CN111135190A - 一种牙龈间充质干细胞外泌体制剂及其应用 - Google Patents
一种牙龈间充质干细胞外泌体制剂及其应用 Download PDFInfo
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Abstract
本发明公开了一种牙龈间充质干细胞外泌体制剂及其应用,属于生物技术领域。本发明公开了牙龈间充质干细胞来源的外泌体在制备治疗银屑病药物中的应用。本发明的牙龈间充质干细胞外泌体制剂可局部涂于银屑病病变部位,可缓解或抑制病变部位炎症细胞浸润和炎症因子表达,从而达到疾病的治疗和病情缓解作用;本发明的外泌体制剂还可作为银屑病药物添加剂或联合用药制剂用于银屑病治疗。
Description
技术领域
本发明涉及生物技术领域,更具体的说是涉及一种牙龈间充质干细胞外泌体制剂及其应用。
背景技术
牙龈间充质干细胞(Gingival mesenchymal stem cells,GMSC)是一种从人体牙龈组织中分离出来的具有干细胞特性的前体细胞,其具有自我更新、多向分化及免疫调节的功能。尤为重要的是,牙龈间充质干细胞能够分泌一种直径约为30~150nm的胞外囊泡,常被称为外泌体(Exosome)。外泌体广泛存在于动植物细胞,但其功能与细胞类型及种类密切相关。其核心作用主要包括细胞间通讯、组织修复与再生、免疫调节、机体发育、疾病诊断等方面。目前,牙龈间充质干细胞已被证实在以小鼠为模型的自身免疫性疾病、神经性疾病及神经修复中具有一定的作用,包括糖尿病性创伤、诱导性关节炎、接触性皮炎、炎性肠病等。然而,牙龈间充质干细胞及牙龈间充质干细胞外泌体在银屑病中作用国内外均未见报道。
银屑病是一种慢性免疫介导的炎性红斑鳞状皮肤病,典型的临床表现为皮损处红色斑块或斑块上覆盖银白色鳞屑,病理特征以角化细胞过度增殖、异常分化、血管形成和炎性细胞浸润为主,影响了全球约3%的人口。目前银屑病的治疗均只能缓解病情且易复发,无法彻底痊愈。因此,开发银屑病治疗药物成为当前的研究热点。基于此,提供一种牙龈间充质干细胞外泌体制剂及其应用是本领域技术人员亟需解决的问题。
发明内容
有鉴于此,本发明提供了一种牙龈间充质干细胞外泌体制剂,并评价了其在治疗银屑病中的应用。
为了实现上述目的,本发明采用如下技术方案:
一种牙龈间充质干细胞外泌体制剂,由牙龈间充质干细胞来源的外泌体制备得到。
进一步,一种牙龈间充质干细胞外泌体制剂的制备方法,具体步骤如下:
选择生长状态良好的从牙龈组织分离的牙龈间充质干细胞接种到细胞培养瓶,待细胞融合度至90%时,且流式细胞术检测细胞表型为CD73、CD90、CD105、CD44和CD29阳性表达,CD14、CD19、CD45和CD34阴性表达后,采用无外泌体血清条件培养基,连续培养48-72h后收集细胞培养上清,300g离心10min,取上清,去除细胞;4℃2000g条件下离心10min,取上清,去除死亡细胞;4℃10000g离心30min,取上清,去除细胞碎片;用0.22μm过滤器过滤上清后,4℃100000g离心70min,弃上清,保留沉淀,加入10mL PBS重悬;4℃100000g离心70min,弃上清,保留沉淀,沉淀即为牙龈间充质干细胞来源的外泌体,用100μL磷酸缓冲盐溶液(phosphate buffer saline,PBS)重悬,-80℃冰箱保存待用。
外泌体液体制剂主要成分包括牙龈间充质干细胞外泌体和PBS,其制剂中牙龈间充质干细胞外泌体含量为5×107颗粒/每毫升PBS。
其中10×PBS配方如下:
NaCl 80g,Na2HPO4·12H2O 32.3g,NaH2PO4·2H2O 4.5g,加双蒸水900mL左右,用HCl/NaOH调pH至7.4,最后定容为1000mL。
进一步,牙龈间充质干细胞来源的外泌体在制备治疗银屑病药物中的应用。
