CN112094804B - 一种异质性干细胞群、其制备方法及用途 - Google Patents
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Abstract
本发明涉及一种异质性干细胞群、其制备方法及用途。具体地,本发明涉及一种异质性干细胞群,其特征在于所述异质性干细胞群中的干细胞表达干性基因MYC、KLF4、GMNN、SOX2和NANOG,并且在所述异质性干细胞群中,表达CD146的干细胞的比率为1%‑50%。
Description
技术领域
本发明涉及一种新的异质性干细胞群,其特征在于成体组织内找到的由胚胎发育后存留的干细胞群体,其表达全能基因c-Myc、Gmnn和Klf4,并且在该混合的异质性干细胞群中,CD146的表达率为1-100%。本发明还涉及所述异质性干细胞群的制备方法及其用途。
背景技术
间充质干细胞(MSC),或者可以定义为多能间充质基质细胞,是异质细胞群(Uccelli等,2008)。MSC的发现通常归功于A.J.的工作。20世纪60年代后期的弗里登斯坦及其同事观察到在塑料培养皿中培养人骨髓(BM)细胞悬浮液导致造血对应物逐渐丧失,有利于增殖粘附的成纤维细胞样细胞集落,能够分化成脂肪细胞、体外(Friedenstein等,1968)、体内(Friedenstein等,1974)、软骨细胞和骨细胞。
首字母缩略词“MSC”在A.I.的工作后开始流行。Caplan等人在1991年提交的文章中提出在成人BM中,干细胞群可以分化成源自中胚层的不同组织的谱(Caplan,1991)。他们将这些细胞称为“间充质干细胞”。后来,Pittenger明确证明了MSC的多向分化能力(Pittenger等,1999)。从这些开创性的研究结果来看,培养扩增的MSC成为许多研究的对象,甚至对这些细胞的确切表征还有待阐明,并且在不同的实验室中没有应用标准化方案。
因此,报告的数据有时是有争议的,并且MSC生物学的许多方面仍然不清楚,这是因为分离程序的多样性、培养方法和组织来源的选择。为了更好地阐明围绕MSC的争议,我们提出了间充质干细胞***的概念,该***由来自胚胎发育的不同阶段的所有MSC组成,从胚胎后的后代细胞干细胞到祖细胞。MSC***的顶部是胚胎样干细胞,即使在胎儿形成后仍留在许多组织中,我们将它们定义为胚胎后多能干细胞(PSC)。
我们鉴定了来自多种人胎儿和成人组织的PSC,并证明这些细胞可以产生内皮细胞、肝上皮细胞、神经细胞、造血细胞、成脂细胞和成骨细胞系。PSC现已被其他团体证实。2010年,Kuroda Y等人在单细胞水平证明成体间充质干细胞(MSC)含有一种独特类型的干细胞,能够产生具有所有三个胚层特征的细胞。这些细胞被称为多谱系分化胁迫耐受(Muse)细胞。从脂肪组织中分离出高度纯化的Muse细胞群,并报道其自发分化为间充质、内胚层和外胚层细胞谱系。
所有这些研究都表明成人组织中存在PSC。我们假设MSC***中的其他细胞如周细胞和一般MSC是PSC的衍生物。PSC的定义是从干细胞分化的角度出发的。通过多年对这种细胞类型的研究,我们强烈提倡干细胞功能定义的思想。在这里,我们将PSC定义为培养活化的胚后亚多能干细胞(CAPPSC),其具有三个重要的生物学特征:干细胞特性包括多能性和自我更新,低免疫原性和免疫调节功能,微环境和组织平衡。
发明内容
本发明人的研究发现,本发明人提供的新干细胞群与以前报道的MSC不同,其是一组干性更强混合的异质性的干细胞群体;其处于具有表观遗传多能性的常染色质状态,并表达多潜能标记物MYC、KLF4和GMNN。与胚层规范相关的大多数基因由H3K4me3修饰或由H3K4me3和H3K27me3共修饰。通过单细胞RNA-seq解析新干细胞向功能性肝脏细胞分化的过程,也发现在分化的很早阶段,三胚层早期分化相关基因都先后上调表达,当细胞定向肝脏谱系分化后,其它胚层分化相关基因下调表达。
本研究中,单细胞数据分析显示,我们体外分离培养获得的新干细胞群体,表达全能基因c-Myc、Gmnn和Klf4,表达三胚层早期发育相关基因,如外胚层的Nes和Hes1、中内胚层的Pdgfra和Gsc,限定性内胚层的Hhex和Sox17,内胚层器官肝脏发育关键基因ATF5、Tle3、Hnf4a和Krt18的表达较高,中胚层Mesp2、Gata4、Hand1和Tbx6表达很弱,但成骨分化相关基因Wnt5a、Runx2、TAZ明显表达,成脂分化相关的上游调控基因Cebpb、Cebpd、Gsk3a、Gsk3b显著表达,Mapk7中等表达,成脂分化关键转录因子Pparg和Cebpa低或不表达。
