CN111494417B - 诱导性细胞外囊泡在制备治疗肿瘤药物中的应用 - Google Patents
诱导性细胞外囊泡在制备治疗肿瘤药物中的应用 Download PDFInfo
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Abstract
本发明公开了间充质干细胞来源的诱导性细胞外囊泡在制备治疗肿瘤的药物中的应用。本申请的发明人发现，MSCs产生的IEVs能够在体外促进多发性骨髓瘤细胞系5TGMI细胞的凋亡，而体内注射IEVs能够显著延长多发性骨髓瘤小鼠的寿命，并缓解其骨质丧失等症状，因此，通过优化MSCs来源的IEVs来治疗多发性骨髓瘤将具有良好的应用前景。采用本发明的方法治疗多发性骨髓瘤，疗效显著，而且能显著延长患者寿命，副作用很小。
Description
技术领域
本发明涉及肿瘤治疗技术领域，尤其是一种间充质干细胞来源的诱导性细胞外囊泡的用途。
背景技术
细胞外囊泡(extracellular vesicles,EVs)是细胞分泌的含有蛋白质、核酸及各种细胞因子的纳米级载体。细胞外囊泡可以通过内分泌或旁分泌的方式作用于靶细胞，在细胞间物质传递和信息交流过程中发挥了重要作用。研究发现，细胞外囊泡所介导的信息交流在机体生理或病理过程中发挥了重要的调控作用，涉及到免疫调节、肿瘤生长、血管生成、损伤修复等。目前该领域的研究主要集中在外泌体(exosomes)方向。外泌体是直径在30-150nm左右的细胞外囊泡，其内含有RNA、脂质和蛋白质等成分。外泌体广泛参与了机体的各种生理/病理性调控，能够用作多种疾病的诊断、治疗和预后评估。
迄今为止，间充质干细胞(mesenchymal stem cells，MSCs)被认为是产生外泌体能力最强的细胞。众多的研究发现MSCs来源的外泌体能模拟MSCs的生物学功能，在促进细胞生长和分化，修复组织缺损等方面发挥了重要的调控作用。因此，近年来以MSCs来源的外泌体为基础的细胞囊泡疗法取得了显著的发展。然而，目前以外泌体为基础的细胞囊泡治疗仍然存在诸多问题，主要表现在外泌体的提取和纯化过程复杂，耗时长，对设备和试剂的要求较高，生理性外泌体产量较低等等，这些缺陷都限制了外泌体治疗的临床转化和应用。
细胞凋亡是细胞程序性死亡，其特征包括明显的细胞收缩、染色质浓缩和细胞膜出泡。细胞凋亡产生大量的凋亡小体，含有多种细胞成分。星形孢菌素(staurosporine，STS)能够在体外诱导MSCs的细胞凋亡，在此过程中会产生大量的细胞外囊泡。以往研究主要集中在细胞凋亡本身，对伴随产生的细胞外囊泡缺乏系统和深入的研究。本申请的发明人/课题组的前期研究证实，STS体外诱导产生的细胞外囊泡对MSCs的稳态有重要的调节作用，能够显著改善骨质疏松小鼠的骨质破坏情况。与外泌体相比，这种诱导性细胞外囊泡(induced extracellular vesicles,IEVs)更具有优势，主要体现在：IEVs的产量更加丰富，单个MSC能够产出300-1000个IEVs；IEVs的制备流程简单，耗时短，对试剂和设备的要求低，治疗效果好。因此，以MSCs来源的IEVs为基础的细胞外囊泡疗法具备良好的应用前景。
多发性骨髓瘤(MM，multiple myeloma)是一种血液系统恶性肿瘤，发病率已经超过急性白血病。常见临床表现为骨痛、贫血、肾功能不全、感染。我国骨髓瘤发病率约为1/10万，低于西方工业发达国家(约4/10万)，但有逐年增加的趋势。其发病年龄多见于50-60岁之间的中年和老年，男女比例约为2：1。目前WHO将其归为B细胞淋巴瘤的一种，称为浆细胞骨髓瘤/浆细胞瘤。其特征为骨髓浆细胞异常增生伴有单克隆免疫球蛋白或轻链(M蛋白)过度生成，由于正常免疫球蛋白的生成受抑制，因此容易出现各种细菌性感染。
在造血系统恶性肿瘤中,多发性骨髓瘤迄今仍然不可治愈。传统化疗是治疗多发性骨髓瘤的标准方法，化疗能杀死大多数病患的多发性骨髓瘤细胞，这也是消灭肿瘤的最直接方法之一，但由于血液肿瘤的特殊发病机制，消灭整个血液系统中的癌细胞往往需要大剂量的化疗，对应的副作用十分强烈。此外，在绝大部分病例中，不论时间长短，骨髓瘤细胞最终都会对化疗产生抗药性。由于患病早期没有明显症状，治疗后的多发性骨髓瘤复发率又相当高，因此亟需开发针对多发性骨髓瘤的新的治疗策略。
发明内容
基于上述问题，本发明的目的在于克服上述现有技术的不足之处而提供一种能有效治疗多发性骨髓瘤的方法，其能显著延长患者寿命，副作用很小。
