CN111269883A - 一种线粒体mt.3243A>G突变人群中高突变尿液干细胞的制备方法 - Google Patents
一种线粒体mt.3243A>G突变人群中高突变尿液干细胞的制备方法 Download PDFInfo
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Abstract
本发明提供了一种线粒体mt.3243A>G突变人群中高突变尿液干细胞的制备方法,包括一个收集线粒体mt.3243A>G突变人群的尿液细胞的步骤,一个采用离心和加入PBS溶液吹打混匀的方式得到沉渣红管的步骤,一个获得P1代高突变尿液干细胞的步骤,一个通过消化传代和突变率检测获得线粒体mt.3243A>G突变95%以上高突变的尿液干细胞的步骤。本发明的优点在于它可以无创获取,并且无论患者的性别、年龄和健康状况都可以获取。而且它作为原代细胞,保有原有细胞的基本性质。本发明对未来mt.3243A>G突变细胞的发病机制研究和针对mt.3243A>G的新药筛选提供了细胞应用平台。
Description
技术领域
本发明属于生物化学领域,涉及线粒体mt.3243A>G人群,具体来说是一种线粒体mt.3243A>G突变人群中高突变尿液干细胞的制备方法。
背景技术
干细胞是一种未充分分化,尚不成熟的细胞,具有再生各种组织器官和人体的潜在功能,医学界称为“万用细胞”。间充质干细胞是一种多能干细胞,它具有干细胞的所有共性,即自我更新和多向分化能力。间充质干细胞临床应用于解决多种血液系统疾病,心血管疾病、肝硬化、神经系统疾病、膝关节半月板部分切除损伤修复、自身免疫性疾病等方面取得了重大突破。
尿液来源的干细胞是一种新型的间充质干细胞,具有取材容易,无创,供应充足可反复取材,且没有伦理上的争议。线粒体DNA3243A>G突变(以下简称mt.3243A>G突变)是最常见的一种单基因突变糖尿病,由于线粒体DNA突变的细胞杂胞质性(不同细胞之间线粒体DNA突变比例存在差异)。线粒体基因tRNALeu(UUR)A3243G突变(以下简称mt.3243A>G突变)糖尿病是一种以糖尿病伴神经性耳聋为主要临床表现的母系遗传单基因突变糖尿病,该疾病是由于编码线粒体亮氨酸tRNA基因——tRNALeu(UUR)发生突变、即线粒体DNA第3243位核苷酸序位发生A→G突变所致。
mt.3243A>G突变容易累及ATP阈值高的组织,主要临床特点表现为:40岁以前起病的早发糖尿病。患者多消瘦,病程中胰岛功能进行性衰退,糖尿病确诊后2年内常需改用胰岛素治疗,其病理生理机制是胰岛β细胞线粒体功能损害致胰岛素分泌不足。超过75%患者有感觉神经性耳聋。其他临床症状包括线粒体脑肌病伴高乳酸血症和脑卒中样发作、视网膜色素变性、骨骼肌病、心肌疾病、肾病等,且病变呈母系遗传。该病在不同患者中的临床表现呈明显组织器官异质性,疾病严重程度不一,给临床防治带来巨大挑战[Murphy R,Turnbull DM,Walker M,Hattersley AT.Clinical features,diagnosis and managementof maternally inherited diabetes and deafness(MIDD)associated with the 3243A>G mitochondrial point mutation.Diabet Med.2008 Apr;25(4):383-99.]。患者的外周血和尿液具有杂胞质性的特征,即突变细胞和正常细胞在不同组织中共同存在且比例不同。尿液源的干细胞也存在这个问题。