经由上述的技术方案获得的牙龈间充质干细胞外泌体制剂,与现有技术相比,本发明公开提供了牙龈间充质干细胞外泌体制剂在治疗银屑病中的应用。牙龈间充质干细胞外泌体制剂可局部涂于银屑病病变部位,可缓解或抑制病变部位炎症细胞浸润和炎症因子表达,从而达到疾病的治疗和病情缓解作用。此外,本发明的外泌体制剂还可作为银屑病药物添加剂或联合用药制剂用于银屑病治疗。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据提供的附图获得其他的附图。
图1附图为本发明原代牙龈间充质干细胞在第一天,第三天,第五天和第七天的镜下形态(比例尺为100nm);
图2附图为本发明流式细胞术检测牙龈间充质干细胞表面标志物CD14的结果图;
图3附图为本发明流式细胞术检测牙龈间充质干细胞表面标志物CD34的结果图;
图4附图为本发明流式细胞术检测牙龈间充质干细胞表面标志物CD45的结果图;
图5附图为本发明流式细胞术检测牙龈间充质干细胞表面标志物CD73的结果图;
图6附图为本发明流式细胞术检测牙龈间充质干细胞表面标志物CD105的结果图;
图7附图为本发明流式细胞术检测牙龈间充质干细胞表面标志物CD44的结果图;
图8附图为本发明流式细胞术检测牙龈间充质干细胞表面标志物CD90的结果图;
图9附图为本发明流式细胞术检测牙龈间充质干细胞表面标志物CD29的结果图;
图10附图为本发明流式细胞术检测牙龈间充质干细胞表面标志物CD19的结果图;
图11附图为本发明牙龈间充质干细胞成骨诱导四周后茜素红染色镜下形态;
图12附图为本发明牙龈间充质干细胞成脂诱导后油红O染色镜下形态;
图13附图为本发明牙龈间充质干细胞外泌体电镜图;
图14附图为本发明牙龈间充质干细胞外泌体粒径大小分布图;
图15附图为本发明Westernblot检测牙龈间充质干细胞外泌体标志蛋白质CD9,CD63的表达情况;
图16附图为本发明银屑病小鼠模型各组的皮损情况;
图17附图为本发明牙龈间充质干细胞外泌体治疗组(GMSCs-exo)PASI总评分(total score);
图18附图为本发明牙龈间充质干细胞外泌体治疗组(GMSCs-exo)皮肤厚度程度(thickness intensity)评分;
图19附图为本发明牙龈间充质干细胞外泌体治疗组(GMSCs-exo)鳞屑程度(scalling intensity)评分;
图20附图为本发明牙龈间充质干细胞外泌体治疗组(GMSCs-exo)红斑程度(redness intensity)评分;
图21附图为本发明银屑病小鼠模型各组皮损组织HE染色结果(比例尺为50nm);
图22附图为本发明银屑病小鼠模型各组脾脏组织;
图23附图为本发明银屑病小鼠模型各组脾指数;**:p<0.01;***:p<0.001;
图24附图为本发明免疫组织化学技术检测银屑病小鼠模型各组皮损组织中F4/80,CD163,CD206,TNF-α,IL-10的表达情况;
图25附图为本发明F4/80的光密度结果;**:p<0.01;
图26附图为本发明CD163的光密度结果;*:p<0.05;
图27附图为本发明CD206的光密度结果;***:p<0.001;
图28附图为本发明TNF-α的光密度结果;***:p<0.001
图29附图为本发明IL-10的光密度结果;***:p<0.001。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1牙龈间充质干细胞的分离培养与检测
(1)牙龈间充质干细胞的培养和传代
