CN115386543A - 一种鹿茸干细胞外泌体及其制备方法和应用 - Google Patents
一种鹿茸干细胞外泌体及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种鹿茸干细胞外泌体及其制备方法和应用,属于生物技术领域;具体步骤为:S1采集鹿茸间充质组织,分离鹿茸干细胞;S2将所述鹿茸干细胞于完全培养基中进行培养,待细胞密度生长至65‑80%时,替换无血清培养基继续培养,其后收集培养上清液,并过滤;S3将过滤后的上清液进行离心,离心后所得沉淀即为所述鹿茸干细胞外泌体;该鹿茸干细胞外泌体对酒精性肝损伤具有一定的预防性保护以及保护作用,能显著降低肝细胞病变程度,以及肝细胞坏死程度;可增强机体对氧自由基的清除,有效改善酒精性肝损伤小鼠脂质过氧化;同时还对肝脏组织的氧化损伤具有一定的保护作用,且能提高肝脏的抗氧化能力,降低脂质过氧化水平,有效预防氧化应激的发生。
Description
技术领域
本发明涉及生物技术领域,特别是一种鹿茸干细胞外泌体及其制备方法和应用。
背景技术
外泌体是指包含了复杂RNA和蛋白质的小膜泡(30-150nm),现今,其特指直径在40-100nm的盘状囊泡。1983年,外泌体首次于绵羊网织红细胞中被发现,1987年Johnstone将其命名为“exosome”。多种细胞在正常及病理状态下均可分泌外泌体。其主要来源于细胞内溶酶体微粒内陷形成的多囊泡体,经多囊泡体外膜与细胞膜融合后释放到胞外基质中。
干细胞来源的外泌体可通过囊泡,将干细胞的精华部分——mRNA、miRNA、IncRNA及蛋白质等生物活性物质,打包运出干细胞体外。通过“细胞间高速公路”来“快递”到人体各个组织内。干细胞外泌体可以通过改变细胞外基质,改变受体细胞的转录组和蛋白质组,来调节细胞凋亡、生长、增殖和分化途径。因此,干细胞外泌体具有减少细胞凋亡、减轻炎症反应、促进血管生成、抑制纤维化、提高组织修复潜力等重要生物学功能,在调控组织再生方面存在良好的临床应用前景。
鹿茸是唯一可以完全再生的哺乳动物器官,与目前所常用的骨髓间充质干细胞、脐带间充质干细胞等干细胞相比,鹿茸干细胞更易于获取,增殖速度快,分泌大量的细胞因子和生物活性因子,因此,鹿茸干细胞外泌体容易获得。现有技术中,还没有有关鹿茸干细胞外泌体在治疗和预防肝损伤方面中的相关报道。
发明内容
本发明的目的在于提供一种鹿茸干细胞外泌体及其制备方法和应用;
通过将鹿茸间充质组织中的鹿茸干细胞进行分离,于完全培养基中进行培养一段时间后,替换无血清培养基继续培养,收集培养上清液,并过滤、离心,获得鹿茸干细胞外泌体,该鹿茸干细胞外泌体在治疗和预防肝损伤方面发挥极为显著的作用,且效果好于骨髓间充质干细胞外泌体。
本发明的目的是通过以下技术方案来实现的:
一种鹿茸干细胞外泌体的制备方法,包括如下步骤:
S1采集鹿茸间充质组织,分离鹿茸干细胞;
S2将所述鹿茸干细胞于完全培养基中进行培养,待细胞密度生长至65-80%时,替换无血清培养基继续培养,其后收集培养上清液,并过滤;
S3将过滤后的上清液进行离心,离心后所得沉淀即为所述鹿茸干细胞外泌体。
本发明先从鹿茸间充质组织中分离鹿茸干细胞,其后将鹿茸干细胞依次在完全培养基、无血清培养基中进行培养,培养结束后收集培养上清液并过滤上清液中的细胞碎片,最后经离心后得到鹿茸干细胞;本发明制备的鹿茸干细胞在治疗和预防肝损伤方面具有极佳的效果。
优选的,所述完全培养基中加入10%胎牛血清。通过加入该浓度下的胎牛血清,能够使其内的各种血浆蛋白、多肽、脂肪、碳水化合物、生长因子、激素、无机物等最大程度促进鹿茸干细胞生长。
优选的,所述步骤S2中,用无血清培养基培养时间为70-75h。