mt.3243A>G突变研究缓慢的一个重要原因就是建造此突变的生物模型十分困难。目前,人工修饰线粒体mt.3243A>G仍然十分困难,还没有很好的手段对突变基因进行编辑。国际上,以往研究用mt.3243A>G突变线粒体与缺失线粒体(rho°)的人类骨肉瘤细胞株形成的融合胞质杂交细胞来模拟线粒体突变。[de Andrade PB,Rubi B,Frigerio F,van denOuweland JM,Maassen JA,Maechler P.Diabetes-associated mitochondrial DNAmutation A3243G impairs cellular metabolic pathways necessary for beta cellfunction.Diabetologia.2006Aug;49(8):1816-26.]但是由于这种细胞是胞质杂交细胞,并不能完全的反映患者自身的病理生理特点。因此,在临床研究中,如何从突变患者中分离出高突变的尿液间充质干细胞是目前科研的难题,也是急需的疾病模型,应用此疾病模型可以用于疾病机制研究以及新药的靶点筛选等。构建体现患者疾病进程和疾病严重程度的细胞平台是开展高质量mt.3243A>G突变分子机制研究的重要条件。
2012年以来,国际上陆续有学者利用mt.3243A>G突变患者皮肤成纤维细胞制备诱导多能干细胞(Induced Pluripotent Stem Cells,iPS),从同一个mt.3243A>G突变糖尿病患者中建立了高突变(80-90%突变)和无突变(野生型)iPS细胞。[Hatakeyama H,GotoY.Concise Review:Heteroplasmic Mitochondrial DNA Mutations and MitochondrialDiseases:Toward iPSC-Based Disease Modeling,Drug Discovery,and RegenerativeTherapeutics.Stem Cells.2016Apr;34(4):801-8.]此类研究提示,来自患者相同基因背景的高突变iPS细胞可很好的再现患者组织或器官特异性疾病发展进程和临床表型的严重程度,是开展疾病机制研究、进行新药筛选的重要手段。
然而,采用iPS细胞平台的研究也面临许多问题,例如诱导效率较低、无法完全绕开伦理问题、存在致瘤风险和安全性问题等等。因此,寻找更为安全可行、制备简便、安全无创的患者来源细胞平台对mt.3243A>G突变分子机制研究至关重要。
发明内容
针对现有技术中的上述技术问题,本发明提供了一种线粒体mt.3243A>G突变人群中高突变尿液干细胞的制备方法,所述的这种线粒体mt.3243A>G突变人群中高突变尿液干细胞的制备方法要解决现有技术中线粒体mt.3243A>G突变的生物模型建立困难的技术问题。
本发明提供了一种线粒体mt.3243A>G突变人群中高突变尿液干细胞的制备方法,包括如下步骤:
1)将青霉素、链霉素和两性霉素B的混合溶液加入T75瓶子,每ml混合溶液中含有青霉素10kU,链霉素10mg,两性霉素B 25μg;
2)选择线粒体mt.3243A>G突变人群的尿液,采用步骤1)的T75瓶子进行尿液收集;
3)打开T75瓶子,选择4个50ml离心红管,每一个离心红管中各加50ml尿液样本,离心机离心;
4)每个红管中用移液管吸去上清至至5ml以下,第一个红管加入15ml~20ml PBS,吹打混匀,然后将重悬液转移至下一个红管中,再次吹打混匀,以此类推,直至最后所有管的沉渣汇集于一管,离心;吸去上清,留至少于5ml的溶液,得到沉渣红管;
5)采用质量百分比浓度为0.1-1%的明胶包被过的96孔培养板;
6)将步骤5)的96孔板放入超净台,吸净明胶,吸取培养基加入步骤4)的沉渣红管,加入15ml~20ml的PBS溶液吹打混匀,形成重悬液,然后加入96孔板,100ul/孔,在第3天或第4天,每孔再加100ul培养基;
7)第5或第6天半量换液;