①培养:在超净工作台中使用碘伏浸泡牙龈组织30s,PBS洗涤牙龈组织3次,用手术刀将洗涤后的牙龈组织切至3mm2大小。将切碎的组织块转移至装有4mg/mL IV型胶原蛋白酶的EP管中,37℃振荡消化2h。2h后,于1000r/min条件下离心5min,弃上清,用含10%FBS的α-MEM完全培养基重悬,并转移至6cm的培养皿中,37℃,5%CO2的条件下培养。前3天静置,第4天换液,之后每3天换液一次。于显微镜下观察原代牙龈间充质干细胞在第一天(day1),第三天(day3),第五天(day5)和第七天(day7)的镜下形态,结果见图1。结果显示分离得到的牙龈间充质干细胞形态均一,表现出成纤维细胞样的细胞,并能稳定生长增殖。
②传代:待细胞融合度至80%后,用0.05%胰蛋白酶消化传代,细胞置于75cm2的培养瓶培养。
(2)牙龈间充质干细胞表面标志物的检测
①待牙龈间充质干细胞融合度至80%后,消化细胞,并收集细胞悬液至离心管中;
②每管细胞悬液500μL,细胞密度为2×105个/mL。除了空白对照管外,其余分别加入2μL的抗体(分别为PE标记的CD14,CD34,CD45,CD73,CD105;FITC标记的CD44,CD90;APC标记的CD29;PerCP-cy5.5标记的CD19),室温避光孵育30min;
③用PBS将细胞洗涤2遍后,转移至流式管中,上机检测,结果见图2-10。结果显示牙龈间充质干细胞CD73、CD90、CD105、CD44和CD29阳性表达,CD14、CD19、CD34和CD45阴性表达,说明分离得到的细胞具有干细胞特点。
(3)牙龈间充质干细胞多向分化能力检测
1)成骨分化能力检测
①将5×105个牙龈间充质干细胞接种于六孔板中,每孔加入2mL含10%FBS的α-MEM完全培养基,置于37℃,5%CO2的培养箱中进行培养;
②当细胞融合度达到60%-70%时,轻轻将孔内完全培养基吸走,向六孔板中加入2mL的成骨诱导分化完全培养基(购自ScienCell公司);
③每隔3天换用新鲜的成骨诱导分化完全培养基;
④诱导4周后,吸走六孔板中的成骨诱导分化完全培养基,用PBS冲洗2次;每孔加入2mL 4%中性甲醛溶液,固定30min;
⑤吸走中性甲醛溶液,用PBS冲洗2次;每孔中加入1mL茜素红染液染染色5min;
⑥吸走茜素红染液,用PBS冲洗2次;
⑦将培养板置于显微镜下观察并拍照,结果见图11。结果显示牙龈间充质干细胞经成骨诱导分化后表现出很强的橘红色钙沉积现象,提示该细胞具有成骨诱导分化能力。
2)成脂分化能力检测
①将5×105个牙龈间充质干细胞接种于六孔板中,每孔加入2mL含10%FBS的α-MEM完全培养基,置于37℃,5%CO2的培养箱中进行培养;
②每隔三天换液,直到细胞融合度达到100%或者过融合;
③轻轻将α-MEM完全培养基吸走,向六孔板中加入2mL成脂诱导分化培养基A液(购自ScienCell公司);
④诱导3天后,吸走六孔板中的A液,加入2mL成脂诱导分化培养基B液(购自ScienCell公司);
⑤24h后,吸走B液,换回A液进行诱导;
⑥A液和B液交替作用4次后(16天),继续用B液维持培养6天直到脂滴变得足够大、圆;B液维持培养期间,每隔3天需要换用新鲜的B液;
⑦成脂诱导分化结束后,吸走六孔板中的间质干细胞成脂诱导分化培养基,用PBS冲洗2次;每孔加入2mL 4%中性甲醛溶液,固定30min;
⑧吸走中性甲醛溶液,用PBS冲洗2次;每孔中加入1mL油红O染液,染色30min;
⑨吸走油红O染液,用PBS冲洗2次;
⑩将培养板置于显微镜下观察并拍照,结果见图12。结果显示牙龈间充质干细胞经成脂诱导分化后可见明显的脂滴富集,提示该细胞具有成脂诱导分化能力。
实施例2牙龈间充质干细胞来源的外泌体的提取与检测
(1)超速离心法提取牙龈间充质干细胞来源的外泌体
选择生长状态良好的第2代牙龈间充质干细胞,待细胞融合度至90%时,采用无外泌体血清条件培养基(购自ScienCell公司),连续培养48h后收集细胞培养上清,300g离心10min,取上清,去除细胞;4℃2000g条件下离心10min,取上清,去除死亡细胞;4℃10000g离心30min,取上清,去除细胞碎片;用0.22μm过滤器过滤上清后,4℃100000g离心70min,弃上清,保留沉淀,加入10mL PBS重悬;4℃100000g离心70min,弃上清,保留沉淀,沉淀为牙龈间充质干细胞来源的外泌体,用100μL PBS重悬,-80℃冰箱保存待用。
