或与其他药物联合应用于药品。
为了使本发明的目的、技术方案以及优点更加清晰明白,以下结合附图和实施例,对本发明进行进一步详细说明。此处所描述的具体实施例,仅用于解释本发明,并不用于限定本发明。
实例1鱼腥草叶片类外泌体的制备
取鱼腥草叶片,自来水冲洗干净,超纯水润洗后加入PBS,匀浆,汁液开展以下步骤:
(1)4℃离心20 min,收集上清液,所述离心力为700 g;
(2)步骤(1)的上清在4℃离心30 min,收集上清液,所述离心力为20000 g;
(3)步骤(2)的上清进行10kDa超滤膜超滤,取滤液;
(4)步骤(3)的滤液进行3kDa超滤膜浓缩,所得浓缩液即为鱼腥草叶片类外泌体(HLNVs)。
取步骤(4)所得类外泌体进行粒径检测,设备为马尔文纳米粒子跟踪仪,平均粒径为187.6±6.4 nm。取步骤(4)的类外泌体进行透射电子显微镜观察,如图1。
实例2经典差速离心法提取鱼腥草叶片类外泌体
取新鲜鱼腥草叶片,自来水冲洗干净,超纯水润洗后加入PBS,匀浆,汁液开展以下步骤:
(1)4℃离心20 min,收集上清液,所述离心力为700 g;
(2)步骤(1)的上清在4℃离心60 min,收集上清液,所述离心力为4000 g;
(3)步骤(2)的上清在4℃离心60 min,收集上清液,所述离心力为20000 g;
(4)步骤(3)的上清在4℃离心120 min,弃去上清,沉淀重悬于1 mL PBS,即为鱼腥草类外泌体,所述离心力为100000 g。
取步骤(4)所得类外泌体进行粒径检测,设备为马尔文纳米粒子跟踪仪,平均粒径为234.3±1.7 nm。
实例3青梅果来源类外泌体的制备
取新鲜青梅果,洗净,切块后加适量预冷PBS匀浆。将新鲜汁液加入1倍体积PBS溶液进行稀释,依次经1000 g和10000 g离心60 min去除杂质,上清经100kDa超滤膜超滤,取滤液,10kDa超滤膜浓缩,即得青梅果来源类外泌体。马尔文纳米粒子跟踪仪测得平均粒径为147.6±4.1 nm。
实例4天冬根部来源类外泌体的制备
取天冬根部,洗净,切块后加适量预冷PBS匀浆。将新鲜汁液加入1倍体积PBS溶液进行稀释,依次经500 g离心30 min,3000 g离心120 min去除杂质,上清经100kDa超滤膜超滤,取滤液,5kDa超滤膜浓缩,即得天冬根部来源类外泌体。马尔文纳米粒子跟踪仪测得平均粒径为153.2±1.8 nm。
实例5鱼腥草叶片来源类外泌体冻干粉的制备
取鱼腥草叶片,自来水冲洗干净,超纯水润洗后加入PBS,匀浆,汁液开展以下步骤:
(1)4℃离心20 min,收集上清液,所述离心力为700 g;
(2)步骤(1)的上清在4℃离心30 min,收集上清液,所述离心力为20000 g;
(4)步骤(3)的上清进行10kDa超滤膜超滤,取滤液;
(5)步骤(4)的滤液进行3kDa超滤膜浓缩,所得浓缩液即为鱼腥草叶片类外泌体。
(6)将步骤(5)得到的类外泌体加入甘露醇使得终浓度为5 %,不加甘露醇的类外泌体原液作为对照组。分装后进行第一预冻过程,即降温至-55℃,保持3 h;再进行第二预冻过程,即继续降温至-80℃,保持6 h;然后开启真空泵,控制真空度为10 Pa,冷凝器温度控制在-50℃,搁板温度逐步上升至25℃,冷冻干燥24 h,得到鱼腥草类外泌体冻干粉。
取步骤(6)冻干粉用PBS复溶制备水分散液,马尔文纳米粒子跟踪仪测得甘露醇组的平均粒径为193.1±2.9 nm,不加甘露醇组的粒径为272.3 ±12.7 nm。取步骤(6)冻干粉的PBS复溶液进行透射电子显微镜观察,如图2和图3。
实例6鱼腥草叶片来源类外泌体及其冻干粉的抗菌活性评价
-80℃冰箱取出金黄色葡萄球菌冻存液(ATCC-29213,南京二院提供),用接种环蘸取少量菌液,在LB固体平板上划线,接着倒置在37℃恒温培养箱内培养24 h复苏。挑取单菌落至LB液体培养基中,于恒温摇床(37℃,200rpm/min)中进行培养16 h。紫外分光光度计于600nm检测金黄色葡萄的生长情况(OD600,OD600越大,金黄色葡萄球菌生长得越好)。接着用LB液体培养基将菌液稀释至3 mL且OD600=0.02,备用。
在96孔板内,分别考察差速离心与超滤结合法提取的鱼腥草叶片类外泌体及其冻干粉末对金黄色葡萄球菌生长的抑制作用。即100 μL上述样品加入100 μL OD600=0.02的细菌悬液置于96孔板内,以各样品与100 μL培养基为对照组,排除各样品自身吸光度的影响。将96孔板放入37℃恒温培养箱中培养16 h。用酶标仪在600 nm下检测其吸光度。如图4所示,差速离心与超滤结合提取的鱼腥草叶类外泌体及其冻干粉末对金黄色葡萄球菌的生长均有抑制作用,冻干操作使得鱼腥草类外泌体的抗菌活性有一定丧失,但加入冻干保护剂甘露醇的鱼腥草类外泌体保持了冻干前的活性。此外,超高速离心法提取的鱼腥草叶类外泌体未表现出明显的抗金黄色葡萄球菌的作用。
实例7含鱼腥草叶片来源类外泌体的水剂浓缩液制备