有趣的是,我们发现新干细胞中,上皮——间质转换相关关键基因Tjp1、Ctnnb1、Cdh2、Fn1、Vim、Zeb1和Twist1均明显高表达,表明新干细胞处于上皮——间质转换的中间阶段,可以随着不同微环境信号刺激,发生快速的功能转换。
在本发明的一个方面中,提供了一种异质性干细胞群,其特征在于所述异质性干细胞群中的干细胞表达干性基因MYC、KLF4、GMNN、SOX2和NANOG,并且在所述异质性干细胞群中,表达CD146的干细胞的比率为1-100%,优选地,表达CD146的干细胞的比率为1-50%。
进一步地,根据本发明提供的异质性干细胞群,在用含有50%-99%DMEM/F12、0.1-30ng/m1表皮生长因子和0.1-2%B27、0.1-10%FBS的培养基中传代所述异质性干细胞群1代、2代、3代、4代、5代、6代、7代、8代、9代、10代或10代以上后,表达CD146的干细胞的比率为1-99%,优选地,表达CD146的干细胞的比率为1-50%。
另一方面,根据本发明提供的异质性干细胞群,在用含有FBS的培养基传代所述异质性干细胞群1代、2代、3代、4代、5代、6代、7代、8代、9代、10代或10代以上后,表达CD146的干细胞的比率为50-100%,优选地,表达CD146的干细胞的比率为55-100%。
进一步地,根据本发明提供的异质性干细胞群,其中表达CD146+弱阳性(其中,表达CD146的干细胞的比率为1-50%,不包括50%为CD146+弱阳性)的干细胞中的干性基因MYC、KLF4、GMNN、SOX2和NANOG的表达水平显著高于表达CD146+++强阳性(其中,表达CD146的干细胞的比率为50-100%为CD146+++强阳性)的干细胞。一种体外诱导根据本发明提供的异质性干细胞群分化成网状血管内皮细胞的方法,所述方法包括在含有50%-99%DMEM/F12、0.1-30ng/m1表皮生长因子和0.1-2%B27、0.1-10%FBS的培养基中诱导本发明提供的异质性干细胞群分化成网状血管内皮细胞。
一种体外促进T细胞活化的方法,所述方法包括将根据本发明提供的异质性干细胞群与分离的PBMC在含有LPS的培养基中共培养的步骤。
一种体外抑制T细胞活化的方法,所述方法包括将根据本发明提供的异质性干细胞群与分离的PBMC在含有PolyIC或IFN-γ+TNF-α(I+T)的培养基中共培养的步骤。
根据本发明提供的异质性干细胞群在制备用于修复组织损伤的药物中的用途,优选地,所述组织损伤为肝细胞损伤。
根据本发明提供的异质性干细胞群在制备用于治疗cGVHD的药物中的用途。
一种制备本发明提供的异质性干细胞群的方法,所述方法包括以下步骤:
(1)获得干细胞群;
(2)在含有50%-99%DMEM/F12、0.1-30ng/m1表皮生长因子和0.1-2%B27、0.1-10%FBS的培养基中培养步骤1)获得的干细胞群。
另一方面,本发明涉及根据本发明的异质性干细胞群在制备用于构建血脑屏障的试剂中的用途。
另一方面,本发明涉及根据本发明的异质性干细胞群在制备用于诱导分化米色脂肪细胞的试剂中的用途。
另一方面,本发明人发现一个小分子可将新干细胞群体诱导成抑炎型干细胞群体,新干细胞群体在小分子CZ的诱导下可分化成为抑炎型的MSC2,结合体外实验的结果,我们以单细胞测序进一步研究新干细胞群体转化成为抑炎型的MSC2,新的细胞群体经过CZ处理以后,细胞进入了类似于MSC2的抑炎功能活化状态,将用于用于自身免疫性疾病的临床治疗。
因而,本发明进一步提供了一种体外诱导本发明的异质性干细胞群成抑炎型干细胞群体的方法,所述方法包括使本发明的异质性干细胞群与小分子CZ接触。
另一方面,本发明提供了根据以上方法得的抑炎型干细胞群体在制备用于治疗自身免疫性疾病的药物中的用途。
附图说明
图1A表示通过本发明的间充质干细胞培养方法(MSC-AB)与现有技术中的间充质干细胞培养方法(MSC-FBS)获得的异质性干细胞群中MYC、KLF4、GMNN、SOX2和NANOG基因的表达水平;
图1B表示通过本发明的间充质干细胞培养方法(MSC-AB)与现有技术中的间充质干细胞培养方法(MSC-FBS)连续培养获得的间充质干细胞10代中表达CD146的细胞百分比;
图2表示通过本发明的方法获得的异质性干细胞群中,CD146+++强阳性细胞与CD146+弱阳性细胞中CD146、MYC、KLF4、GMNN、SOX2和NANOG基因的表达水平;
图3A表示本发明的培养体系(AB)和现有技术中的培养体系(FBS)培养的异质性干细胞群在诱导血管内皮细胞中的结果图;
图3B表示本发明的培养体系(AB)和现有技术中的培养体系(FBS)培养的异质性干细胞群中血管生成相关基因的相对表达;
图3C表示本发明的方法获得的异质性干细胞群中,CD146+++强阳性细胞与CD146+弱阳性细胞中血管生成相关基因的相对表达;