8)第10~12天,将出现密集成单克隆集簇细胞的96孔板内的培养基吸去,孔内加入PBS缓冲液冲洗;
9)在96孔板内每孔加入100ul胰酶,在显微镜下观察细胞是否消化分离,若细胞分离,吸去胰酶,在96孔板内每孔先加入200ul培养基;加入PBS缓冲液吹打混匀使细胞重悬,传代为P1代;
10)后续细胞培养每2~3天换液,待细胞达80%~90%融合时进行消化传代;
11)细胞培养后进行突变率检测,挑选出线粒体mt.3243A>G突变95%以上高突变的尿液干细胞。
进一步的,上述的培养基为间充质干细胞增殖培养基[Zhang Y,McNeill E,TianH,et al.Urine derived cells are a potential source for urological tissuereconstruction.J Urol.2008 180:2226]。
本发明还提供了采用上述方法制备的线粒体mt.3243A>G突变95%以上高突变的尿液干细胞。
本发明还提供了上述线粒体mt.3243A>G突变95%以上高突变的尿液干细胞在筛选用于治疗线粒体mt.3243A>G突变的疾病的药物中的用途。
本发明的创新之处在于在细胞单克隆的时候进行挑选,分别培养后进行焦磷酸定量测序,筛选出需要的高突变细胞。
本发明和已有技术相比,其技术进步是显著的。本发明获得的这种细胞的方法在于它可以无创获取,并且无论患者的性别、年龄和健康状况都可以获取。而且它作为原代细胞,保有原有细胞的基本性质。从突变患者的尿液中分离提取出干细胞,通过细胞单克隆分离出高突变的尿液干细胞,对未来mt.3243A>G突变细胞的发病机制研究和针对mt.3243A>G的新药筛选提供了细胞应用平台。
附图说明
图1是焦磷酸测序的拟合方程曲线。
图2是高突变细胞和正常细胞的膜电位比较。
图3是高突变细胞和正常细胞的ROS比较。
图4是高突变细胞和正常细胞的ATP比较。
图5是高突变细胞和正常细胞的基础氧耗比较。
图6是高突变尿液干细胞成脂分化和成骨分化前后的突变率比较。
图7是正常尿液干细胞和高突变干细胞成骨分化21天后的成骨标志物比较。
具体实施方式
实施例1
从突变患者的尿液中分离培养干细胞并单克隆分离培养,包括如下步骤:
1)将青霉素、链霉素和两性霉素B的混合溶液5ml(其中青霉素含量10kU/ml,链霉素含量10mg/ml,两性霉素B含量25μg/ml)加入T75瓶子,将T75瓶口周围喷上消毒水;
2)选择线粒体mt.3243A>G突变人群的尿液,采用步骤1)的T75瓶子进行尿液收集;
3)打开T75瓶子,在4个50ml离心红管中各加50ml尿液样本,离心机离心;
4)每个红管中用移液管吸去上清至至5ml以下,第一个红管加入15ml~20ml PBS,吹打混匀,然后将重悬液转移至下一个红管中,再次吹打混匀,以此类推,直至最后所有管的沉渣汇集于一管,1500转离心10min;吸去上清,留至少于5ml的溶液,得到沉渣红管;
5)采用质量百分比浓度为0.1-1%的明胶包被过的96孔培养板;
6)将步骤5)的96孔板放入超净台,吸净明胶,吸取培养基加入步骤4)的沉渣红管,加入15ml~20ml的PBS溶液吹打混匀,形成重悬液,然后加入96孔板,100ul/孔,在第3天或第4天,每孔再加100ul培养基;
7)第5或第6天半量换液;
8)第11天左右将出现密集成单克隆集簇细胞的96孔板内的培养基吸去,孔内加入PBS缓冲液冲洗,若孔内出现2个或以上克隆则弃去不要。
9)加入100ul胰酶,立即在显微镜下观察细胞是否消化分离,若细胞分离,吸去胰酶。在孔内先加入200ul培养基,吹打混匀使细胞重悬,传代为P1代。
10)后续细胞培养每2~3天换液,待细胞达80%~90%融合时进行消化传代。
