CN112410286A - 一种利用诱导多能干细胞构建妊娠期致流产药物筛查模型的方法及应用 - Google Patents
一种利用诱导多能干细胞构建妊娠期致流产药物筛查模型的方法及应用 Download PDFInfo
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Abstract
本发明公开了一种利用诱导多能干细胞构建妊娠期致流产药物筛查模型的方法,通过三维悬浮培养的方式对诱导多能干细胞进行培养,形成拟胚体;向拟胚体的培养基中施加待筛选药物暴露,通过分析拟胚体在含有待筛选药物的培养基中的发育指标筛选和/或评价该药物。本发明还公开了上述方法在妊娠期致流产药物筛查中的应用,该方法构建的模型基于拟胚体类器官的发育,动态模拟了整体胚胎早期妊娠流产的过程,观察其发育形态学和胚层分化的改变,检测拟胚体直径大小变化、拟胚体内部细胞凋亡变化及拟胚体胚层的发育水平,以此为标准确定药物流产毒性评价的指标,为后续药物筛选提供参考。
Description
技术领域
本发明涉及生物医学领域,涉及一种利用诱导多能干细胞构建妊娠期致流产药物筛查模型的方法及应用。
背景技术
随着我国“全面两孩”政策的开放,高龄和高危产妇增多,妊娠期用药往往不可避免。妊娠期药物的系统评价显示,60%~90%妊娠期妇女需要使用药物,平均用药2~4种,最多达8种。其中约79%孕妇服用过对胎儿影响不明的药物,增加了自发流产、早产和低体重儿的发生概率。因此,有效地筛查具有生殖毒性的药物,对实现妊娠期妇女安全用药这一目的至关重要。传统的药物生殖毒性风险评估往往是基于怀孕的实验动物模型或细胞模型筛选的结果。动物模型通常以小鼠、家兔和斑马鱼等作为实验动物。利用此模型筛选药物通常面临构建怀孕动物模型耗时费力、实验成本高昂等问题。动物与人类还存在种属差异性问题,其结果可能无法准确地预测药物在人体的不良反应。细胞模型是一种动物替代的实验方法,其中胚胎干细胞实验(Embryonic stem cell test,EST)被正式批准用于体外筛选生殖毒性药物和化合物。但是它丧失了组织、细胞发育的微环境,破坏了组织和器官的结构,不利于对整体胚胎发育的毒理学分析。因此,寻求一种简便、灵敏、准确、经济的妊娠期药物筛选模型势在必行。
通过干细胞重编程技术,利用诱导多能干细胞(Induced pluripotent stemcells,iPS细胞)对药物进行筛选逐步得到广大药物和医务工作者的认可。iPS细胞是通过特定基因转染或小分子化合物诱导等方式,使正常体细胞重编程为胚胎干细胞(embryonicstem cells,ES细胞)式的多潜能细胞。利用人类源性诱导多能干细胞建立发育毒性药物筛选存在以下优势:1.两者在细胞形态、生长特性、细胞标志物表达类似,具有体外发育成三胚层的潜力;2.iPS细胞取材方便,制备的来源细胞类型很多,如成纤维细胞、外周血细胞和脂肪干细胞等,规避了破坏胚胎等伦理学问题;3.人类来源的iPS细胞模型无物种差异性问题。目前利用诱导多能干细胞构建模型实现对药物的筛选已广泛应用于神经发育毒性和心血管发育毒性等研究。
发明内容
为了克服现有技术的不足,本发明的目的之一在于提供一种利用诱导多能干细胞构建妊娠期致流产药物筛查模型的方法,该方法可有效确定药物流产毒性评价指标,并对后续药物筛选具有参考价值。
本发明的目的之二在于提供该方法在妊娠期致流产药物筛查中的应用。
本发明的目的之一采用如下技术方案实现:
一种利用诱导多能干细胞构建妊娠期致流产药物筛查模型的方法,包括以下步骤:
1)拟胚体的培养:通过三维悬浮培养的方式对人诱导多能干细胞(iPS细胞)进行培养,形成拟胚体(EBs);
2)施加待筛选药物;
3)通过分析拟胚体发育的指标筛选和/或评价所述药物。