图4A和图4B表明与对照组人胚肺成纤维细胞(MRC-5)相比,本发明的异质性干细胞能够显著增加T细胞的活化比例;
图4C表明T细胞与本发明方法获得的异质性干细胞群共培养以后CD28表达上调;
图4D表示用磁珠分选方法分选的CD28+、CD28-和未分选群分别与本发明的异质性干细胞群共培养后,CD28的表达;
图4E表示用磁珠分选方法分选的CD28+、CD28-和未分选群分别与本发明的异质性干细胞群共培养后,用CD单抗体激活的T细胞活化;
图4F和图4G表明用LPS诱导本发明的异质性干细胞群后转变为促炎亚型,能够在体外促进T细胞活化;而用PolyIC或IFN-γ+TNF-α(I+T)诱导本发明的异质性干细胞群转变为抑炎亚型,能够抑制T细胞活化;
图4H和图4I表明在大鼠急性肾损伤模型的体内实验中,给予大鼠促炎亚型的异质性干细胞群输注,将会显著增加尿肌酐水平;
图5-1A至5-1C表明利用单细胞测序的方法,经过小分子CZ处理后,MSC的细胞聚类有明显的改变;
图5-1D表明细胞周期相关的基因在细胞群中的表达;
图5-1E表明细胞周期蛋白基因CCNI、组蛋白基因HIST1H4C、着丝粒蛋白基因CENPF、DNA拓扑异构酶基因TOP2A和细胞骨架蛋白基因TLN1都在细胞群中的表达;
图5-1F表明小分子CZ处理后的MSC在细胞周期中更多地处于G2/M期,而G0/G1期细胞所占比例则有所减少;
图5-2A表明对单细胞测序数据进行分析,与天然免疫相关的基因表达;
图5-2B表明转运蛋白基因AP2B1、整合素β1基因ITGB1、EIF4A1、PSMB3和PSMB7基因的表达;
图5-2C表明PBMC与经过小分子CZ处理后的MSC共培养后活化率更低;
图5-2D表明PBMC与经过小分子CZ处理后的MSC共培养后增殖更慢;
图5-3表示不同方式处理的MSC治疗大鼠肾脏损伤后尿肌酐水平;
图6A表示C57BL/6小鼠腹腔注射CCl4诱导ALI后,ALI小鼠注射CD146+弱阳性干细胞、CD146+++强阳性干细胞或PBS后第1、4或7天的炎性细胞浸润;
图6B表示C57BL/6小鼠腹腔注射CCl4诱导ALI后,ALI小鼠注射CD146+弱阳性干细胞、CD146+++强阳性干细胞或PBS后第1、4或7天的ALT水平;
图6C表示C57BL/6小鼠腹腔注射CCl4诱导ALI后,ALI小鼠注射CD146+弱阳性干细胞、CD146+++强阳性干细胞或PBS后第1、4或7天的AST水平;
图6D表示小鼠的存活百分比;
图7A至7D表示本发明的异质性干细胞群治疗cGVHD的实验结果;
图7A表示皮肤总体疗效评分;
图7B表示关节P-ROM评分;
图7C表示随访期1年内主要疗效指标有效性分析(FAS);其中,总体疗效评分的评分依据:皮肤评分、关节筋膜评分和总体等级评分中任意一个评价有效即为总体疗效有效;在治疗后1、2、6和12个月,试验组的部分有效以上例数均大于对照组,差异有统计学意义;试验组总体疗效评分明显优于对照组;
图7D表示随访期1年内其他主要疗效指标有效性分析(FAS);其中,总体疗效评分在治疗后1、2、6和12个月,试验组的部分有效以上例数均大于对照组,差异有统计学意义;试验组总体等级评分明显优于对照组;
图7E表示本发明的异质性干细胞群治疗cGVHD的实验流程图;
图8A表示利用Transwell体外培养血脑屏障的模型示意图。脑微血管内皮细胞培养在上侧,其下部为周细胞,而星形胶质细胞培养在Transwell底部;
图8B表示脑微血管周细胞、内皮细胞及星形胶质细胞免疫荧光染色鉴定。周细胞α-SMA和NG2表达阳性,而vWF和GFAP表达阴性;内皮细胞vWF表达阳性;星形胶质细胞GFAP表达阳性;
图8C和图8D分别表示正常及IL-1β作用下notch3在三细胞中的表达及在IL-1β作用下MMP-9在三细胞中的表达;
图8E表示在IL-1β与DAPT和PDTC分别作用下,notch3、MMP-9、TIMP-1及NF-κB在周细胞中的表达变化;其中,表达变化是相对于对照的倍数变化;
图8F表示明胶酶谱分析对照组和不同处理组(IL-1β、IL-1β+DAPT、IL-1β+PDTC)MMP-9和MMP-2活性变化;
图8G和图8H表示利用Na-F来检测对照组和不同处理组(IL-1β、IL-1β+DAPT、IL-1β+PDTC)BBB通透性的变化。
图9表示IRISIN诱导亚多能干细胞分化为米色脂肪细胞,实时荧光定量和蛋白质印迹检测蛋白UCP1表达(P<0.05)。
具体实施方式
实施例1干细胞群分离和体外扩增培养的方法