具体的,上述的培养基为培养基:间充质干细胞增殖培养基[Zhang Y,McNeill E,Tian H,et al.Urine derived cells are a potential source for urological tissuereconstruction.J Urol.2008 180:2226]
11)抽提细胞DNA并对样本DNA做浓度测定。
12)对抽提到的DNA进行PCR扩增
正向引物ttcacaaagcgccttccccc;反向引物ccattgcgattagaatgggtaca。
13)焦磷酸测序,96孔板,每孔各加70uL binding buffer mix,各孔对应PCR产物10uL,振荡器打开,25℃,1300rpm震荡15Min,加入引物(ggtttgttaagatggcag)和缓冲液,24孔圆板置于振荡器,80℃,5min,0rpm。记录A>G的突变率。
14)建立定量焦磷酸测序分析拟合曲线方程(图1)
15)计算出每个克隆的突变率,挑选出95%以上突变率的单细胞进行扩增。
M1和M2为2个线粒体3243A>G突变携带者,采用上述的方法,M1共培养出9个单克隆,M2共培养出19个单克隆,其中全突变的单克隆各为1和4个。
实施例2膜电位检测
JC-1染色工作液的量为1ml,取适量JC-1(200X),按照每50μl JC-1(200X)加入8ml超纯水的比例稀释JC-1。剧烈Vortex充分溶解并混匀JC-1。然后再加入2ml JC-1染色缓冲液(5X),混匀后即为JC-1染色工作液。
a.对于六孔板的一个孔,吸除培养液,根据具体实验如有必要可以用PBS或其它适当溶液洗涤细胞一次,加入1ml间充质干细胞培养液。细胞培养液中可以含有血清和酚红。
b.加入1ml JC-1染色工作液,充分混匀。细胞培养箱中37℃孵育20分钟。
c.在孵育期间,按照每1ml JC-1染色缓冲液(5X)加入4ml蒸馏水的比例,配制适量的JC-1染色缓冲液(1X),并放置于冰浴。
d.37℃孵育结束后,吸除上清,用JC-1染色缓冲液(1X)洗涤2次。
e.加入2ml细胞培养液,培养液中可以含有血清和酚红。
如图2所示,经检测,高突变细胞的膜电位显著低于正常细胞(P<0.05)
实施例3 ROS检测
收集尿液间充质干细胞后装载探针:按照1:1000用无血清培养液稀释DCFH-DA,使终浓度为10umol/L。
细胞600g,4min(或3000rpm/5min)离心,去上清,将细胞悬浮于稀释好的DCFH-DA(1ml)中,细胞浓度为10^6-2*10^7/毫升,37℃细胞培养箱内孵育20分钟。每隔3-5分钟颠倒混匀一下,使探针和细胞充分接触。将细胞600g,4min离心,用无血清细胞培养液洗涤(离心、弹散、吹打)细胞三次,以充分去除未进入细胞内的DCFH-DA。加入约500ul(视细胞密度而定)培养液吹打混匀,涂抹10ul样品至载玻片上均匀涂开,荧光显微镜下观察,每个样品三个视野,每个视野先拍绿光,再白光。
如图3所示,经检测,高突变细胞的ROS显著高于正常细胞(P<0.05)
实施例4 ATP检测
用离心管离心沉淀尿液间充质干细胞3000rpm/5min,弃上清,轻轻弹散细胞,按照6孔板每孔的细胞量加入200微升裂解液的比例加入裂解液,裂解细胞。裂解后4℃12000g离心5-10分钟,取上清,用于后续的测定。冰浴上溶解待用试剂,把ATP标准溶液用ATP检测裂解液稀释成适当的浓度梯度。按照每个样品或标准品需100微升ATP检测工作液的比例配制适当量的ATP检测工作液。把待用试剂在冰浴上溶解。取适当量的ATP检测试剂,按照1:100的比例用ATP检测试剂稀释液稀释ATP检测试剂。例如50微升ATP检测试剂可以加入5毫升ATP检测试剂稀释液配制成5毫升ATP检测工作液。稀释后的ATP检测试剂即为用于后续实验的ATP检测工作液。ATP检测工作液可在冰浴上暂时保存。加100微升ATP检测工作液到检测孔或检测管内,避光。室温放置3-5分钟,在检测孔内分别加上20ul标准品及样品,根据标准曲线计算出样品中ATP的浓度。