优选的,所述步骤S2中,用无血清培养基培养时间为72h。通过进一步限定培养时间,能够提高外泌体的总蛋白以及制备产量,同时提供制备效率。
优选的,所述步骤S2中的过滤采用梯度过滤法,具体为:将所述上清液依次依次通过孔径为1-8μm、300-500nm和180-250nm的滤膜进行过滤。通过采用梯度过滤法,能够将上清液中的细胞碎片去除更加干净,同时去除细胞释放的微囊泡和凋亡小体,进而提高外泌体的纯度,进而提高其在预防和治疗肝损伤方面的效果;同时,采用梯度过滤法能够提高分级过滤,提高过滤效率,避免采用一次过滤导致滤膜堵塞的缺陷。
优选的,所述步骤S3中的离心的条件为4-20℃,100000-1200 000g,4-6h。通过限定离心条件,能够提高外泌体的分离速率以及成品质量。
基于上述方法制备得到的鹿茸干细胞,本发明还提供了一种鹿茸干细胞外泌体在制备治疗和预防肝损伤药物中的应用。
本发明的有益效果是:
1.本发明选择增殖速度极快的鹿茸干细胞作为培养对象,能够加快鹿茸干细胞外泌体的获取,从而提高鹿茸干细胞外泌体的产量;通过优化制备条件,能够使制备的鹿茸干细胞外泌体纯度更高,使其在预防和治疗酒精性肝损伤方面发挥更为显著的效果;经实验,证明其在预防和治疗酒精性肝损伤方面的作用远胜于骨髓间充质干细胞外泌体,进而说明不同干细胞外泌体在预防和治疗酒精性肝损伤方面的作用效果均不同,而鹿茸干细胞外泌体在治疗酒精性肝损伤方面的作用更为显著。
2.本发明制备的鹿茸干细胞外泌体对酒精性肝损伤具有一定的预防性保护以及保护作用,能显著降低肝细胞病变程度,以及肝细胞坏死程度;可增强机体对氧自由基的清除,有效改善酒精性肝损伤小鼠脂质过氧化;同时还对肝脏组织的氧化损伤具有一定的保护作用,且能提高肝脏的抗氧化能力,降低脂质过氧化水平,有效预防氧化应激的发生。
附图说明:
图1:小鼠体重及肝脏指数比较图;
其中:1a为小鼠体重;1b为肝指数(****P<0.0001);
图2:肝脏病理组织切片观察图;
图3:血清指标AST、ALT、MDA、GSH-Px、SOD及肝组织中MDA、GSH-Px、SOD的测定比较图;
其中:3a为实验鼠血清ALT值(****P<0.0001,*P<0.05);3b为实验鼠血清AST值(****P<0.0001,**P<0.01);3c为实验鼠肝血清MDA值(****P<0.0001,***P<0.001,**P<0.01);3d为实验鼠肝血清GSH-PX值(****P<0.0001,***P<0.001,*P<0.05);3e为实验鼠肝血清SOD值(****P<0.0001,**P<0.01);3f为实验鼠组织MDA值(****P<0.0001,**P<0.01,*P<0.05);3g为实验鼠组织GSH-PX值(****P<0.0001,***P<0.001,*P<0.05);3h为实验鼠组织SOD值(****P<0.0001,**P<0.01,*P<0.05)。
具体实施方式
下面结合实施例进一步详细描述本发明的技术方案,但本发明的保护范围不局限于以下。
实施例1
S1采集鹿茸间充质组织,分离鹿茸干细胞;
S2将所述鹿茸干细胞于含有10%胎牛血清的完全培养基中进行培养,待细胞密度生长至65%时,替换无血清培养基继续培养70h,其后收集培养上清液,并将所述上清液依次经孔径为8μm、500nm和200nm的滤膜进行过滤;
S3将过滤后的上清液在4℃,1200 000g下离心4h,离心后所得沉淀即为所述鹿茸干细胞外泌体。
实施例2
S1采集鹿茸间充质组织,分离鹿茸干细胞;
S2将所述鹿茸干细胞于含有10%胎牛血清的完全培养基中进行培养,待细胞密度生长至80%时,替换无血清培养基继续培养72h,其后收集培养上清液,并将所述上清液依次经孔径为1μm、300nm和180nm的滤膜进行过滤;