本发明的方法包括获得分离的人体组织,包括但不限于:外周血、骨髓、羊膜、脂肪组织、胎盘、脐带、肌肉、皮肤。用胶原酶消化,经梯度离心、过滤,随后在体外培养体系中进行扩增培养多至10代。由该方法分离、培养获得的新干细胞群体具有c-Myc、Gmnn和Klf4及CD146+弱阳性;所述体外培养体系含有50%-99%DMEM/F12、0.1-30ng/m1表皮生长因子和0.1-2%B27、0.1-10%FBS。
具体培养步骤:
1)从胎盘、脐带、肌肉、皮肤中分离提取脂肪的MSC;
2)将获得的脂肪组织分装至离心管中,加入相应体积PBS混匀后800rpm离心3min,重复清洗2次,之后每管加入相应体积0.2%胶原酶37度摇床消化30min;
3)加入PBS终止消化,100微米筛网过滤后1500rpm离心10min,弃去脂肪及上清后获得细胞沉淀之后继续加入PBS清洗细胞2次,1500rpm离心8min,根据脂肪体积每20ml放入一个T75的方式种细胞;
4)传代:观察细胞形态及密度后倒掉旧培养基,用PBS清洗细胞2遍后加入10ml 1x胰酶消化半分钟左右,用几滴血清终止消化,1200rpm离心5min,弃去上清用相应体积培养基重悬细胞种于培养皿中培养。
通过本发明人建立的异质性干细胞群的培养方法,本发明人发现使用我们实验室的培养体系,与普通的异质性干细胞群培养体系(FBS体系)培养的干细胞群相比,在体外高表达干细胞干性相关基因MYC、KLF4、GMNN、SOX2和NANOG(参见图1A)。换用FBS培养体系培养的异质性干细胞群中CD146阳性率明显升高,在7代后达到90%以上,出现类似异质性干细胞群的表型。而使用我们的培养体系培养的异质性干细胞群中,CD146的阳性率保持在50%弱阳性(图1B)。
单克隆抗体检测细胞表型:
收集细胞,计数后以相应浓度重悬细胞,之后在细胞中加入需要检测的相应抗体,根据使用量计算加入,充分混匀后4度孵育30min,之后加入PBS洗涤细胞2次,1000rpm离心5min弃去上清,再加入相应体积PBS重悬细胞,然后上机检测。
实施例2通过本发明的方法获得的异质性干细胞群的分选
接下来我们通过磁珠分选的方法,将我们的培养体系培养的新干细胞中CD146+++(细胞表面标志阳性率表达50%至100%)和CD146+(细胞表面标志阳性率表达1%至50%,不包括50%)细胞群体分选出来进行比较。
具体的分选步骤如下详述:
使用Vario—MACS***,CD146+正向选择免疫磁珠分选脐带来源间充质干细胞:
(1)收集细胞沉淀,弃上清。
(2)取缓冲液(含D-Hanks、0.5%BSA、2mMolEDTA)重悬细胞沉淀,按每60ul缓冲液107个细胞比例,向细胞悬液中分别加入FcR和CD146+磁珠,均按20ul每107个细胞比例充分吹打混匀,置于4度冰箱,孵育15min。
(3)按照每1ml缓冲液洗涤107个细胞的比例洗涤细胞。收集沉淀。
(4)按照107个细胞/500u l缓冲液的比例重悬细胞。
(5)把LP分选柱放入Vario-MACS***的磁性区域,加入3ml缓冲液冲洗分选柱3次。待缓冲液完全流出分选柱后,加入细胞悬液。
使用适当量的缓冲液冲洗柱子,待所有液体全部流出分选柱后,将LP分离柱移出MACS***的磁场,置于收集管上,加入5ml缓冲液,***活塞快速冲出细胞,获得被CD146+磁珠标记的CD146+++强阳性细胞亚群。我们发现,CD146+弱阳性细胞中上述干性基因的表达水平明显高于CD146+++强阳性细胞(参见图2)。这些结果提示,本发明的方法获得的异质性干细胞群中,CD146+++强阳性细胞的干性低于CD146+弱阳性细胞,在分化谱系上处于CD146+弱阳性细胞下游的干细胞亚群。而在我们的培养体系中培养的异质性干细胞群能够在体外传代中维持高比例CD146+弱阳性细胞,我们将我们的体系称为异质性干细胞群的干性维持体系。
实施例3体外成血管内皮诱导实验
在本实验中,实验步骤如下详述:
1)提前预冷融化matrigol(BD,低生长因子,354230),体积小可提前一小时融化,若体积大需过夜,96孔板(也可在冰冻板背面直接操作)。
2)垂直加入凝胶,每孔40-55ul,注意不要有气泡,先在常温下平衡10分钟再放入37℃静置半小时。
3)在凝胶等待中准备细胞。(也可铺好胶后备用,一周内用完)
4)加入细胞悬液,每孔150ul。
本发明的干细胞干性维持体系培养的异质性干细胞群能够顺利诱导出连接成网状的血管内皮,而现有技术中的MSC培养体系的细胞则不能(参见图3A)。进一步检测血管生成相关基因Ang-1、VASH1、FLK、VEGF、bFGF,显示本发明的干性维持体系培养的异质性干细胞群在诱导过程中高表达成血管相关基因。对分选后的CD146+++强阳性和CD146+弱阳性细胞的比较也得到了类似的结果(参见图3B和图3C)。
如上述结果所示,本发明的培养体系(AB)获得的异质性干细胞群的干性更强,成血管内皮能力更强。