如图4所示,经检测,高突变细胞的ATP显著小于正常细胞(P<0.05)
实施例5
正常对照和突变患者全突变的尿液干细胞氧耗比较,采用Seahorse XF代谢分析仪测定活细胞——USCs的耗氧率(oxygen consumption rate,OCR),反映其细胞线粒体呼吸及氧化磷酸化功能。
1.细胞培养及细胞传代;
所述尿液干细胞为突变患者的人尿液干细胞。人尿液干细胞从200ml以上的人尿液中提取,并采用0.1-1%明胶包被过的96孔培养板进行培养。
1)将青霉素、链霉素和两性霉素B的混合溶液5ml(其中青霉素含量10kU/ml,链霉素含量10mg/ml,两性霉素B含量25μg/ml)加入T75瓶子,将T75瓶口周围喷上消毒水;
2)选择线粒体mt.3243A>G突变人群的尿液,采用步骤1)的T75瓶子进行尿液收集;
3)打开T75瓶子,在4个50ml离心红管中各加50ml尿液样本,离心机离心;
4)每个红管中用移液管吸去上清至至5ml以下,第一个红管加入15ml~20ml PBS,吹打混匀,然后将重悬液转移至下一个红管中,再次吹打混匀,以此类推,直至最后所有管的沉渣汇集于一管,1500转离心10min;吸去上清,留至少于5ml的溶液,得到沉渣红管;
5)采用质量百分比浓度为0.1-1%的明胶包被过的96孔培养板;
6)将步骤5)的96孔板放入超净台,吸净明胶,吸取培养基加入步骤4)的沉渣红管,加入15ml~20ml的PBS溶液吹打混匀,形成重悬液,然后加入96孔板,100ul/孔,在第3天或第4天,每孔再加100ul培养基;
7)第5或第6天半量换液;
8)第11天左右将出现密集成单克隆集簇细胞的96孔板内的培养基吸去,孔内加入PBS缓冲液冲洗,若孔内出现2个或以上克隆则弃去不要。
9)加入100ul胰酶,立即在显微镜下观察细胞是否消化分离,若细胞分离,吸去胰酶。在孔内先加入200ul培养基,吹打混匀使细胞重悬,传代为P1代。后续细胞培养每2~3天换液,待细胞达80%~90%融合时进行消化传代
10)后续细胞培养每2~3天换液,待细胞达80%~90%融合时进行消化传代;
11)细胞培养后进行突变率检测(见实施例1),挑选出线粒体mt.3243A>G突变95%以上高突变的尿液干细胞。
2.活细胞计数;
3.Agilent Seahorse XF Cell Mito Test Kit检测。
如图5所示,经试验分析,高突变细胞的基础氧耗显著小于正常细胞(P<0.05),表明mt.3243A>G患者高突变USCs的线粒体呼吸功能及氧化磷酸化能力明显降低。
实施例6
从实施例1获得的尿液间充质干细胞挑选4例高突变细胞,经传代培养7代,高突变细胞突变率稳定。
实施例7
成骨分化:将尿液间充质干细胞置于37℃,5%CO2的培养箱中进行培养,当细胞融合度达到60%-70%时,小心的将孔内完全培养基吸走,向六孔板中加入2mL成人骨髓间质干细胞成骨诱导分化完全培养基(Cyagen,HUXMA-90021)。每2~3天换用新鲜的成人骨髓间质干细胞成骨诱导分化完全培养基(使用前需预热至37℃),诱导2-4周后,视细胞的形态变化及生长情况,用茜素红进行染色并进行相关实验。
成脂分化:将尿液间充质干细胞置于37℃,5%CO2的培养箱中进行培养,当细胞融合度达到100%或者过融合,小心地将间质干细胞完全培养基吸走,向六孔板中加入2mL成人骨髓间质干细胞成脂诱导分化培养基(Cyagen,HUXMA-90031)A液。诱导3天后,吸走六孔板中的A液,加入2mL成人骨髓间质干细胞成脂诱导分化培养基B液。24h后,吸走B液,换回A液进行诱导。A液和B液交替作用3-5次后(交替次数视分化情况而定),继续用B液维持培养4-7天直到脂滴变得足够大、圆。B液维持培养期间,每隔2-3天需要换用新鲜的B液。视细胞的形态变化及分化情况,用油红O进行染色并进行相关实验。