为实现上述目的，本发明采取的技术方案包括以下几个方面：
在一个方面，本发明提供了间充质干细胞来源的诱导性细胞外囊泡在制备治疗肿瘤的药物中的应用。
作为上述方案的进一步优化，所述肿瘤为B细胞淋巴瘤或/和乳腺癌。
作为上述方案的进一步优化，所述间充质干细胞源自口腔颌面部组织、骨髓、脂肪或脐带，或者所述间充质干细胞选自牙髓间充质干细胞、牙龈间充质干细胞和根尖牙乳头间充质干细胞。更优选地，所述间充质干细胞来自小鼠、人、兔或猴；最优选地，所述间充质干细胞来自小鼠或人。
作为上述方案的进一步优化，所述肿瘤为多发性骨髓瘤。
另一个方面，本发明提供了一种治疗肿瘤的药物，所述药物含有间充质干细胞来源的诱导性细胞外囊泡。
作为上述方案的进一步优化，所述肿瘤为B细胞淋巴瘤或/和乳腺癌。
作为上述方案的进一步优化，所述肿瘤为多发性骨髓瘤。
作为上述方案的进一步优化，所述间充质干细胞源自口腔颌面部组织、骨髓、脂肪或脐带，或者所述间充质干细胞选自牙髓间充质干细胞、牙龈间充质干细胞和根尖牙乳头间充质干细胞。
本申请的发明人还发现了一种用于鉴定MSCs来源的IEVs的分子标志物组合，所述分子标志物组合包括Annexin V，Flotillin-1，Cadherin 11(钙粘附蛋白-11)，Integrinalpha 5(整合素α5)和Syntexin 4。由此，所述分子标志物组合可作为区分MSCs来源的IEVs和外泌体(exosomes)的特征性蛋白标志物。
另外，本发明还提出Fas配体作为靶点在制备或筛选治疗多发性骨髓瘤的药物中的应用。
综上所述，本发明的有益效果为：
采用本发明的方法治疗多发性骨髓瘤，疗效显著，而且能显著延长患者寿命，副作用很小；本申请的发明人发现，MSCs产生的IEVs能够在体外促进多发性骨髓瘤细胞系5TGMI细胞的凋亡，而体内注射IEVs能够显著延长多发性骨髓瘤小鼠的寿命，并缓解其骨质丧失等症状，因此，通过优化MSCs来源的IEVs来治疗多发性骨髓瘤将具有良好的应用前景。
附图说明
图1为Kaplan-Meier生存曲线，其中图1A显示，DPSC来源的IEVs注射能显著提升多发性骨髓瘤小鼠存活率，延长其寿命；图1B显示，BMMSC来源的IEVs注射能显著提升多发性骨髓瘤小鼠存活率，延长其寿命，MM为多发性骨髓瘤(multiple myeloma)；
图2为肿瘤实体的照片，其显示IEVs注射能显著减小多发性骨髓瘤小鼠腿骨区域肿瘤的大小；
图3为Tunel染色结果图和细胞存活率统计结果图，其中显示，体外IEVs处理能显著诱导多发性骨髓瘤5TGM1细胞的凋亡，抑制其生存；
图4为Kaplan-Meier生存曲线，其中显示，相较于野生型小鼠MSCs来源的IEVs而言，FasL缺陷小鼠(GLD小鼠)MSCs来源的IEVs未能提升多发性骨髓瘤小鼠的存活率及寿命；
图5为乳腺癌细胞在显微镜下的照片和相应的细胞存活率统计结果图，其中显示，人牙髓间充质干细胞(DPSC)来源的IEVs体外处理能显著抑制和杀伤乳腺癌细胞MCF7，抑制其存活率；
图6为MCF7癌细胞经IEVs处理后在多孔板中的照片，其中显示牙龈间充质干细胞(GMSC)、根尖牙乳头间充质干细胞(SCAP)、骨髓间充质干细胞(BMMSC)来源的IEVs，体外处理能显著抑制和杀伤乳腺癌细胞MCF7，降低其存活率；
图7为Brdu染色后，细胞MCF7在显微镜下的照片，其中显示MSCs来源IEVs体外处理能显著抑制乳腺癌细胞MCF7的增殖；
图8为Tunel染色后，细胞MCF7在显微镜下的照片，其中显示MSCs来源IEVs体外处理能显著诱导乳腺癌细胞MCF7的凋亡。
具体实施方式
为更好的说明本发明的目的、技术方案和优点，下面将结合附图和具体实施例对本发明作进一步说明。如无特别说明，本发明中的实验方法均为常规方法。如无特别说明，本发明中的试剂、材料、细胞或实验动物均可从市场上或其它公开渠道获得。
实施例1 MSCs来源的IEVs的提取和检测
(1)IEVs的提取：
体外培养MSCs(口腔颌面部组织、骨髓、脂肪、脐带来源的MSCs)，细胞汇合80％-100％时，体外采用星形孢菌素、紫杉醇、紫外照射等多种方法诱导处细胞，37℃孵育16-24h，收集细胞上清液，4℃下800g离心10分钟，收取上清液4℃下2000g离心10分钟，再次收集上清液4℃下16000g离心30分钟，所得沉淀物即为IEVs。1ml PBS重悬清洗沉淀，4℃下16000g再次离心30分钟，即得到清洗后的IEVs。使用时，取适量PBS等缓冲液重悬IEVs。