称取鱼腥草叶片来源类外泌体冻干粉,加入适量生理盐水,混合后过0.3 μm滤膜,按质量分数添加2%甘油后制成鱼腥草叶片来源类外泌体水剂浓缩液,可以添加到喷雾、润肤水、面膜、精华液、啫喱、乳液等产品中,具有消炎、抗菌作用,配合无菌分装工艺可部分或完全替代化学防腐剂。
实例8天冬根部来源类外泌体用于阿霉素的装载
取天冬根部来源类外泌体 1mL与盐酸阿霉素溶液1mg/mL 100μL混合,置于37℃水浴中孵育6 h,超声1 min。葡聚糖凝胶色谱去除游离药物,得载阿霉素的天冬类外泌体制剂。动态光散射仪测得平均粒径158.1±2.5 nm,Zeta电位-22.5 mV,包封率73.8%。
实例9青梅果来源类外泌体对溃疡性结肠炎小鼠的治疗作用
雌性C57BL/6小鼠,6-8周龄,体重20-22 g。将小鼠随机分为正常组、模型组、青梅果来源类外泌体(PMDNs,0.5×10 10/mL和1.5×10 10/mL)和5-氨基水杨酸(5-ASA, 200 mg/kg)组。除正常对照组外,其余组小鼠均自由饮用2.5%葡聚糖硫酸钠(DSS)溶液连续7天,随后换成单蒸水自由饮用3天。开始造模当天起开始灌胃给予青梅果来源类外泌体和5-ASA,每天1次,连续10天,给药体积为0.2 mL/20 g。正常组和模型组小鼠给予等量溶媒。末次给药后1 h,于距离***1 cm处取结肠,测量长度,观察形态变化,见图5。制备组织切片,染色,观察病理变化,见图6。
上面结合附图和具体实例对本发明的实施方式作了详细的说明,但是本发明不限于上述实施方式,在所属技术领域普通技术人员所具备的知识范围内,还可以在不脱离本发明宗旨的前提下做出各种变化。
Claims (6)
1.一种植物来源类外泌体制品的制备方法,包括以下步骤:
(1)取植物适宜部位,冲洗表面污渍,超纯水润洗后加入适量生理盐水或PBS浸泡,匀浆,于4℃离心20~60 min,收集上清液,离心力为500~1000 g;
(2)取步骤(1)的上清液4℃离心30~120 min,收集上清液,离心力为3000~20000 g;
(3)取步骤(2)的上清液超滤取滤液,滤膜截留分子量10~100kDa;
(4)取步骤(3)的滤液,超滤浓缩,滤膜截留分子量3~10kDa,获得植物来源类外泌体的浓缩液。
2.如权利要求1所述的植物来源类外泌体制品的制备方法,进一步包括以下步骤:
(5)在步骤(4)所得类外泌体浓缩液中加入冻干保护剂和适量水溶解,制得冻干用溶液;
(6)将步骤(5)得到的冻干用溶液分装后进行冻干,得到植物类外泌体冻干粉。
3. 如权利要求2所述的植物来源类外泌体制品的制备方法,其中所述冻干过程包括如下步骤:第一预冻过程,即降温至-40~-55℃,保持1~3 h;再进行第二预冻过程,即继续降温至-75~-85℃,保持3~6 h;然后开启真空泵,控制真空度为10~20 Pa,冷凝器温度控制在-50℃~-60℃,搁板温度逐步上升至25℃,冷冻干燥20~40 h。
4.如权利要求2所述的植物来源类外泌体制品的制备方法,其中所述冻干保护剂包括乳糖、甘露醇、海藻糖、葡萄糖、蔗糖中的一种或几种。
5.如权利要求2所述的植物来源类外泌体制品的制备方法,其中所述冻干保护剂按质量百分比计,冻干保护剂总量为5%~25%。
6.如权利要求1至5中任一项所述的植物来源类外泌体制品的应用,所述植物来源类外泌体制品单独或与其他成分联合用于护肤品、药品或药物递送***。
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CN115634254A (zh) * | 2022-10-10 | 2023-01-24 | 重庆生物智能制造研究院 | 一种藠头植物外泌体的制备方法及应用 |
CN115634254B (zh) * | 2022-10-10 | 2024-04-30 | 重庆生物智能制造研究院 | 一种藠头植物外泌体的制备方法及应用 |
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CN115671394A (zh) * | 2022-11-23 | 2023-02-03 | 国纳之星(上海)纳米科技发展有限公司 | 一种负载植物外泌体的可注射型磷酸钙骨水泥的制备及其产品和应用 |
CN115778890A (zh) * | 2022-12-05 | 2023-03-14 | 上海水大技术转移有限公司 | 洋甘菊胞外囊泡在制备化妆品中的应用 |
CN115778890B (zh) * | 2022-12-05 | 2023-07-21 | 上海水大技术转移有限公司 | 洋甘菊胞外囊泡在制备化妆品中的应用 |
WO2024125343A1 (zh) * | 2022-12-16 | 2024-06-20 | 浙江养生堂天然药物研究所有限公司 | 来源于桦树原料的外泌体及其制备方法 |
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