如图6所示,成脂成骨分化后高突变细胞的突变率保持稳定。
如图7所示,RT-PCR检测显示,成骨分化21天后成骨标记物BMP2、RUNX2、OCN、ALP在高突变USCs组表达均显著降低(P<0.01)。上述结果提示mt.3243A>G患者来源的高突变USCs的成骨分化能力显著低于正常对照组USCs。
序列表
<110> 上海市东方医院(同济大学附属东方医院)
<120> 一种线粒体mt.3243A>G突变人群中高突变尿液干细胞的制备方法
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
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<212> DNA
<213> 人工序列(Artificial Sequence)
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ccattgcgat tagaatgggt aca 23
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<212> DNA
<213> 人工序列(Artificial Sequence)
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ggtttgttaa gatggcag 18
Claims (4)
1.一种线粒体mt.3243A>G突变人群中高突变尿液干细胞的制备方法,其特征在于包括如下步骤:
将青霉素、链霉素和两性霉素B的混合溶液加入T75瓶子,每ml混合溶液中含有青霉素10kU,链霉素10mg,两性霉素B 25µg;
选择线粒体mt.3243A>G突变人群的尿液,采用步骤1)的T75瓶子进行尿液收集;
打开T75瓶子,选择4个50ml离心红管,每一个离心红管中各加50ml尿液样本,离心机离心;
每个红管中用移液管吸去上清至至5ml以下,第一个红管加入15ml~20ml PBS,吹打混匀,然后将重悬液转移至下一个红管中,再次吹打混匀,以此类推,直至最后所有管的沉渣汇集于一管,离心;吸去上清,留至少5ml的溶液,得到沉渣红管;
采用质量百分比浓度为0.1-1%的明胶包被过的96孔培养板;
将步骤5)的96孔板放入超净台,吸净明胶,吸取培养基加入步骤4)的沉渣红管,加入15ml~20ml的 PBS溶液吹打混匀,形成重悬液,然后加入96孔板,100ul/孔,在第3天或第4天,每孔再加100ul培养基;
第5或第6天半量换液;
第10~12天,将出现密集成单克隆集簇细胞的96孔板内的培养基吸去,孔内加入PBS缓冲液冲洗;
在96孔板内每孔加入100ul胰酶,在显微镜下观察细胞是否消化分离,若细胞分离,吸去胰酶,在96孔板内每孔先加入200ul培养基;再加入PBS缓冲液吹打混匀使细胞重悬,传代为P1代;
后续细胞培养每2~3天换液,待细胞达80%~90%融合时进行消化传代;
细胞培养后进行突变率检测,挑选出线粒体mt.3243A>G 突变95%以上高突变的尿液干细胞。
2.根据权利要求1所述的一种线粒体mt.3243A>G突变人群中高突变尿液干细胞的制备方法,其特征在于:所述的培养基为间充质干细胞增殖培养基。
3.采用上述方法制备的线粒体mt.3243A>G 突变95%以上高突变的尿液干细胞。
4.权利要求3所述的线粒体mt.3243A>G 突变95%以上高突变的尿液干细胞在筛选用于治疗线粒体mt.3243A>G 突变的疾病的药物中的用途。
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