进一步地,所述步骤1)中拟胚体的培养,具体步骤为:
S1.通过将外源因子组合OCT4、SOX2和KLF2导入正常人的尿上皮细胞重新编程获得诱导多能干细胞;
S2.将步骤(1)中的诱导多能干细胞转移至以Matrigel为基质代替饲养层细胞的无血清STEMdiffTM的培养基中,培养4~5天后,用EDTA消化液直接消化细胞;
S3.将步骤(2)中消化后的细胞转移至含y-27632诱导因子的拟胚体培养基,在超低黏附96孔板中进行悬浮培养,细胞浓度为1×104~1×105cell/mL;
S4.次日,添加不含y-27632诱导因子的拟胚体培养基,每日半量换液;
S5.体外培养3~4天,即得拟胚体。
进一步地,所述步骤2)将得到的拟胚体体外培养2天后,移入含有待筛选药物的拟胚体培养基中。
进一步地,所述步骤3)中拟胚体发育指标包括拟胚体直径大小变化、拟胚体内部细胞凋亡变化、拟胚体胚层发育水平。
进一步地,所述步骤1)得到的拟胚体可继续分化。若分析妊娠中晚期的流产或死产,则需要对大脑皮质类器官进行观察。通过添加特异性的神经信号分子等诱导剂促使拟胚体向神经系统方面发育。
进一步地,所述拟胚体向神经系统的诱导培养,具体步骤为:
(1)拟胚体培养5天后,加入1.5mL神经上皮诱导培养基,诱导培养48h;
(2)更换步骤(1)中培养基为2.0mL神经上皮扩大培养基,培养96h;
(3)更换步骤(2)中培养基为2.0~4.0mL大脑皮质成熟培养基,每隔3~4天换液,培养至50-52天。
本发明的目的之二是提供上述方法在妊娠期致流产药物筛查中的应用。
相比现有技术,本发明的有益效果在于:
本发明提供一种利用诱导多能干细胞构建妊娠期致流产药物筛查模型的方法,通过对人诱导多能干细胞三维培养形成拟胚体EBs作为药物筛查模型。本发明使用的是尿上皮来源性的iPS细胞,其在细胞形态、多能基因的表达、三胚层分化能力等方面与胚胎干细胞高度相似,且取材方便、无伦理性和种族差异性问题。EBs在结构和功能上与胚胎早期发育阶段的胚泡相似,可以体外模拟整个早期胚胎从内细胞团向三胚层体系的分化过程。向拟胚体的培养基中施加待筛选药物暴露,通过分析拟胚体在含有待筛选药物的培养基中的发育指标筛选和/或评价该药物。本申请的药物筛选模型的实验操作简单,观察周期短,且测量指标易获得,在一般药物毒性实验室都可以进行操作。本发明还提供了上述方法在妊娠期致流产药物筛查中的应用,该方法构建的模型基于EBs类器官的发育,动态模拟了整体胚胎早期妊娠流产的过程,观察其发育形态学,细胞凋亡和胚层分化的改变,以此为标准确定药物流产毒性评价的指标,为后续药物筛选提供参考。
附图说明
图1为本发明iPS细胞的拟胚体和大脑皮质类器官的发育过程;
图2为本发明施加米非司酮暴露后EBs直径大小的变化;
图3为本发明施加米非司酮暴露后EBs的凋亡情况;
图4为本发明施加米非司酮暴露后不同胚层细胞的分化情况;
图中*和△表示P<0.05,**和△△表示P<0.01。
具体实施方式
下面,结合附图以及具体实施方式,对本发明做进一步描述,需要说明的是,在不相冲突的前提下,以下描述的各实施例之间或各技术特征之间可以任意组合形成新的实施例。
实施例
1.拟胚体的培养
1)iPS细胞的来源:本研究的来源细胞购买于中国科学院(UE017C1)iPS细胞系,通过电转的方法将外源因子组合OCT4、SOX2和KLF2导入正常人的尿上皮细胞重编程获得iPS细胞,经过各项干性指标检测符合人类多能干细胞的标准。
2)iPS细胞三维悬浮培养为EBs:
将上述步骤中得到的细胞转移至以Matrigel基质代替饲养层细胞的无血清STEMdiffTM培养基中,每天更换iPS细胞培养基,培养4~5天使iPS细胞克隆团长到每孔70%~80%时,用0.5mM的EDTA消化液(品牌:Life technologies,15575020)直接消化细胞,37℃孵育5~8min后,终止消化;
