CN113528576A - 含有nlrp7纯和突变的复发性葡萄胎患者特异性诱导多能干细胞系及构建方法 - Google Patents
含有nlrp7纯和突变的复发性葡萄胎患者特异性诱导多能干细胞系及构建方法 Download PDFInfo
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Abstract
本发明公开了一种含有NLRP7纯和突变的复发性葡萄胎患者特异性诱导多能干细胞系及构建方法。首先收集NLRP7基因突变患者的外周血,然后分离和培养外周血单个核细胞,最后非整合病毒方法重编程为诱导干细胞,获得含有NLRP7突变的复发性葡萄胎患者特异性诱导多能干细胞。该诱导多能干细胞表达多能性分子标记,有形成畸胎瘤三个胚层分化的能力,通过突变检测、STR鉴定明确其患者来源,为NLRP7突变相关疾病的发病机制研究和治疗探索提供干细胞模型。
Description
技术领域
本发明属于生物医药领域,具体涉及一种含有NLRP7纯和突变(c.1261C>T)的复发性葡萄胎患者特异性诱导多能干细胞系及其构建方法。
背景技术
葡萄胎是一种异常的人类妊娠,以绒毛间质水肿同时缺乏胚胎发育或异常胚胎发育为特征。根据大体、镜下病理特点、核型分析以及临床表现,可以将葡萄胎分类为完全性葡萄胎和部分性葡萄胎。遗传学研究发现双亲来源的葡萄胎是一种特殊类型,约占完全性葡萄胎的20%,与家族复发性葡萄胎相关,多因基因突变导致,其中最主要的突变基因就是NLRP7,约占全部复发性葡萄胎的48-80%。
NLRP家族属于胞浆组合蛋白,包括三个结构:分别是PYD区域、NACHT区域和LRR区域。NLRP7,又名NALP7、PYPAF3、NOD12、PAN7、CLR19.4,该基因位于人类常染色体19q13.42上,是NLRP家族14个成员之一,表达于滋养细胞、卵巢、睾丸、心脏、肺脏、肝脏等器官。到目前为止已发现超过200个NLRP7基因变异与复发性葡萄胎相关。尽管NLRP7基因变异与葡萄胎发病的相关性是基本明确的,但是其突变发生后导致疾病的分子机制尚不清楚,也缺乏有效的治疗手段。并且NLRP7在生殖系统、妊娠发育以及胎盘形成等过程中都有非常重要的作用,但是其机制研究也因缺乏合适的模型进展非常缓慢。
随着体细胞重编程技术的发展,获取患者特异性的诱导多能干细胞成为现实。研究表明,诱导多能干细胞具有向机体多种细胞分化的能力,并且携带患者特有的遗传信息,可以建立患者特异性的体外培养细胞模型进行疾病模拟,以研究发病机制或者进行治疗方法的探索。采用血液中的单个核细胞进行重编程,避免既往取成纤维细胞重编程的有创性,降低尿液重编程的污染风险。
因此,构建含有NLRP7纯和突变的复发性葡萄胎患者特异性诱导多能干细胞系,可以为NLPR7基因突变相关疾病的发病机制和治疗研究提供新的疾病模拟模型。
发明内容
本发明的目的是针对现有技术的不足,提供一种含有NLRP7纯和突变(c.1261C>T)的复发性葡萄胎患者特异性诱导多能干细胞系及构建方法,本发明构建含有NLRP7纯和突变的复发性葡萄胎患者特异性诱导多能干细胞系,可以为NLPR7基因突变相关疾病的发病机制和治疗研究提供新的疾病模拟模型。
本发明的目的是通过以下技术方案来实现的:一种含有NLRP7纯和突变的复发性葡萄胎患者特异性诱导多能干细胞系的构建方法,包括以下步骤:在无菌条件下取NLRP7基因突变患者的静脉血,分离外周血单个核细胞,体外扩增后重编程为诱导多能干细胞,获得含有纯和突变的复发性葡萄胎患者特异性诱导多能干细胞系;所述的NLRP7基因突变是NLRP7基因的第1261位碱基由C突变为T(氨基酸第421位由Gln突变位终止密码子),为纯和突变。
进一步地,所述的外周血单个核细胞是在收集静脉血后采用密度梯度离心方法,获得单个核细胞。使用PBS洗涤,重悬于CD34阳性细胞扩增培养基中悬浮培养6-8天;所述的扩增培养基为500ml StemSpanTM SFEM II加入10ml StemSpanTM CD34+ExpensionSupplement和1×双抗(青链霉素混合液)。
进一步地,所述的血液细胞重编程为诱导多能干细胞是在扩增的CD34+细胞中通过非整合病毒转入OCT4、SOX2、c-MYC和KLF4四因子,将细胞转移至由Matrigel包被的培养板上,E7培养基隔天换液,10-12天左右出现克隆样细胞,改为mTeSR1培养基培养;再经过传代培养,获得含有NLRP7纯和突变的复发性葡萄胎患者特异性诱导多能干细胞系。