(2)外泌体电镜检测
采用透视电镜分析牙龈间充质干细胞来源的外泌体形态及大小,结果见图13。结果显示分离得到的外泌体为直径大约为30~100nm的具有脂质双层膜的微小膜泡,即为外泌体。
(3)外泌体粒径检测
将上述提取的外泌体稀释100倍后,采用NanoFCM技术,分析外泌体的浓度以及大小,结果见图14。结果显示NanoFCM技术分析所得外泌体粒径大小为50~100nm,溶度为:1.34×109颗粒/mL。
(4)Westernblot检测外泌体标志蛋白质
取一定体积外泌体,加入计算量5×上样缓冲液。将上述样品经SDS-PAGE电泳后,于300mA,1.5h条件下电转至PVDF膜上。用2%脱脂奶粉室温封闭1h后,TBST洗涤3次。将一抗(CD9,CD63)按1:1000稀释后,4℃振荡孵育过夜。TBST洗涤3次后,用1:2000稀释的山羊抗兔IgG-HRP二抗室温孵育1h,TBST洗涤3次后,加入适量发光液于PVDF膜上,置于Azure C400成像系统中进行发光检测,结果见图15。结果显示分离得到的牙龈间充质干细胞来源的外泌体表达外泌体标志物CD9和CD63。
实施例3银屑病小鼠模型的建立
选用SPF级雌性C57BL/6小鼠(6~8周龄,体质量16~18g)18只。小心剃去小鼠背部中央区域被毛,再用温和型脱毛膏脱去表面毳毛。脱毛后第二天起于小鼠背部(62.5mg/cm2)涂抹5%咪喹莫特乳膏,小鼠持续造模6d。
将造模成功的银屑病小鼠模型随机分为3组:阴性对照组(NC),阳性药物治疗组(CP),牙龈间充质干细胞外泌体治疗组(GMSCs-exo)组,每组6只。NC组在病变处涂100μL的PBS,CP组在病变处涂20mg/cm2的阳性药物(丙酸氯倍他索软膏),GMSCs-exo组在病变处涂100μL的GMSC来源的外泌体(1mg/mL)。每日一次,连续5d,观察小鼠背部皮损变化,结果见图16。结果显示牙龈间充质干细胞外泌体治疗组(GMSCs-ex)与阴性对照组相比,红斑消失、大多数皮肤无鳞屑覆盖、皮损与正常皮肤齐平。
实施例4银屑病小鼠模型的评估
1)皮损评分
参考临床银屑病面积与严重性指数(PASI)评分标准,给药第1天开始,每日对背部皮损处红斑、鳞屑、厚度按0~4评分,计三项总分。PASI评分标准:(1)红斑:无红斑为0分,淡红色为1分,红色为2分,深红色为3分,极深为4分;(2)鳞屑:无鳞屑为0分,部分皮损表面上覆有鳞屑为1分,大多数皮损表面覆盖有鳞屑为2分,皮损部位几乎全部被鳞屑覆盖为3分,皮损部位全部被鳞屑覆盖为4分;(3)厚度:皮损与正常皮肤齐平为0分,皮损较正常皮肤表面稍高为1分,皮损中度隆起为2分,皮损肥厚且隆起明显为3分,皮损高度肥厚且明显凸起为4分。结果见图17-图20。结果显示,牙龈间充质干细胞外泌体治疗组(GMSCs-exo)与阴性对照组(NC)相比,PASI总评分(total score)显著降低、皮肤厚度程度(thicknessintensity)、鳞屑程度(scalling intensity)和红斑程度(redness intensity)显著降低,提示牙龈间充质干细胞来源的外泌体对银屑病具有很好的治疗效果。
2)小鼠背部皮损组织HE染色
①处死小鼠后,取背部皮肤损伤处组织,用10%甲醛溶液固定,经石蜡包埋并切片;
②将石蜡切片浸泡于二甲苯溶液中,微波炉加热5min;再次将石蜡切片浸泡入二甲苯溶液中,微波炉加热5min;再分别浸泡于无水乙醇、95%乙醇、85%乙醇、75%乙醇溶液1min、75%乙醇溶液再1min;然后用自来水进行冲洗;
