CN107177555A - 一种H9C2心肌细胞培养液诱导iPSCs定向心肌分化的方法 - Google Patents
一种H9C2心肌细胞培养液诱导iPSCs定向心肌分化的方法 Download PDFInfo
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Abstract
本发明公开一种H9C2心肌细胞培养液诱导iPSCs定向心肌分化的方法,属于医药技术领域。该方法是将去除了LIF的常规iPSCs细胞培养液和H9C2心肌细胞培养液混合后培养iPSCs。本发明证实H9C2心肌细胞培养液可有效诱导iPSCs心肌定向分化。促进多向分化潜能基因OCT‑4荧光猝灭,细胞免疫荧光实验证实可提高心肌α‑横纹肌辅肌动蛋白(α‑actinin)表达,qPCR结果表明β‑MHC、GATA4、Mef2C、NCX‑1等mRNA表达显著增加。
Description
技术领域
本发明属于医药技术领域,具体涉及一种H9C2心肌细胞培养液诱导iPSCs定向心肌分化的方法。
背景技术
心肌梗死(myocardial infarction,MI)是引起死亡最常见的疾病之一,且心肌细胞一旦损伤缺乏再生能力,体外诱导培养心肌细胞进行心脏移植已成为治疗心血管疾病重要的研究热点。诱导性多能干细胞(induced pluripotent stem cells,iPSCs)具有多向分化的潜能,可分化为多种细胞。目前研究虽证实iPSCs体外可分化心肌细胞,但其自然分化效率不高。IPSCs分化为心肌细胞受多种因素调控,特别是细胞外微环境的变化。目前有关H9C2心肌细胞培养液诱导干细胞分化的研究鲜有报道,且不同类型细胞对于相同的刺激因素表现不同的诱导效应。
发明内容
为了克服现有技术的缺点与不足,本发明的首要目的在于提供一种H9C2心肌细胞培养液诱导iPSCsiPSCs定向心肌分化的方法。
目前诱导iPSCs心肌定向分化的方法各异,本研究为了推进iPSCs的临床应用,旨在提供一种有效安全的诱导心肌分化方法。
本发明的目的通过下述技术方案实现:
一种H9C2心肌细胞培养液诱导iPSCs定向心肌分化的方法,包括如下步骤:
通过将去除了LIF的常规iPSCs细胞培养液和H9C2心肌细胞培养液混合后培养iPSCs,倒置显微镜、细胞免疫技术以及qPCR检测其心肌定向分化情况。实验证实不同浓度H9C2分化培养液均可在第7天显著减少小鼠诱导性多能干细胞OCT-4表达,但对各心肌基因表达诱导作用不一,以1:2体积比的H9C2分化培养液最佳。
所述的H9C2心肌细胞培养液,是将H9C2心肌细胞常规培养3d,传代前吸取培养液过滤即可。
优选的,所述的去除了LIF的常规iPSCs细胞培养液和H9C2心肌细胞培养液的体积比为1:1~1:2;更优选为1:2。
优选的,所述的培养iPSCs的时间为10~14d。
本发明的机理是:本申请通过研究H9C2心肌细胞培养液对诱导性多能干细胞(induced pluripotent stem cells,iPSCs)定向心肌分化的影响,以建立一种安全有效的体外诱导iPSCs分化为心肌细胞的实验方法,iPSCs作为心肌定向分化的理想细胞,具有广阔的应用前景,目前虽然诱导其心肌定向分化的方法很多,但仍未发现一种极为有效的诱导方法,造成其治疗效果受限。
本发明相对于现有技术,具有如下的优点及效果:
本发明证实H9C2心肌细胞培养液可有效诱导iPSCs心肌定向分化。促进多向分化潜能基因OCT-4荧光猝灭,细胞免疫荧光实验证实可提高心肌α-横纹肌辅肌动蛋白(α-actinin)表达,qPCR结果表明β-MHC、GATA4、Mef2C、NCX-1等mRNA表达显著增加。
附图说明
图1是诱导性多能干细胞EGFP表达变化情况。
图2是免疫荧光分析检测诱导性多能干细胞拟胚体中心肌特异标志物α肌动蛋白(α-actin)的表达情况。