进一步地,干细胞传代培养后,干细胞扩增密度达到约80%,使用ACCUTASE进行消化和1:5-6传代。
进一步地,所述的非整合病毒优选为仙台病毒。
一种含有NLRP7纯和突变的复发性葡萄胎患者特异性诱导多能干细胞系由上述制备方法制得。
本发明的有益效果是:本发明获取的复发性葡萄胎患者特异性诱导干细胞系,填补了国内NLRP7研究在干细胞研究领域的空白。为体外模拟因NLRP7突变导致的疾病模型的建立提供重要细胞来源。本发明获得了含有NLRP7纯和突变的复发葡萄胎患者特异性诱导多能干细胞系,与NLRP7突变患者具有相同的遗传背景,可以分化成为多种细胞类型,包括滋养细胞,可以为NLRP7突变相关疾病的发病机制和治疗研究提供新的疾病模型。
附图说明:
图1是外周血单个核细胞体外悬浮培养照片;
图2中的(A)是病毒转染后形成的克隆特征照片,图2中的(B)是干细胞克隆碱性磷酸酶染色图;
图3是OCT4的转录组表达情况图;
图4是SOX的转录组表达情况图;
图5是NANOG的转录组表达情况图;
图6中的(A)是畸胎瘤HE染色图的鳞状上皮,图6中的(B)是畸胎瘤HE染色图的神经组织,图6中的(C)是畸胎瘤HE染色图的软骨,图6中的(D)是畸胎瘤HE染色图的肠腔上皮。
具体实施方式
实施例1:外周血单核细胞分离和体外扩增实验
①患者为38岁女性,既往5次葡萄胎病史,其中三次妊娠产物核型分析确定为双亲来源完全性葡萄胎。随后外周血Sanger测序证实其NLRP7基因有纯和致突变。在无菌条件下抽取静脉血5ml分离外周血单个核细胞。
②试剂及耗材具体为:
1)Ficoll-Paque,GE:#17144002;
2)PBS缓冲液,HyClone:#SH30256.01B;
3)15ml离心管康宁:#339650;
4)50ml离心管康宁:#339652;
5)巴氏吸管。
③所用仪器为:
1)Eppendorf离心机5804R;
2)二氧化碳培养箱Thermo Fisher 371;
3)倒置显微镜Olympus CKX41。
④具体步骤如下:
1)按1:1等体积加入PBS,并转移至干净的15ml/50ml离心管;
2)用巴氏吸管将15ml/50ml管中的样品吹打混匀,每管吸打时间不少于8min;
3)按照步骤2)中血样的总量准备新的15ml离心管,每管用巴氏吸管加约3ml的Ficoll-Pague,再将吹打混匀的血样缓缓加在Ficoll上层,加4ml血样;
4)加样完毕,将离心管在25℃下按离心转速为1410rpm离心30min,离心时将下降速率调至0档(自然降速);升速也可以降到7-8档;
5)将离心后离心管取出,用巴氏吸管轻轻将最上层吸出一部分,剩余约5-6ml,不要碰到白细胞层;
6)将巴氏吸管伸至白细胞层转圈并吸出白细胞,将每一管吸出的白细胞合并至新的15ml离心管;
7)加PBS至10ml处混匀几次后,在25℃下按离心转速为1410rpm离心10min,此时下降速度可改为7-8档;
8)离心后取出离心管,将上面的液体用巴氏吸管吸出丢弃,留约2ml;
9)再加PBS至8ml处,轻轻混匀后在25℃下按离心转速为1410rpm离心8min;
10)去除上清液,扩增培养基500ul悬浮,置入12孔板一孔,37℃二氧化碳孵箱悬浮培养6-8天,如图1所示,小圆细胞分散悬浮于培养基中,隔日换液。所述的扩增培养基由StemSpanTM SFEM II 500ml加入StemSpanTM CD34+Expension Supplement 10ml和1×双抗(青链霉素混合液)组成。
实施2:血液重编程与扩增传代实验
①细胞:采用外周血单个核细胞。
②试剂及耗材具体为:
1)PBMC基础培养基:StemSpanTM SFEM II,Stem cell:#09605;
2)CD34+扩增培养基:StemSpanTM CD34+Expansion Supplement(10X),Stemcell:#02691;
3)ReproTeSRTM Medium,Stem cell:#05920;
4)mTeSRTM1,Stem cell:#05850;
5)仙台病毒,Thermo Fisher Scientific:#A16517(包括OCT4、SOX2、c-MYC和KLF4四因子);
6)Matrigel,BD Sciences:#354277;
7)DMEM/F12,GIBCO:#11330;
③所用仪器为:
1)Eppendorf离心机5804R;
2)二氧化碳培养箱Thermo Fisher 371;
3)倒置显微镜Olympus CKX41;
4)细胞计数器英潍捷基Countess II;