③苏木素染色5min后,用流水冲洗,1%盐酸酒精分化,再次用流水冲洗;伊红染色1min;再次浸泡于75%乙醇、85%乙醇、95%乙醇、无水乙醇溶液1min、无水乙醇溶液再1min,于二甲苯溶液中浸泡5min两次后,用中性树脂封片;
④显微镜下观察并拍照,光学显微镜下观察比较各组小鼠皮肤组织病理学改变,如表皮角化程度、棘层厚度、炎性细胞浸润等,结果见图21。结果显示牙龈间充质干细胞外泌体可通过影响表皮细胞过度增殖、角化不全、炎症细胞浸润及血管增生而改善咪喹莫特诱导的小鼠银屑病样皮损变化。
3)各组小鼠脾指数计算
于给药第5天,处死小鼠并取脾脏,观察脾脏组织,结果见图22。按以下公式计算脾指数:脾指数=脾质量(g)/小鼠体质量(g)。结果见图23。结果显示牙龈间充质干细胞外泌体治疗组小鼠脾指数降低,提示牙龈间充质干细胞外泌体有效抑制了银屑病小鼠炎症过程。
4)小鼠背部皮损组织免疫组织化学检测
处死小鼠后,取背部皮肤损伤处组织,用10%甲醛溶液固定,经石蜡包埋并切片。将石蜡切片浸泡于二甲苯溶液中,微波炉加热5min;再次将石蜡切片浸泡于二甲苯溶液中,微波炉加热5min;分别浸泡于无水乙醇、95%乙醇、85%乙醇、75%乙醇溶液1min、75%乙醇溶液再1min;用自来水冲洗。将切片置于3%H2O2溶液中室温浸泡10min,用PBST洗涤3次。浸泡于柠檬酸盐缓冲液中,高压修复10min,用PBST洗涤3次。浸泡于5%BSA溶液中封闭20min。将切片上液体甩掉,免疫组化笔圈出切片上组织,加50μL一抗(F4/80,CD163,CD206,TNF-α,IL-10)(按1:200稀释)于组织上,4℃孵育过夜。PBST洗涤切片3次后,在组织处加IgG-HRP二抗,室温孵育1h,PBST洗涤3次。滴加DAB显色液于组织上,显色5min。将切片用流水冲洗,用苏木素复染1min;再次用流水冲洗后,用1%盐酸酒精分化,用流水冲洗;再次浸泡于75%乙醇、85%乙醇、95%乙醇、无水乙醇溶液中5min、无水乙醇溶液中再5min,于二甲苯溶液中浸泡5min、二甲苯溶液中再浸泡5min,用中性树脂封片。显微镜下观察染色拍照,结果见图24。其中F4/80的光密度结果见图25;CD163的光密度结果见图26;CD206的光密度结果见图27;TNF-α的光密度结果见图28;IL-10的光密度结果见图29;图25-图29结果显示,牙龈间充质干细胞外泌体治疗组F4/80、CD163、TNF-α表达降低,CD206和IL-10表达升高,提示牙龈间充质干细胞外泌体可能通过调节M2/M1型巨噬细胞比例改善咪喹莫特诱导的小鼠银屑病样皮损变化。
对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (3)
1.一种牙龈间充质干细胞外泌体制剂,其特征在于,由牙龈间充质干细胞来源的外泌体制备得到。
2.根据权利要求1所述的一种牙龈间充质干细胞外泌体制剂的制备方法,其特征在于,具体步骤如下:
选择生长状态良好的牙龈间充质干细胞,待细胞融合度至90%时,且流式细胞术检测细胞表型为CD73、CD90、CD105、CD44和CD29阳性表达,CD14、CD19、CD45和CD34阴性表达后,采用无外泌体血清条件培养基,连续培养48-72h后收集细胞培养上清,300g离心10min,取上清,去除细胞;4℃2000g条件下离心10min,取上清,去除死亡细胞;4℃10000g离心30min,取上清,去除细胞碎片;用0.22μm过滤器过滤上清后,4℃100000g离心70min,弃上清,保留沉淀,加入10mL PBS重悬;4℃100000g离心70min,弃上清,保留沉淀,沉淀为牙龈间充质干细胞来源的外泌体,用100μL PBS重悬,-80℃冰箱保存待用。
3.牙龈间充质干细胞来源的外泌体在制备治疗银屑病药物中的应用。
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