实施例4本发明的异质性干细胞群具有较强的免疫调节能力
在本实验中,实验方法和步骤如下详述:
(1)将干细胞在AB液培养体系中传代至3-5代。
(2)在体外与静息的外周血单个核细胞(PBMC)共培养(1640+10%FBS)7天后,撤除干细胞,并对T细胞进行CD3抗体活化72h。
(3)与对照组人胚肺成纤维细胞(MRC-5)共培养相比,我们的干细胞共培养组能够显著增加T细胞CD28的表达和T细胞的活化比例。
已经报道了MSC具有较强的免疫调节作用,已有许多实验证明MSC能够在体外抑制T细胞的活化和增殖。本发明的干性维持体系培养的异质性干细胞群具有很强的免疫调节作用。
首先,将本发明的方法获得的异质性干细胞群在体外与静息的PBMC共培养7天后,撤除干细胞,并对T细胞进行CD3/CD28抗体共刺激活化。我们发现,与对照组人胚肺成纤维细胞(MRC-5)相比,本发明的异质性干细胞能够显著增加T细胞的活化比例(参见图4A和4B),而普通MSC通常是抑制T细胞活化的。
结果提示,在本发明的异质性干细胞群与T细胞共培养的过程中,该干细胞群可能增强T细胞的活化潜能。为了证明这种可能,我们进一步检测了T细胞在与本发明的异质性干细胞群共培养前后共刺激活化途径CD3和CD28基因的表达,发现跟我们预期的一样,CD28基因在共培养后有显著的上调(参见图4C)。
为了进一步明确上调的CD28分子的来源,我们用磁珠分选的方法将CD28+/-的T细胞分离开来,分别与本发明的异质性干细胞群共培养,与对照组相比,我们发现CD28-的T细胞群和未分选的T细胞群中CD28分子的上调显著(参见图4D)。对分选后再共培养的T细胞群进行CD单抗体激活(去除CD28抗体的影响),我们发现与本发明的异质性干细胞群共培后各群T细胞的活化率都显著增强(参见图4E)。
此外,本发明的异质性干细胞群也具有与普通MSC相似的免疫可塑性。用LPS诱导本发明的异质性干细胞群后转变为促炎亚型,能够在体外促进T细胞活化;而用PolyIC或IFN-γ+TNF-α(I+T)诱导本发明的异质性干细胞群转变为抑炎亚型,能够抑制T细胞活化(参见图4F和图4G)。
在大鼠急性肾损伤模型的体内实验中,我们发现给予大鼠促炎亚型的异质性干细胞群输注,将会显著增加尿肌酐水平,且病理切片显示肾脏炎性细胞浸润增加,纤维素样坏死加重。而给予大鼠抑炎亚型输注,炎性细胞浸润则大大降低,且系膜细胞溶解和增殖减轻,其中I+T组主要表现为炎性细胞浸润减少,PolyIC组主要表现为细胞增殖减轻(参见图4H和4I)。
结论:本发明的方法获得的新干细胞是一群混合的异质性干细胞群,其能够在本发明人的干性维持培养体系中保持多能性至少达10代。这是一个区分于普通MSC的重要特性,使得本发明的异质性干细胞群能够在较长的窗口期被诱导分化为下游功能细胞,如血管内皮细胞,这种在血管衰老中重要的指示细胞。
CD146(MCAM)是一种细胞黏附分子,通常被用作内皮细胞和周细胞的标记物。最近也有研究显示,CD146是神经神经生长因子(netrin)的受体和血管生成因子受体2(VEGFR2)的共受体。我们的实验结果证实了CD146分子与本发明的异质性干细胞群的干性之间存在着一种紧密的相关性,它是否能成为新干细胞的潜在标记物,需要未来进行进一步的研究。
CD28是T细胞表面的一种免疫共激活分子,出生时几乎所有的T细胞均为CD28+。随着机体在成长和衰老过程中的抗原暴露,T细胞逐渐丢失CD28表型,并失去活化的能力。因此CD28成为一种指示免疫***衰老的分子。本发明的新干细胞群能够在与PBMC共培后显著提高T细胞群体表达CD28的比例,因此我们认为新干细胞能够增强T细胞的活化潜能和免疫功能,特别是针对年龄在65岁以上的人,他们的T细胞中50-60%的CD8+T细胞和5-10%CD4+T细胞缺乏CD28分子。这个发现使得新干细胞治疗免疫***衰老成为一种极具希望的细胞治疗方式。
实施例5小分子CZ可将本发明的异质性干细胞群诱导成抑炎型干细胞群体
小分子CZ的产品名称为氯唑沙宗,目录编号:TCZ;CAS号码:95-25-0;其分子式为C7H4ClNO2;分子量为169.57。存储:-80℃的溶剂下保存2年;-20℃的粉剂下3年。
氯唑沙宗是一种中枢作用的肌肉松弛剂,用于治疗肌肉痉挛和由此引起的疼痛或不适。通过抑制反射作用于脊髓。氯唑沙宗目前被用作体外/体内研究的标记物底物,以量化人体中的细胞色素P4502E1(CYP2E)活性。
其物理特性:
沸点:181℃~192℃
熔点:N/A
溶解度:二甲基甲酰胺(DMF),34mg/ml(200.5mM)
二甲基亚砜(DMSO),34mg/ml(200.5mM)
水
该研究目的仅用于诊断或治疗用途。