S3将过滤后的上清液在4℃,1000 000g下离心5h,离心后所得沉淀即为所述鹿茸干细胞外泌体。
实施例3
S1采集鹿茸间充质组织,分离鹿茸干细胞;
S2将所述鹿茸干细胞于含有10%胎牛血清的完全培养基中进行培养,待细胞密度生长至80%时,替换无血清培养基继续培养75h,其后收集培养上清液,并将所述上清液依次经孔径为4μm、350nm和250nm的滤膜进行过滤;
S3将过滤后的上清液在10℃,1000 000g下离心6h,离心后所得沉淀即为所述鹿茸干细胞外泌体。
实验例
1.动物分组
将40只C57BL/6小鼠随机分为四组,分别为CTRL组、PBS组、BMSC-exos组和AnSC-exos组。
2.模型制备
(1)CTRL组:小鼠尾静脉注射PBS 7天,第8天注射PBS后2小时用PBS灌胃;灌胃后饲养24小时后取眼球血离心收取上清,处死小鼠并取出肝组织;
(2)PBS组:小鼠尾静脉注射PBS 7天,第8天注射PBS后2小时用50%的酒精灌胃,灌胃后饲养24小时后取眼球血离心收取上清,处死小鼠并取出肝组织;
(3)BMSC-exos组:小鼠尾静脉注射骨髓干细胞外泌体7天,第8天注射小鼠骨髓干细胞外泌体后2小时用50%的酒精灌胃,灌胃后饲养24小时后取眼球血离心收取上清,处死小鼠并取出肝组织;
(4)AnSC-exos组:小鼠尾静脉注射鹿茸干细胞外泌体(选自实施例2中的鹿茸干细胞外泌体)7天,第8天注射鹿茸干细胞外泌体后2小时用50%的酒精灌胃,灌胃后饲养24小时后取眼球血离心收取上清,处死小鼠并取出肝组织。
3.具体检测实验
(1)小鼠称重、眼球取血、肝脏取样及肝指数的计算
在灌胃24h后,需先记录小鼠体重,其后眼球取血,血样放血样室室温静置备用;眼球取血后将小鼠进行麻醉,小鼠平躺在操作台上,腹部消毒,剪开腹部找到肝脏,将肝脏整叶取下并用生理盐水洗去肝脏表面残血,滤纸吸干表面水分,肝脏称重并计算肝脏指数。其中,肝脏指数=肝脏质量(g)/小鼠体重(g)×100%。
小鼠的体重及肝脏指数如图1所示。
由图1a和1b表明:与CTRL组比较,PBS组肝脏指数略有升高,说明酒精能够引起肝脏组织的损伤;与PBS组比较,AnSC-exos和BMSC-exos组肝脏指数均显著降低,其中AnSC-exos组下降程度更大。
通过图1可知:鹿茸干细胞外泌体对酒精性肝损伤具有一定的预防性保护作用。
(2)肝脏病理组织切片观察
将取出的小鼠肝脏组织置于10%的甲醛溶液中固定,常规脱水、透明、石蜡包埋、切片、HE染色,进行肝脏病理组织学检查。酒精性肝损伤受试小鼠肝脏的病理变化以肝细胞脂肪变性为主。
根据实验动物肝组织细胞出现不同的病理改变分为四级:一级肝小叶结构正常,肝细胞索、肝窦和肝细胞均正常;二级肝小叶结构较正常,肝细胞轻度水肿,有点状坏死,可见少量炎性细胞浸润;三级肝小叶结构变形,肝细胞中度水肿,细胞核固缩溶解,肝细胞脂肪变性和灶性坏死,可见多个炎性细胞浸润明显;四级肝小叶结构破坏,肝细胞重度水肿,可见大面积脂肪空泡和灶性坏死,细胞核固缩溶解,大量炎性细胞浸润。
病理切片结果如图2所示:CTRL组小鼠肝小叶结构清楚,肝细胞索呈放射状排列,肝窦和肝细胞均未见异常病理改变;PBS组小鼠肝小叶结构破坏,大多数肝细胞肿胀,大量的肝细胞脂肪变性和坏死,并出现炎细胞浸润现象;与PBS组比较,BMSC-exos组小鼠肝细胞病变程度变轻,肝细胞肿胀及脂肪变性程度略有降低,肝细胞坏死程度相对较轻,AnSC-exos组小鼠肝细胞病变程度较轻,肝细胞肿胀及脂肪变性不明显,肝细胞坏死程度明显减轻,且AnSC-exos组效果明显好于BMSC-exos组。
通过肝脏病理组织切片观察实验可知:鹿茸干细胞外泌体能显著降低小鼠肝细胞病变程度,以及肝细胞坏死程度。