图3是qPCR检测诱导性多能干细胞拟胚体中心肌特异性标志物的表达情况。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。
下列实施例中未注明具体实验条件的实验方法,通常按照常规实验条件或按照制造厂商所建议的实验条件。未注明具体来源的试剂及生物材料,均为市售产品。
实施例1制备iPSCs
1)滋养层(MEF)的制备:0.1%(体积分数)的明胶加入T25培养瓶,于37℃细胞培养箱静置20min后将其吸除,加入5~6mL预热37℃的MEF培养液,与此同时将小鼠胚胎成纤维细胞(MEF)(购自上海中科院细胞库)从液氮中快速取出,置于37℃水浴中快速融解并立即用体积分数为75%的酒精擦拭冻存管后拿到超净台内,将冻存管内的细胞悬液转移至含MEF培养液的15mL离心管内,以1000rpm离心5min,弃上清液重悬后加入到T25培养瓶中,放置到CO2恒温培养箱中,培养24h后得到滋养层即可加入iPSCs;
2)iPSCs的培养以及传代:步骤(1)中培养24h后得到的滋养层,4倍显微镜下可见已经均匀铺满T25培养瓶,此时可迅速从液氮中取出iPSCs(中科院上海生科院生化细胞所和中科院上海生科院生化细胞所干细胞平台提供)冻存管,复苏过程类似小鼠胚胎成纤维细胞,将复苏的iPSCs直接传入MEF铺板的T25培养瓶,置于37℃细胞培养箱,每日换液并且观察细胞集落形态的变化。待细胞集落长到合适大小及时传代,PBS(不含钙镁离子)轻轻冲洗一遍后加入0.25%(体积分数)胰酶(含EDTA)消化,小鼠iPSCs培养液终止消化,以1000rpm离心5min,弃上清加培养液吹打制备成单细胞悬液。
实施例2H9C2心肌细胞培养液诱导iPSCs分化
含15%FBS、50U/mL青霉素及10μg/mL链霉素的DMEM培养液重悬H9C2心肌细胞后以适当细胞密度接种于T25培养瓶中培养,1~2d传代。将培养3d的传代前的培养液吸出,经0.22μm滤过膜过滤后,即诱导培养液,即H9C2心肌细胞培养液,备用。
取生长对数期的iPSCs,以100×106cells/mL iPSCs悬滴培养48h后,接种于0.1%明胶预处理1小时的六孔板,将预先配制的去除LIF的常规iPSCs细胞培养液与H9C2心肌细胞培养液按1:0、1:1、1:2比例混合配制成15%胎牛血清的分化培养液培养iPSCs。常规未处理的iPSCs作为对照组。
常规iPSCs细胞培养液:80%DMEM、15%胎牛血清、1%谷氨酰胺、1%非必须氨基酸、1%核苷、1000U/mL LIF、0.25%巯基乙醇、1%双抗。
实施例3倒置显微镜以及细胞免疫荧光检测多向分化潜能基因Oct-4-EGFP和α-横纹肌肌动蛋白(α-sarcomeric actinin,α-actin)表达情况
诱导培养14d后,PBS洗涤3次,以4%多聚甲醛固定,PBS冲洗,5%BSA封闭,滴加兔抗鼠的单克隆一抗α-actin(1:500),4度过夜,PBS冲洗,滴加PE标记的羊抗兔IgG(1:50),37℃避光孵育30min,PBS漂洗后DAPI复染细胞核10min,树脂胶封片固定。荧光显微镜下观察,每孔观察10个视野共30个视野。
诱导性多能干细胞EGFP表达变化情况,如图1所示,结果表明:iPSCs细胞培养液和H9C2心肌细胞培养液以1:2的体积比诱导其分化,可见代表细胞干性的Oct-4-EGFP荧光显著减,细胞形态在第7d即可见形态改变。
免疫荧光分析检测诱导性多能干细胞拟胚体中心肌特异标志物肌动蛋白α-actin,结果如图2所示,结果表明:三组均可见α-actin的表达,证实H9C2心肌细胞培养液可诱导iPSCs心肌定向分化,具体诱导分化效率还有待qPCR分析。
实施例4qPCR检测iPSCs经诱导预处理后心肌特异性蛋白表达情况