将消化后的细胞转移至含(10μM)ROCK抑制剂y-27632(品牌:stemcell,72302)的拟胚体培养基(品牌:stemcell,08574)进行培养,吹打重悬上述步骤得到的iPS细胞,用血小板计数器进行细胞计数,调整细胞浓度为1×104~1×105cell/mL备用;将处理后的iPS细胞接种至超低黏附U型底96孔板中,每孔100μL;分散的iPS细胞迅速聚集成iPS克隆(如图1A所示)次日,每孔再添加75μL不含y-27632因子的新鲜拟胚体培养基,每日半量换液;体外培养第2天(图1B),iPS细胞克隆继续聚集成具有三维结构的EBs。继续培养至第4天,EBs边界清晰,边缘呈半透明环状(图1C),即得EBs。
1.1.EBs的继续分化:若对类皮质进行观察,可拟胚体的培养基中添加特异性的神经信号分子等诱导剂促使EBs向神经系统发育,实现EBs继续分化,表明尿上皮细胞诱导得到的iPS细胞可维持多向分化潜能,与ES细胞高度相似。EBs的继续分化如下:
(1)EBs培养5天后,加入1.5mL神经上皮诱导培养基(品牌:StemdiffTM cerebralorganoid basal medium-1,stemcell,08572;Supplement B,stemcell,08575),诱导培养48h;
(2)更换步骤(1)中培养基为2.0mL神经上皮扩大培养基(品牌:StemdiffTMcerebral organoid basal medium 2,stemcell,08573;Supplement C,stemcell,08576;Supplement D,stemcell,08577),培养96h;
(3)更换步骤(2)中培养基为2.0~4.0mL大脑皮质成熟培养基(品牌:StemdiffTMcerebral organoid basal medium 2,stemcell,08573;Supplement E,stemcell,08578),每隔3天或4天换液,培养至52天。
对类皮质进行观察时,需要添加特异性的神经信号分析诱导剂促使EBs向神经系统发育。培养至第8天,将EBs转移到Martrigel基质胶做的液滴中,Martrigel基质为类器官三维悬浮培养提供支架(图1D);培养至第15~20天(图1E和图1F),EBs外围出现数个玫瑰花环状半透明的芽突(图中*所示),初级类器官的形态可见;继续培养观察第42天(图1G)EBs发育情况可以看出,类器官表面玫瑰花状结构会逐渐融合或退化,只有少数会形成放射状的结构的大脑皮质类器官;EBs继续分化至第52天时(图1H),发现皮质类器官出现典型的层次化分布特征(图中↑所示)。人尿源细胞诱导形成的iPS细胞具备胚胎干细胞的继续分化功能,可通过添加诱导因子可诱导EBs定向分化。
2.施加待筛选药物
2.1)药物的选择
目前临床指南推荐终止早期妊娠的药物是米非司酮和米索前列醇,尤其是米非司酮对早期胚胎发育尤为敏感,是目前临床上使用最广泛的流产药物。本研究选用药物为米非司酮(品牌:Sigma,M8046,规格:100mg)。以倒置显微镜下观察施加该药物导致的EBs体积固缩、球体破裂和周围散在分布的死亡细胞增多为死亡观察指标,利用SPSS软件的回归模型计算米非司酮暴露EBs的半数致死剂量LD50(Lethal Dose,50%)。数据显示LD50为31.70μg/mL(95%CI:18.98~44.35μg/mL),根据LD50数值确定本实验中米非司酮用量,以无水乙醇为溶剂溶解,分别设置浓度为低剂量(10μg/mL)、高剂量(20μg/mL)和空白剂量(0μg/mL)。
2.2)实验建模