④具体步骤如下:
1)感染仙台病毒的前一天,48孔板一孔铺Matrigel备用;
2)感染仙台病毒当天从超低温冰箱中取出仙台病毒,冰上融化;
3)计数1.5x105悬浮细胞(如出现细胞成团,建议滤网过滤后计数细胞),加入单次病毒量。
4)感染病毒一天后,将全部悬浮细胞离心,按离心速率为300g离心7min,去除仙台病毒,将细胞转移至铺有Matrigel的24孔板的一孔,培养2天。
5)第三天可见部分细胞贴壁,轻柔吸取培养基,按离心速率为300g离心7min,丢弃上清液,继续加入扩增培养基500ul。
6)第四天更换为PBMC基础培养基培养,可将悬浮细胞离心后转移至新孔内,培养2天。
7)第6-10天采用ReproTeSR半换液,按离心速率为300g离心7min,保留细胞。
8)第11天起采用ReproTeSR每日换液,至镜下见克隆后改为mTeSR每1-2日换液一次。克隆特征表现为细胞集落样生长,边界清晰,细胞核质比高,如图2所示,图2中的(A)是病毒转染后形成的克隆特征照片,图2中的(B)是干细胞克隆碱性磷酸酶染色图。
9)待克隆增至200个细胞左右,可挑出12个左右单个克隆转移到Matrigel铺底的12孔板中,命名为第1代(P1),并继续用mTeSR培养液培养7-10天后。
10)7-10天后,克隆逐渐生长扩增,以0.5ml Accutase消化传代至Matrigel铺底的6孔板的1孔中,命名为第2代(P2),并继续用mTeSR培养液培养。
11)干细胞再传代培养3-5天左右,干细胞扩增密度达到约80%,使用ACCUTASE进行消化和1:5-6传代
干细胞特征鉴定实验:诱导干细胞突变鉴定
①细胞:NLRP7突变诱导多能干细胞
②试剂及耗材具体为:
1)TIANamp Genomic DNA Kit,TIANGEN:#DP304;
2)2XPCR Master Mix,碧云天:#D7228;
③所用仪器具体为:
1)离心机Eppendorf 5424R;
2)NANOdrop Thermo ND2000;
3)PCR仪安捷伦EasyCycler Gradient 96;
④具体步骤如下:
1)取6孔板1孔细胞,去除上清液,PBS漂洗;
2)加入1mlAccutase,室温孵育5分钟;
3)加入DMEM/F12,将细胞转移至15ml离心管中,按离心速率为200g离心5min,去上清;
4)根据试剂盒说明提DNA,NANOdrop定量;
5)设计引物,根据PCR扩增说明书配制50ul反应体系,反应参数:起始变性94℃3min,变性94℃30sec,退火55℃30sec,延伸72℃1min,30个循环后最终延伸72℃10min,然后4℃保存;
6)对PCR产物进行测序,测序结果显示建立的患者来源的诱导干细胞系,携带目的突变NLRP7(c.1261C>T),与患者外周血测序结果一致;
7)扩增引物及测序引物见表1。
干细胞特征鉴定实验:免疫荧光染色
①细胞:NLRP7突变诱导多能干细胞
②试剂及耗材具体为:
1)多聚甲醛,Biosharp:#BL539A;
2)Triton X-100,普利莱:#B1014;
3)BSA,Sigma:#V900933-100g;
4)抗体清单如表1所示;
5)抗淬灭封片剂,Biosharp:#BL701A;
③仪器具体为:荧光共聚焦显微镜Nikon;
④具体步骤如下:
1)吸走培养基,用温浴过的PBS漂洗1次;
2)常温4%的多聚甲醛(PFA)固定15min;
3)用温浴过的PBS漂洗3次;
4)0.2%的Triton X-100/PBS常温下孵育5min,;
5)用温浴过的PBS漂洗3次;
6)常温下3%的BSA/PBS封闭1h;
7)孵一抗,4℃过夜;
8)用温浴过的PBS漂洗3次,每次5min;
9)常温下孵二抗和DAPI,2h;
10)用温浴过的PBS漂洗3次,每次5min;
11)荧光显微镜下观察细胞,可见克隆样生长的细胞,DAPI蓝染,目的蛋白OCT4、NANOG、SOX2为细胞核染色,SSEA4细胞膜染色阳性。
表1:抗体和引物信息
干细胞特征鉴定实验:RT-PCR
①细胞:NLRP7突变诱导多能干细胞
②试剂及耗材具体为:
1)TRIzol,Life Technologies:#15596-026;
2)High Capacity cDNA Reverse Transcription Kit,Applied Biosystems:#4368814;
3)SYBR Green PCR Master Mix,Takara:#DRR820A;
③所用仪器具体为:
1)Applied Biosystems StepOne(Thermo Fisher Scientific);
2)台式离心机Eppendorf 5424R;
④具体步骤如下:
1)取6孔板1孔细胞,去除上清液,PBS漂洗;
2)加入1mlAccutase,室温孵育5分钟;
3)加入DMEM/F12,将细胞转移至15ml离心管中,按离心速率为200g离心5min,去上清层;
4)加入1ml TRIzol,室温放置5min,使其充分裂解;
5)加入200ul氯仿,震荡混匀后室温放置15min;
6)在4℃下按离心速率为12000g离心15min;
7)吸取上层水相至另一离心管中,加入0.5ml异丙醇,混匀,室温放置5min;
8)在4℃下按离心速率为12000g离心15min,丢弃上清液;
9)加入无水乙醇1ml,震荡离心,悬浮沉淀;
10)在4℃下按离心速率为7500g离心5min,弃上清层,室温干燥10min,挥发无水乙醇,得到沉淀即RNA样本;
11)50ul RNAse-free水溶解步骤10)制得的RNA样本,NANOdrop定量;
12)根据逆转录试剂盒High Capacity cDNA Reverse Transcription Kit说明步骤完成RNA逆转录为cDNA;
13)再根据SYBR Green PCR Master Mix说明上样,Applied Biosystems StepOne完成定量,每个RNA样本最少4个复孔,降低误差;
14)结果见图3、4、5,转录组水平上OCT4、SOX2和NANOG均明显高于外周血单个核细胞,与以往建立的诱导干细胞水平相似。
干细胞特征鉴定实验:畸胎瘤成瘤实验
①细胞:NLRP7突变诱导多能干细胞;动物:NOD-SCID小鼠
②试剂及耗材具体为:
1)Matrigel,康宁:#354277;
2)福尔马林;
3)苏木素伊红染色液,Solarbio:#G1120;
③所用仪器具体为:
1)石蜡切片机:Leica RM2235;
2)显微镜:ZEISS;
④具体步骤如下:
1)将含有1-2x106的目的干细胞悬浮于mTeSR中,再与Matrigel按体积比1:1混合;
2)注射到小鼠四肢内侧皮下及背部;
3)移植后4周开始隔日查看肿瘤,待直径超过0.5cm可以处死小鼠,取畸胎瘤组织福尔马林固定;
4)组织脱水,蜡块包埋,常规切片;
5)石蜡切片进行HE染色;
6)显微镜观察,将外胚层(鳞状上皮、神经组织)、中胚层(软骨)、内胚层(肠腔腺体)分别拍照,如图6所示,图6中的(A)是畸胎瘤HE染色图的鳞状上皮,图6中的(B)是畸胎瘤HE染色图的神经组织,图6中的(C)是畸胎瘤HE染色图的软骨,图6中的(D)是畸胎瘤HE染色图的肠腔上皮。
干细胞特征鉴定实验:仙台病毒去除鉴定
①细胞:NLRP7突变诱导多能干细胞;
②试剂及耗材具体为;
1)RNeasy Mini Kit(Qiagen);
2)High Capacity cDNA Reverse Transcription Kit,Applied Biosystems:#4368814;
3)Ex Taq DNA polymerase(Takara);
③仪器为:PCR仪Easycycler Thermocycler(Analytic Jena)
④具体步骤如下:
1)根据试剂盒说明提RNA并逆转录为cDNA;
2)使用Ex Taq DNA polymerase进行PCR扩增,扩增条件为30周期,变性95℃30sec,退火55℃30sec,延伸72℃30sec;
3)PCR产物进行1%琼脂糖凝胶电泳,阳性对照组可见100-200bp之间条带,患者特异干细胞系组无条带,确认仙台病毒已去除。
获得含有NLRP7基因纯和突变的复发性葡萄胎患者特异性诱导多能干细胞,表达干性标志物(OCT4、NANOG、SSEA4、SOX2),具有三胚层分化能力,核型正常,Sanger测序证实其含有NLRP7纯和突变(c.1261C>T)。STR鉴定结果显示其与患者血液细胞具有相同的短重复序列位点如表2所示。
表2:STR分型检测
综上,本发明获取的复发性葡萄胎患者特异性诱导干细胞系,填补了国内NLRP7研究在干细胞研究领域的空白。为体外模拟因NLRP7突变导致的疾病模型的建立提供重要细胞来源。本发明获得了含有NLRP7纯和突变的复发葡萄胎患者特异性诱导多能干细胞系,与NLRP7突变患者具有相同的遗传背景,可以分化成为多种细胞类型,包括滋养细胞,可以为NLRP7突变相关疾病的发病机制和治疗研究提供新的疾病模型。
Claims (6)