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本发明人发现异质性干细胞群体在小分子CZ的诱导下可分化成为抑炎型的MSC2(干细胞群体),结合体外实验的结果,我们以单细胞测序进一步研究本发明的异质性干细胞群体转化成为抑炎型的MSC2,该细胞群体经过CZ处理以后,细胞进入了类似于MSC2的抑炎功能活化状态,将用于自身免疫性疾病的临床治疗。
小分子CZ能够增加MSC的增殖能力
利用单细胞测序的方法,我们发现经过小分子CZ预处理48h后,MSC的细胞聚类有明显的改变(图5-1A)。与细胞周期相关的基因表达显示,经过小分子CZ处理以后的MSC更多地表达S期和G2/M期的基因(图5-1B)。对对照组和小分子CZ处理组进行仔细聚类分析以后,我们发现细胞群2(cluster2)是其中变化最大的细胞群体,细胞群2在CZ处理以后的样本中完全消失,且仅表达G1期和S期的基因,而不表达G2/M期的基因(图5-1A、5-1B、5-1C)。因此接下来我们对细胞群2进行了细胞周期相关基因的仔细分析。我们在细胞群2中发现,与细胞周期相关的基因在细胞群2中的表达高低与其他群体细胞有明显的差异(图5-1D)。其中细胞周期蛋白基因CCNI,组蛋白基因HIST1H4C、着丝粒蛋白基因CENPF、DNA拓扑异构酶基因TOP2A和细胞骨架蛋白基因TLN1都在细胞群2中相对低表达而在其他细胞群体中高表达(图5-1E)。
体外实验也显示,用小分子CZ处理后的MSC在细胞周期中更多地处于G2/M期,而G0/G1期细胞所占比例则有所减少(图5-1F)。
小分子CZ能够增加MSC的免疫调节功能
进一步对单细胞测序数据中的进行分析,我们发现,与天然免疫相关的基因表达中细胞群2和其他群体细胞也有明显不同(图5-2A)。在差异最明显的蛋白中,我们发现,转运蛋白基因AP2B1和整合素β1基因ITGB1在细胞群2中低表达,其中前者可参与TGFβ受体介导的胞吞作用而后者参与IL1β受体介导的IL1β信号通路,且在微生物感染免疫中参与重要作用。而相反的是,参与组成20s蛋白酶复合体的基因PSMB3和PSMB7基因则在细胞群2中高表达,表明细胞群2以外的细胞泛素化的蛋白水解较慢,因此蛋白的功能普遍较强(图5-2B)。
体外实验也表明,PBMC与经过小分子CZ处理后的MSC共培养后活化率更低(图5-2C),增殖更慢(图5-2D)。因此,小分子CZ能够促进MSC的免疫抑制功能。
结论:经过单细胞数据的多方面分析,我们认为,细胞群2细胞是一类较为静息的细胞群体,它们在未经小分子活化的状态下处于功能原始状态。而在经过小分子CZ处理后,新的干细胞群体进入一个功能活化的状态,细胞周期加快,增殖速度加快,且与免疫调节功能相关的基因表达也随之改变。
MSC的免疫抑制功能已经广为人知,目前已成功进入临床治疗自身免疫疾病,如GvHD等。但在治疗过程中有少部分病例出现疗效不如预期甚至效果相反的现象。MSC是一个异质性的细胞群体,我们认为MSC群体中能够有效抑制免疫反应的细胞比例不够,那么在体内的疗效就会大打折扣。
目前已有理论提出MSC分为促炎型的MSC1和抑炎型的MSC2,已知LPS能诱导促炎型的MSC1,IFN-γ+TNF-α、poly(I:C)能够诱导MSC向抑炎型的MSC2转变,但由于经过这样方式处理以后的MSC免疫原性显著增强,不能广泛用于临床治疗,故我们希望找到一种临床级小分子能够增强MSC的免疫抑制功能,或者增加MSC群体中的MSC2的比例,但不会明显改变MSC的免疫原性。
通过大数据挖掘和功能筛选,我们找到了一种代号为CZ的小分子化合物,它是一种FDA批准的临床药物。在大鼠急性肾损伤模型中,我们发现,给予大鼠促炎亚型(LPS处理组)的MSC1输注,将会显著增加尿肌酐水平;而给予大鼠抑炎亚型的MSC2(I+T、poly(I:C)、CZ组)输注,尿肌酐水平明显降低,其中小分子CZ的效果最显著(图5-3)。在病理组织切片中,促炎型MSC1增加了肾脏炎性细胞浸润增加,加重了肾小球纤维素样坏死;而抑炎型MSC2使得炎性细胞浸润大大降低,系膜细胞溶解和增殖减轻。
小分子CZ的筛选过程:
1.大数据挖掘人MSC免疫调节中的关键分子IDO的相互作用蛋白;
2.在获得的蛋白中进行功能筛选,从而得到了能够增强MSC免疫抑制功能的CZ分子。
实施例6本发明的异质性干细胞群与组织损伤修复
本实施例研究了来自本发明异质性干细胞群体在急性肝损伤(ALI)中的不同疗效。