挑选外观大小均匀的EBs进行培养,2天后开始施加待筛选药物进行暴露。根据米非司酮用药浓度的不同,将其分成3组。(1)低剂量组:将EBs移入含有10μg/mL米非司酮的拟胚体培养基中培养;(2)高剂量组:EBs移入含有20μg/mL米非司酮的拟胚体培养基培养;(3)对照组:将EBs移入含有0μg/mL米非司酮的拟胚体培养基培养。为消除不同组别间无水乙醇用量不同对EBs的影响,各组根据米非司酮加入量的不同分别加入相应体积的无水乙醇。每天半量换液,观察并定期收集EBs。
3.通过分析EBs发育的指标筛选和/或评价米非司酮
利用倒置显微镜,定期观察EBs直径和形态学改变,通过免疫荧光技术来观察不同组不同时期EBs胚层细胞发育和分化情况,并收集图片。使用SPSS 21对所得结果进行统计,测量所得的数据利用单因素方差分析和t检验进行分析。结果采用均值±标准差表示,P<0.05时为差异具有统计学意义。
3.1)用药后拟胚体直径大小变化
以EBs直径(μm)为测量指标表征用药后拟胚体直径大小变化。结果如图2所示,利用倒置显微镜对EBs的生长动力学参数进行观察,通过观察不同培养阶段EBs直径、体积的变化,旨在探讨该化学物质对胚胎的毒性作用。图2A-2C显示体外培养第5天时,米非司酮暴露后对照组(Control)、低剂量(L-RU486)和高剂量组(H-RU486)间EBs直径变化不明显。图2D-2F显示第8天时,三组间EBs的直径随着米非司酮剂量增加而减小,各组间EBs直径与剂量呈依赖性关系(P<0.01)。将收集到的图片用Image J 1.48软件进行数据处理,得到的数据如图2M所示,三组间EBs的直径差异显著,对照组EBs直径约为(636.73±19.48)μm,低剂量组EBs直径约为(564.74±15.95)μm,高剂量组EBs直径约为(522.65±6.03)μm,其中高剂量组EBs直径比对照组缩小约17.92%(P<0.01)。此外,倒置显微镜下还发现EBs周围分布的较小的聚集体逐渐退化消失。图2G-2I显示培养至第11天,三组间EBs直径差异进一步增大,随着剂量增加呈依赖性下降趋势(P<0.01);对照组EBs直径约为(670.68±11.73)μm,低剂量组EBs直径约为(591.19±6.05)μm,高剂量组EB直径约为(538.92±18.55)μm,高剂量组EBs直径比对照组缩小约19.64%(P<0.01)。
分别以Sox2阳性细胞标记胚胎干细胞分化,CXCR4阳性标记中胚层细胞分化。图2J-2L显示EBs培养至11天时胚层的发育情况,图中采用免疫荧光双标记法标记,其中Sox2显示为绿色,CXCR4显示为红色,同组对比结果表明随着体外培养时间的增加,EBs体积逐渐增大。随着米非司酮用药剂量的增加,EBs体积减小,且EBs内部出现囊性空洞的结构。综上,米非司酮的用药剂量与EBs直径大小具有相关性,随着米非司酮剂量增加呈依赖性下降趋势。高剂量组米非司酮暴露造成EBs直径相对于对照组缩小18%以上,可以作为流产药物筛选的一个重要指标。
3.2)用药后拟胚体内部细胞凋亡的变化
以细胞凋亡率为测量指标表征用药后拟胚体内部细胞凋亡的变化。参数公式为:细胞凋亡率(%)=TUNEL阳性细胞数/细胞总数×100%。利用TUNEL和DAPI分析并比较各组EBs内部细胞凋亡情况,结果如图3所示:细胞凋亡是细胞主动激活的程序性死亡过程,早期的凋亡细胞核固缩,染色加深,或呈新月形聚集于核膜一侧,晚期凋亡细胞出现凋亡小体,即细胞膜包裹的内含核碎片、胞质和细胞器的大小不等的圆形小体。图3A-3C表明用DAPI染色显示,培养5天之后,体外培养第5天发现,DAPI染色的凋亡细胞指数分别是对照组约为(18.56±6.70)%,低剂量组约为(25.62±0.80)%,高剂量组约为(32.15±7.73)%。其中,米非司酮暴露组凋亡小体逐渐增多,呈剂量依赖性关系(P<0.05)。