按TRIZOL操作说明书抽提样品细胞的总RNA。取总RNA用反转录酶逆转录得到cDNA;于PCR仪上进行扩增,设计引物:
GATA-4上游引物:5'-CTCCAGCACAGATCCTTTGT-3',
GATA-4下游引物:5'-TTCTCCCGTCTATCACCTTTG-3',片段大小109bp;
Mef2c上游引物:5'-CAAATCTCTCCCTGCCTTCTAC-3',
Mef2c下游引物:5'-GTGGTGTGTTGTGGGTATCT-3',片段大小106bp;
β-MHC上游引物:5'-CCACC CAAGTTCGACAAGAT-3',
β-MHC下游引物:5'-AAGAGGCCCGAGTAGGTATAG-3',片段大小122bp;
NCX-1上游引物:5'-GAGAGGATAGCAGGCAAACTAC-3',
NCX-1下游引物:5'-CCAGCCTTTCTTCTCAGTCTAC-3',片段大小113bp;
β-actin上游引物:5'-GCAGAAGGAGATCACAGCCCT-3',
β-actin下游引物:5'-GCTGATCCACATCTGCTGGAA-3',片段大小136bp。
根据实时定量扩增曲线获取目的基因和内参基因的Ct值,以目的基因表达的相对定量值进行统计学分析,实验重复3次。
RT-PCR检测诱导性多能干细胞拟胚体中心肌特异性标志物mRNA表达情况,结果如图3所示,结果表明:相比对照组而言,GATA-4、Mef2c、β-MHC和NCX-1四个心肌特异性标志物中,预先配制的去除了LIF的常规iPSCs细胞培养液与H9C2心肌细胞培养液按1:0的体积比,仅能有效提高GATA-4和β-MHC的表达量;按1:1体积比诱导分化,GATA-4、β-MHC和NCX-1三个均可有效提高(P<0.05);而按1:2比例配制,四个心肌特异标志物的表达量均有统计学意义。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
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Claims (4)
1.一种H9C2心肌细胞培养液诱导iPSCs定向心肌分化的方法,其特征在于包括如下步骤:
将去除了LIF的常规iPSCs细胞培养液和H9C2心肌细胞培养液混合后培养iPSCs;
所述的H9C2心肌细胞培养液,是将H9C2心肌细胞常规培养3d,传代前吸取培养液过滤。
2.根据权利要求1所述的H9C2心肌细胞培养液诱导iPSCs定向心肌分化的方法,其特征在于:
所述的iPSCs细胞培养液和H9C2心肌细胞培养液的体积比为1:1~1:2。
3.根据权利要求1或2所述的H9C2心肌细胞培养液诱导iPSCs定向心肌分化的方法,其特征在于:
所述的iPSCs细胞培养液和H9C2心肌细胞培养液的体积比为1:2。
4.根据权利要求1所述的H9C2心肌细胞培养液诱导iPSCs定向心肌分化的方法,其特征在于:
所述的培养iPSCs的时间为10~14d。
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CN106244527A (zh) * | 2016-08-29 | 2016-12-21 | 广东依浦赛生物科技有限公司 | 人源iPS干细胞体外定向分化为心肌细胞的试剂盒及方法 |
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CN106244527A (zh) * | 2016-08-29 | 2016-12-21 | 广东依浦赛生物科技有限公司 | 人源iPS干细胞体外定向分化为心肌细胞的试剂盒及方法 |
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