C57BL/6小鼠腹腔注射CCl4诱导ALI,6小时后,ALI小鼠注射5×105CD146+弱阳性干细胞、CD146+++强阳性干细胞或PBS(安慰剂治疗组)。在第1天,对照组中有更多的炎性细胞浸润,并且所有三组均具有轻微的球囊样改变。在第4天,对照组具有广泛的球囊样变化和大量炎性细胞浸润,而在CD 146+++强阳性干细胞移植组中观察到大的坏死病灶和广泛的球状样变化。在第7天,三组恢复正常肝脏组织学(参见图6A),CD 146+++强阳性干细胞移植组具有显着更高水平的ALT(p=0.027)和AST(p=0.012)(参见图6B和图6C)。另外,肝内CD146+++强阳性和CD146+弱阳性干细胞的数量在第4天最高,肝内CD146+++强阳性干细胞的数量低于CD146+弱阳性干细胞。
干细胞(CD 146+弱阳性)治疗组的存活率显着高于安慰剂治疗组(82%对54.5%)(参见图6D)。PSC治疗组血清肝酶丙氨酸氨基转移酶(ALT)和天冬氨酸氨基转移酶(AST)的峰值水平显着低于安慰剂治疗组(P<0.01)因此,PSC给药显着减轻CCl4诱导的急性肝损伤。
实施例7本发明的异质性干细胞群治疗cGVHD的疗效
从2012年1月至2015年10月期间,共招募了35名符合条件的患者。患者分为两组,并进行一线免疫抑制治疗。实验组另外接受4剂骨髓内本发明的异质性干细胞群的输注,其中临床试验的流程参见图7E)。
我们的结果表明,本发明的异质性干细胞群的输注分别显着改善了难治性ScGVHD的临床结果,包括皮肤,关节和筋膜,cGVHD症状的总体严重程度和总体反应(P<0.05)(参见图7A-7D)。实验组的总反应率(ORR)在6个月达到峰值82.1%,而对照组的ORR仅为23.1%。没有观察到与MSC输注相关的早期或晚期安全性问题。
从上述数据来看,关节和筋膜似乎对PSC治疗更敏感,6个月时有90%的RR,而只有一半的患者对PSCs治疗有皮肤反应。为了确定皮肤对PSC治疗反应的预后因素,我们比较了皮肤反应者和非皮肤反应者之间的基线人口统计学和临床特征(包括性别,年龄,疾病,供体,cGVHD持续时间,基线NIH皮肤评分,KPS和PSCs剂量)。只有供体类型在皮肤反应者和非反应者之间具有临界显着性差异(P=0.077)。在6个月时,从匹配的同胞供体接受移植的患者的皮肤反应率为72.7%,而接受半相合供体移植的患者的反应率仅为22.2%。
进行流式细胞术以测量基线和1年随访期间两个臂之间的T淋巴细胞亚群。注射PSC后Treg的百分比和Th1/Th2的比例均升高,并且在2次注射PSC后达到峰值,并且在随访期间逐渐下降。在每个评估点处,在两个组之间没有观察到Treg百分比和Th1/Th2比率的差异(P>0.05)。
向28名患者施用总共112个PSC骨髓内输注。所有输注都耐受良好。未观察到急性输注相关反应和PSC输注相关不良事件。在随访期间,登记的患者中共观察到6例死亡。
实施例8本发明的异质性干细胞群在干预血脑屏障药物中的用途
血脑屏障是由脑微血管内皮细胞、周细胞及星形胶质细胞组成。周细胞在血脑屏障中起到很重的作用,同时它也属于中医三焦结构主要功能细胞-新干细胞群体/周细胞群体,三焦器官关联五脏六腑及周身器官组织,通过气血运行、传导感应、互相协调,已到达人体经络功能的调控,三焦器官/间充质组织***主要负责干细胞多组织增殖分化、组织器官再生修复和功能重建,参与人体免疫监督、应答及复杂免疫网络调控作用等,发挥着脑疾病及神经修复、组织间激素、内分泌调节及组织代谢的***性调节功能。
影像学表现为脑白质病变,提示血脑屏障有破坏,其中炎症是参与原因之一,小血管病脑损伤的发病机制是局部炎症导致的血脑屏障损伤引起。炎症对血脑屏障的损伤机制尚未完全阐明。
我们应用本发明方法获得的异质性干细胞群,定向诱导分化为脑微血管内皮细胞、周细胞及星形胶质细胞,从而构建了血脑屏障。
诱导分化的方法步骤和血脑屏障的确立如下进行:
我们将白细胞介素1β(IL-1β)加入到Transwell体外血脑屏障模型中,体外培养人脑微血管内皮细胞(HBMVECs)、人脑周细胞(HBPs)和人脑星形胶质细胞(HBAs)。我们用免疫荧光染色鉴定了HBMVECs、HBPs和HBAs,其中HBMVEC用vWF染色,HBP用α-SMA和NG2抗体染色,而vWF和GFAP不染色,HBA用GFAP染色(图8B)。在正常情况下,q-PCR结果提示HBP表达NOTCH3基因,而HBMVEC和HBA不表达NOTCH3(图8C)。HBP、HBMVEC和HBA与IL-β共培养24小时,HBP组观察到NOTCH3基因表达,HBMVEC和HBA组NOTCH3基因并没有表达(图8D)。用IL-1β处理HBP30分钟,然后用DAPT 24小时,q-PCR结果表示在图8E中,从图8E中可见,与对照组相比,IL-1β组NOTCH3基因表达增加,而与IL-1β组相比,IL-1β+DAPT组的NOTCH3基因表达减少。同时,MMP-9基因表达的结果与NOTCH3相似,但三组中TIMP-1基因表达的结果变化不大。HBP经IL-1β处理30分钟后,PDTC处理24小时,q-PCR结果显示,IL-1β组与对照组相比,Notch3基因表达显著增加,但与IL-1β组相比,IL-1β+PDTC组变化不大。同时,与对照组相比,IL-1β组MMP-9基因表达结果显著增加,IL-1β+DAPT组MMP-9基因表达结果与IL-1β组相比显著降低,但三组TIMP-1基因表达结果变化不大。用IL-1β处理HBP 30分钟,DAPT处理24小时,q-PCR结果显示,与对照组相比,IL-1β组的NF-κB p65基因表达显著增加,而与IL-1β组相比,IL-1β+DAPT组的表达显著降低。同时,PDTC处理组NF-κB p65基因表达结果与DAPT处理组相似(参见图8E)。