1.一种含有NLRP7纯和突变的复发性葡萄胎患者特异性诱导多能干细胞系的构建方法,其特征在于,包括以下步骤:
(1)无菌条件下收集NLRP7基因突变患者的静脉血,该NLRP7突变的患者是NLRP7基因1261位的碱基由C突变为T,纯和突变;
(2)分离和培养外周血单个核细胞;
(3)采用非整合病毒方法将步骤(2)得到的单个核细胞重编程为诱导干细胞,获得含有NLRP7突变的复发性葡萄胎患者特异性诱导多能干细胞。
2.根据权利要求1所述的含有NLRP7纯和突变的复发性葡萄胎患者特异性诱导多能干细胞系的构建方法,其特征在于,所述步骤(2)具体为:收集静脉血后采用密度梯度离心方法,去除血浆和红细胞,获得外周血单个核细胞;使用PBS洗涤,重悬于CD34阳性细胞扩增培养基中悬浮培养6-8天;所述的扩增培养基由StemSpanTM SFEM II 500ml加入StemSpanTMCD34+Expension Supplement 10ml和1×双抗(青链霉素混合液)组成。
3.根据权利要求1所述的含有NLRP7纯和突变的复发性葡萄胎患者特异性诱导多能干细胞系的构建方法,其特征在于,所述步骤(3)中,所述非整合病毒方法重编程为诱导干细胞具体为:在步骤(2)扩增的CD34+细胞中通过非整合病毒转入OCT4、SOX2、c-MYC和KLF4四因子,将细胞转移至由Matrigel包被的培养板上,E7培养基隔天换液,10-12天出现克隆样细胞,改为mTeSR1培养基培养;再经过传代培养,获得含有NLRP7纯和突变的复发性葡萄胎患者特异性诱导多能干细胞系。
4.根据权利要求3所述的含有NLRP7纯和突变的复发性葡萄胎患者特异性诱导多能干细胞系的构建方法,其特征在于,干细胞传代培养后,干细胞扩增密度达到约80%,使用ACCUTASE进行消化和1:5-6传代。
5.根据权利要求1所述的含有NLRP7纯和突变的复发性葡萄胎患者特异性诱导多能干细胞系的构建方法,其特征在于,所述非整合病毒优选为仙台病毒。
6.一种含有NLRP7纯和突变的复发性葡萄胎患者特异性诱导多能干细胞系,其特征在于,由权利要求1-5中任一权利要求所述方法制备得到。
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108913655A (zh) * | 2018-07-16 | 2018-11-30 | 浙江大学 | 基于多能干细胞技术建立“人源性”心肌肥大模型的方法 |
-
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Non-Patent Citations (4)
Title |
---|
ALICI-GARIPCAN ET AL.: "NLRP7 plays a functional role in regulating BMP4 signaling during differentiation of patient-derived trophoblasts", 《CELL DEATH AND DISEASE》 * |
TINGYU GONG, ET AL: "Reprogramming of human peripheral blood mononuclear cells from a patient suffering from recurrent hydatidiform mole to an iPSC line FAHZUi001-A carrying a homozygous p.Gln421Ter mutation in NLRP7 gene", 《STEM CELL RESEARCH》 * |
ZHAOHUI YE, ET AL.: "Human-induced pluripotent stem cells from blood cells of healthy donors and patients with acquired blood disorders", 《BLOOD》 * |
周薇等: "NLRP 7基因在葡萄胎发生中的作用", 《国际妇产科学杂志》 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108913655A (zh) * | 2018-07-16 | 2018-11-30 | 浙江大学 | 基于多能干细胞技术建立“人源性”心肌肥大模型的方法 |
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