TUNEL法也可以检测细胞凋亡,其原理是通过标记DNA断裂的3’-OH末端来检测凋亡细胞。结果如图3D-3F所示,米非司酮用药组中TUNEL阳性细胞细胞核呈片状分布,细胞排列紊乱。体外培养第5天,对照组细胞凋亡率约为(24.18±4.16)%,低剂量组细胞凋亡率约为(29.80±1.24)%,高剂量组细胞凋亡率约为(50.99±4.89)%。三组间细胞凋亡数存在统计学差异(P<0.05),且与DAPI染色结果相比,TUNEL阳性细胞凋亡数偏高,敏感度更高。图3G-3I表明培养第8天时,三组间EBs内部细胞凋亡率差异显著。具体凋亡率如图3M所示,对照组细胞凋亡率约为(26.66±5.01)%,低剂量组细胞凋亡率约为(48.62±8.90)%,高剂量组细胞凋亡率约为(63.36±7.40)%。其中高剂量组比对照组细胞凋亡率增加43.60%(P<0.01)。图3J-3L表明培养第11天时发现,随着体外培养时间的增加,EBs中心部凋亡区域扩大,凋亡细胞数逐渐增多。对照组EB细胞凋亡率约为(38.56±4.70)%,低剂量组EB细胞凋亡率约为(57.62±0.80)%,高剂量组EB细胞凋亡率约为(82.15±7.73)%。其中高剂量组比对照组细胞凋亡率增加53.07%(P<0.01)。综上,米非司酮暴露可以诱导细胞凋亡,在含有米非司酮药物的培养基培养8天之后,高剂量组细胞凋亡率相对于空白对照组增加约44%,细胞凋亡率也可以作为流产药物筛选的重要指标。
3.3)用药后拟胚体胚层发育水平
以Sox2阳性细胞、NeuN标记成熟神经元、GFAP标记星形神经胶质细胞的密度、Nestin和CXCR4阳性细胞有效分化率为测量指标。参数公式分别为:(1)Sox2阳性细胞密度(百个/mm2)=Sox2阳性细胞数/观测面积);(2)胚层细胞分化率(%)=阳性细胞数/细胞总数×100%(如外胚层源性Nestin和中胚层源性CXCR4阳性细胞)。
EBs是多种类型细胞聚合的三维球体结构,根据不同细胞间作用力的强弱,自发的分化出三胚层结构。我们选用Nestin标记外胚层中神经干细胞,利用膜蛋白CXCR4标记中胚层,而中胚层是结缔组织、肌肉、骨髓和血液的原基。FoxA2可以标记内胚层,内胚层是肝、胰等消化腺以及消化管和和呼吸道上皮的生发原基。同时,我们还试图利用Sox2标记胚胎干细胞、NeuN标记成熟神经元、GFAP标记星形神经胶质细胞。
(1)Nestin阳性外胚层细胞分化:如图4A-4C所示,荧光染色结果显示EBs培养第11天时,大量的Nestin阳性细胞呈双极形态突起并贯穿NR管腔的神经上皮组织。Nestin阳性细胞分化率对比如图4N所示:其中,对照组Nestin阳性细胞有效分化率约为(45.80±4.15)%,低剂量组Nestin阳性细胞有效分化率约为(29.00±1.09)%,高剂量组Nestin阳性细胞有效分化率约为(16.75±4.23)%,其中高剂量组比对照组减少约63.44%(P<0.01)。此外,图4D-4F显示在NR管腔内部发现了Caspase-3阳性的细胞,且随着剂量增加,染色加深。结果表明米非司酮可以抑制神经系统发育,导致神经管内部细胞凋亡。
(2)CXCR4阳性中胚层细胞分化:图4G-4I显示培养至第11天,CXCR4阳性细胞多集中在EBs的中心区域,呈剂量依赖性增加(P<0.05)。随着用药剂量的增加,CXCR4阳性细胞相互缠绕成结。CXCR4阳性细胞分化率对比如图4O所示:对照组CXCR4阳性细胞有效分化率约为(31.49±5.81)%,低剂量组CXCR4阳性细胞有效分化率约为(41.81±5.40)%,高剂量组CXCR4阳性细胞有效分化率约为(43.74±3.10)%,其中高剂量比对照组增加约28.01%(P<0.05)。结果表明米非司酮可能促进心血管系统发育。