(3)血清指标AST、ALT、MDA、GSH-PX、SOD及肝组织中MDA、GSH-PX、SOD的测定
将从小鼠眼球取出的血样室温静置2h,并以3000r/min离心10min,其后取血清,并按照试剂盒的说明分别测定小鼠血清中AST、ALT、MDA、GSH-PX、SOD的含量,结果如图3a-3e所示。
称取肝脏相同部位的肝组织0.2g,加入9倍量生理盐水制成10%肝组织匀浆,以3000r/min离心10min,取上清液,并按试剂盒说明检测MDA、GSH-PX、SOD,结果如图3f-3h所示。
由图3a、3b表明:与CTRL组比较,PBS组AST、ALT水平显著升高(P<0.0001),说明酒精灌胃致小鼠急性肝损伤造模成功;与PBS组比较,BMSC-exos组和AnSC-exos组均降低了AST、ALT水平(P<0.01),但AnSC-exos组效果显著,表明鹿茸干细胞外泌体对酒精性肝损伤具有一定的保护作用。
由图3c和3f表明:造模后PBS组小鼠肝脏和血液MDA水平高于CTRL组,并呈显著性差异(P<0.0001),BMSC-exos组和ANSC-exos组小鼠的肝脏和血液MDA含量显著降低,其中ANSC-exos组肝脏MDA含量与空白对照组相当,说明鹿茸干细胞外泌体可增强机体对氧自由基的清除,有效改善酒精性肝损伤小鼠脂质过氧化。
由图3d、3e、3g和3h表明:与CTRL组相比,PBS组小鼠肝脏和血液GSH-PX、SOD水平均显著降低(P<0.0001),BMSC-exos组和ANSC-exos组小鼠的肝脏和血液GSH-PX、SOD含量均增高,其中给予ANSC-exos组肝脏GSH-PX、SOD含量与空白对照组相当,说明鹿茸干细胞外泌体对肝脏组织的氧化损伤具有一定的保护作用,且能提高肝脏的抗氧化能力,降低脂质过氧化水平,有效预防氧化应激的发生。
以上仅是本发明的优选实施方式,应当理解本发明并非局限于本文所披露的形式,不应看作是对其他实施例的排除,而可用于各种其他组合、修改和环境,并能够在本文构想范围内,通过上述教导或相关领域的技术或知识进行改动。而本领域人员所进行的改动和变化不脱离本发明的精神和范围,则都应在本发明所附权利要求的保护范围内。
Claims (8)
1.一种鹿茸干细胞外泌体的制备方法,其特征在于,包括如下步骤:
S1采集鹿茸间充质组织,分离鹿茸干细胞;
S2将所述鹿茸干细胞于完全培养基中进行培养,待细胞密度生长至65-80%时,替换无血清培养基继续培养,其后收集培养上清液,并过滤;
S3将过滤后的上清液进行离心,离心后所得沉淀即为所述鹿茸干细胞外泌体。
2.根据权利要求1所述的一种鹿茸干细胞外泌体的制备方法,其特征在于,所述完全培养基中加入10%胎牛血清。
3.根据权利要求1所述的一种鹿茸干细胞外泌体的制备方法,其特征在于,所述步骤S2中,用无血清培养基培养时间为70-75h。
4.根据权利要求1所述的一种鹿茸干细胞外泌体的制备方法,其特征在于,所述步骤S2中,用无血清培养基培养时间为72h。
5.根据权利要求1所述的一种鹿茸干细胞外泌体的制备方法,其特征在于,所述步骤S2中的过滤采用梯度过滤法,具体为:将所述上清液依次依次通过孔径为1-8μm、300-500nm和180-250nm的滤膜进行过滤。
6.根据权利要求1所述的一种鹿茸干细胞外泌体的制备方法,其特征在于,所述步骤S3中的离心的条件为4-20℃,100000-1200 000g,4-6h。
7.如权利要求1-6任一项所述的方法制备的鹿茸干细胞外泌体在制备治疗和预防肝损伤药物中的应用。
8.根据权利要求7所述的鹿茸干细胞外泌体在制备治疗和预防肝损伤药物中的应用,其特征在于,所述肝损伤为急性酒精肝损伤。
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