(2)IEVs的检测：
采用流式细胞技术对提取的IEVs进行定量分析；采用透射电镜(TEM)观察，大部分囊泡的直径都在1um以下，200nm左右。
纳米粒子跟踪分析(NTA)结果与透射电镜观察结果相符，1um以下的IEVs颗粒直径平均为244nm。
使用流式细胞技术对IEVs的表面膜蛋白进行分析，MSCs来源的IEVs能够表达和MSCs相似的表面蛋白，即CD29,CD44,CD73,CD166阳性，CD34,CD45阴性。同时，IEVs能够表达细胞外囊泡的普遍性表面蛋白CD9,CD63,CD81和C1q。此外，IEVs的膜表面分布有大量的磷脂酰丝氨酸(Phosphatidylserine，PS)。
利用蛋白DIA定量技术完成MSCs，MSCs-Exosoms(外泌体)，MSCs-IEVs的蛋白组学定量分析。结果显示，170种蛋白在IEVs中特异性高表达，结合差异蛋白的GO富集分析结果，进行western blot验证，发现MSCs-IEVs能特异性高表达Annexin V,Flotillin-1，Cadherin 11，Integrin alpha 5和Syntexin 4。以上5种蛋白有望成为区分MSCs来源的IEVs和exosomes的特征性蛋白标志物。
关于上述诱导性细胞外囊泡(IEVs)的分离和鉴定的具体方法，还可以采用在先申请的一份发明202010066154.6(申请日2020年1月20日，发明名称：一种囊泡及其应用)中提及的分离和鉴定方法。
实施例2 MSCs来源的IEVs治疗多发性骨髓瘤(MM)的应用和机制
(1)利用NOD/SCID(非肥胖糖尿病/重症联合免疫缺陷)小鼠经尾静脉注射多发性骨髓瘤细胞系5TGM1细胞(6×106/10g体重)于200μl PBS中诱导建立小鼠多发性骨髓瘤模型。
实验结果表明，尾静脉注射小鼠骨髓间充质干细胞(mBMMSC)以及人牙髓间充质干细胞(hDPSC)来源的IEVs，可提升多发性骨髓瘤小鼠的存活率，进行Kaplan-Meier生存分析后表明，凋亡体治疗显著延长了小鼠的寿命(参见图1A,B)；同时显著减小了肿瘤的体积(参见图2)。
(2)为了检测IEVs的效果，体外利用MSCs来源的IEVs处理5TGM1多发性骨髓瘤细胞，Tunel染色结果显示，mBMMSC以及hDPSC来源的IEVs能够导致5TGM1细胞凋亡，显著抑制肿瘤细胞生长(参见图3)。
Fas/FasL是介导细胞凋亡的重要信号通路，因此发明人推测IEVs导致的5TGM1多发性骨髓瘤细胞凋亡与其相关。进一步在体内验证发现，相较于野生型小鼠MSCs来源的IEVs而言，FasL缺陷小鼠(GLD小鼠)MSCs来源的IEVs未能提升多发性骨髓瘤小鼠的存活率，延长其寿命(参见图4)，该结果表明FasL在IEVs在多发性骨髓瘤模型治疗中发挥关键作用，提示FasL可以作为多发性骨髓瘤的治疗靶点。
实施例3 MSCs来源的IEVs抑制乳腺癌细胞生长
本申请的发明人还发现，多种组织MSCs，包括人牙髓间充质干细胞(DPSC)，牙龈间充质干细胞(GMSC)、根尖牙乳头间充质干细胞(SCAP)、骨髓间充质干细胞(BMMSC)来源的IEVs，(如实施例1所述，不同细胞来源的IEVs制备方法基本相同)具有抑制乳腺癌细胞MCF7生长的功能。
将MCF7细胞体外培养于六孔板中，使用1×105至1×107个IEVs每孔的剂量，细胞培养液中处理细胞24小时。实验结果显示，IEVs处理能显著降低人乳腺癌细胞系MCF7癌细胞的存活率(参见图5，6)，抑制MCF7癌细胞的增殖(参见图7)，诱导癌细胞凋亡(参见图8)。由此表明MSCs来源IEVs对实体肿瘤如乳腺癌有一定的治疗作用。
最后应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。
Claims (1)
1.间充质干细胞来源的诱导性细胞外囊泡在制备抑制乳腺癌细胞MCF7生长的药物中的应用；
所述间充质干细胞为人牙髓间充质干细胞、牙龈间充质干细胞、根尖牙乳头间充质干细胞或骨髓间充质干细胞；
将所述乳腺癌细胞MCF7体外培养于六孔板中，使用1×106至1×107个所述诱导性细胞外囊泡每孔的剂量。
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