(3)FoxA2阳性内胚层细胞分化:图4J-4L显示EBs内胚层FoxA2阳性细胞的表达较少,与米非司酮用药无明显影响。
(4)Sox2阳性胚胎干细胞分化:图4G-4I显示培养至第11天,Sox2阳性细胞多分布在EBs外围,随着米非司酮用药剂量的增加,Sox2阳性细胞数量逐渐减少(P<0.01)。第11天,EBs外围有大量未分化的Sox2阳性细胞,对照组Sox2阳性细胞密度约(14.35±0.69)百个/mm2,低剂量组Sox2阳性细胞密度约(8.99±0.51)百个/mm2,高剂量组Sox2阳性细胞密度约(4.63±0.62)百个/mm2,Sox2阳性细胞密度与米非司酮呈剂量依赖性关系。其中,高剂量组Sox2阳性细胞密度比对照组减少约67.72%(P<0.01)。图4M显示随着培养时间的延长,对比组细胞Sox2阳性细胞密度增加,低剂量组细胞密度先降低后增加,高剂量组密度逐渐减少,说明高剂量米非司酮可以抑制Sox2阳性细胞增殖。
米非司酮暴露影响胚层的发育,抑制胚胎干细胞增殖、抑制外胚层细胞分化,促进中胚层细胞增多。具体表现为Sox2阳性细胞密度减少高达约68%,Nestin阳性细胞有效分化率减少达约63%,CXCR4阳性细胞有效分化率增加约28%,用药后三胚层的发育水平可以作为流产药物筛选的辅助指标。
综上,米非司酮可导致EBs直径变小、细胞凋亡率增加、抑制胚胎干细胞增殖、抑制外胚层细胞分化、促进中胚层细胞发育。结合试验结果,可以将如下标准建立药物流产毒性评价的指标:EBs直径缩小约18%以上,细胞凋亡率增加约44%以上,是流产药物的筛选重要指标。Sox2阳性胚胎干细胞密度减少高达68%以上,Nestin阳性外胚层细胞有效分化率减少达63%以上,CXCR4阳性中胚层细胞有效分化率增加28%以上,可以作为流产药物筛选的辅助指标。基于此,利用已知指标筛选和/或评价未知药物的流产副作用,为后续药物筛选提供参考。
上述实施方式仅为本发明的优选实施方式,不能以此来限定本发明保护的范围,本领域的技术人员在本发明的基础上所做的任何非实质性的变化及替换均属于本发明所要求保护的范围。
Claims (6)
1.一种利用诱导多能干细胞构建妊娠期致流产药物筛查模型的方法,其特征在于,包括以下步骤:
1)拟胚体的培养:通过三维悬浮培养的方式对人诱导多能干细胞进行培养,形成拟胚体;
2)施加待筛选药物;
3)通过分析拟胚体发育的指标筛选和/或评价所述药物。
2.如权利要求1所述的方法,其特征在于,所述步骤1)中拟胚体的培养,具体步骤为:
S1.通过将外源因子组合OCT4、SOX2和KLF2导入正常人的尿上皮细胞重新编程获得诱导多能干细胞;
S2.将步骤(1)中的诱导多能干细胞转移至无饲养层的培养基中,培养4~5天后,用EDTA消化液消化;
S3.将步骤(2)中消化后的细胞转移至含y-27632诱导因子的拟胚体培养基进行悬浮培养,细胞浓度为1×104~1×105cell/mL;
S4.次日,添加不含y-27632诱导因子的拟胚体培养基,每日半量换液;
S5.体外培养3~4天,即得拟胚体。
3.如权利要求2所述的方法,其特征在于,所述步骤(2)培养基为无血清STEMdiffTM,并以Matrigel为基质。
4.如权利要求1所述的方法,其特征在于,所述步骤2)为将步骤1)得到的拟胚体继续培养2天后,移入含有待筛选药物的拟胚体培养基中。
5.如权利要求1所述的方法,其特征在于,所述步骤3)中拟胚体发育指标包括拟胚体直径大小变化、拟胚体内部细胞凋亡变化、拟胚体胚层发育水平。
6.如权利要求1至5任一项所述方法在妊娠期致流产药物筛查中的应用。
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