图8A表明了利用Transwell体外培养血脑屏障的模型示意图。脑微血管内皮细胞培养在上侧,其下部为周细胞,而星形胶质细胞培养在Transwell底部;
图8B表明了脑微血管周细胞、内皮细胞及星形胶质细胞免疫荧光染色鉴定。周细胞α-SMA和NG2表达阳性,而vWF和GFAP表达阴性;内皮细胞vWF表达阳性;星形胶质细胞GFAP表达阳性;
图8C和8D分别表示正常及IL-1β作用下notch3在三细胞中的表达及在IL-1β作用下MMP-9在三细胞中的表达;
图8E表示在IL-1β与DAPT和PDTC分别作用下,notch3、MMP-9、TIMP-1及NF-κB在周细胞中的表达变化;其中,表达变化是相对于对照的倍数变化;
图8F表示明胶酶谱分析对照组和不同处理组(IL-1β、IL-1β+DAPT、IL-1β+PDTC)MMP-9和MMP-2活性变化;
图8G和8H表示利用Na-F来检测对照组和不同处理组(IL-1β、IL-1β+DAPT、IL-1β+PDTC)BBB通透性的变化。
实施例9本发明的异质性干细胞群调节脂肪细胞组织代谢用途
近几年新发现一种激素IRISIN,是锻炼后分泌的一种肌因子,由它的前体-纤连蛋白III结构包含5(Fndc5)剪切释放,其功能涉及在在肌肉、心血管、神经及能量代谢方面。在能量调节过程中,IRISIN在糖代谢中是一个潜在的调节子。本研究中,我们采用本发明的异质性干细胞群作为种子细胞,IRISIN诱导其分化为米色脂肪细胞(参见图9)。
诱导的方法、步骤如下详述:
脂肪组织用含有双抗生素(青霉素、链霉素)的D-Hanks’洗涤2次,800rpm离心3min;将洗涤后的下层液体用移液管吸出,将脂肪组织转至新的50ml离心管内,加入0.2%胶原酶P消化,于37℃恒温培养震荡器震荡30min;加入适量D-Hanks’液将消化后的脂肪组织,100μm细胞滤网过滤,去除未消化的组织,1500rpm离心10min;将上层油脂用移液管吸出后,弃上清,D-Hanks’重悬细胞沉淀,洗涤1次,1500rpm离心10min;弃上清,用12ml含适量双抗生素hAD-MSC工作液重悬2×106细胞接种于T75培养瓶内,于37℃恒温、5%CO2饱和湿度细胞培养箱培养;原代细胞接种48h后,除去上层未贴壁的细胞,继续培养,每隔2-3天全量换液一次;细胞达80%汇合时,可进行传代或冻存保种。
本发明的方法获得的新干细胞群,并非单指分化潜能上的新,而是信号易感性新,能够接受不同类型的信号,转变为不同的亚型,某方面功能增强。比如,不同免疫微环境刺激下,可以转化为促炎型和抑炎型功能亚类。在高浓度FBS培养条件下,转变为CD146+++强阳性亚群,对T细胞调节功能增强,向成骨分化能力增强,向成脂分化能力减弱,基本丧失成血管能力;而在低浓度FBS培养条件下,转变为CD146+弱阳性亚群,表现为免疫能力下降,成脂及成血管能力增强,成骨能力减弱。在寒冷、运动或者应急信号刺激下,新接收来自肌肉分泌的irisin信号,变为易于分化为米色脂肪的前体细胞。
Claims (1)
1.一种异质性干细胞群在制备用于修复组织损伤的药物中的用途,所述组织损伤为肾损伤,其中所述异质性干细胞群中的干细胞表达干性基因MYC、KLF4、GMNN、SOX2和NANOG,其中表达CD146+弱阳性的干细胞中的干性基因MYC、KLF4、GMNN、SOX2和NANOG的表达水平显著高于表达CD146+++强阳性的干细胞,
所述CD146+弱阳性是指表达CD146的干细胞的比率为1-50%,不包括50%;所述CD146+++强阳性是指表达CD146的干细胞的比率为50-100%;
并且其中
所述异质性干细胞群是在用含有50%-99%DMEM/F12、0.1-30ng/m1表皮生长因子和0.1-2%B27、0.1-10%FBS的培养基中传代1代、2代、3代、4代、5代、6代、7代、8代、9代、10代或10代以上后得到的,表达CD146的干细胞的比率为1-50%。
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