CN107012124B - 钠钙交换体1启动子联合丹参酚酸B诱导iPSCs定向心肌分化的方法 - Google Patents
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Abstract
本发明公开一种钠钙交换体1启动子联合丹参酚酸B诱导iPSCs定向心肌分化的方法，属于医药技术领域。该方法是通过构建NCX1慢病毒颗粒并转染至iPSCs，辅以丹参酚酸B联合诱导。所述的丹参酚酸B的浓度为1～100μM。本发明证实钠钙交换体1启动子联合丹参酚酸B可有效诱导iPSCs心肌定向分化。丹参酚酸B可有效促进NCX1表达，同时促进心肌标志物α肌动蛋白(α‑actin)、心肌肌钙蛋白T(cTnT)表达显著提高。且iPSCs形态可见心肌细胞样梭形改变。iPSCs作为修复急性心肌梗死的种子细胞，具有广阔的应用前景。
Description
技术领域
本发明属于医药技术领域，具体涉及一种钠钙交换体1启动子联合丹参酚酸B诱导iPSCs定向心肌分化的方法。
背景技术
急性心肌梗死的干细胞治疗仍存在诸多方面的困难，如种子细胞的选择问题、心肌坏死范围广泛、细胞移植归巢效率低下及局部心肌微环境缺血缺氧等，因此，其临床应用仍然未能得以广泛的开展。诱导性多能干细胞(induced pluripotent stem cells，iPSCs)是通过重编程体细胞获得的具有与胚胎干细胞(embryonic stem cells，ECSs)相似的高度增殖和多向分化潜能的一种多能干细胞，又因其缺少胚胎干细胞的免疫原性及道德伦理限制，诱导性多能干细胞有望成为修复急性心肌梗死后心肌的具有巨大临床应用前景的种子细胞。
丹参为唇形科植物丹参Salvia miltiorrhiza Bge.的干燥根及根茎，除了传统的活血化瘀、养心安神的功效外，还有抗氧化，抑制血小板聚集、促进血栓溶解，抗肿瘤，调节免疫功能的作用，其在心血管疾病方面的新药理作用、作用机制和药代动力学的研究日趋深入，使其拥有非常广阔的临床应用前景。丹参酚酸B是其中一种单体成份。
作为心肌分化最早的标志物钠钙交换体钠钙交换体启动子广泛分布于心肌细胞膜上，双向转运胞膜内外Na+和Ca2+，参与心肌兴奋-收缩耦联及胞内钙稳态的维持，钠钙交换体1是一个糖基化的具有12个跨膜部分的跨膜蛋白，对维持心肌钙平衡有重要作用。
目前利用iPSCs定向心肌分化的方法很多，本研究旨在提供一种新型的诱导方法，进一步推动iPSCs的临床应用。
发明内容
为了克服现有技术的缺点与不足，本发明的目的在于提供一种钠钙交换体1启动子联合丹参酚酸B诱导iPSCs定向心肌分化的方法。
目前尚无有效诱导iPSCs心肌定向分化的方法，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：
一种钠钙交换体1启动子联合丹参酚酸B诱导iPSCs定向心肌分化的方法，包括如下步骤：
通过构建NCX1慢病毒颗粒并转染至iPSCs，辅以丹参酚酸B联合诱导，倒置显微镜、细胞免疫技术以及Western Blots观察其转染情况、形态学以及心肌标志物表达情况。实验证实钠钙交换体1启动子联合丹参酚酸B可有效诱导iPSCs定向心肌分化。
优选的，所述的丹参酚酸B的浓度为1～100μM；更优选为10～100μM。
优选的，所述的培养的条件为5％CO2、37℃培养15～20d。
本发明的机理是：拟通过钠钙交换体1启动子(sodium-calcium exchangerpromoter，NCX1)联合丹参酚酸B(Salvianolic acid B，Sal B)诱导诱导性多能干细胞(induced pluripotent stem cells，iPSCs)定向心肌细胞分化，iPSCs作为修复急性心肌梗死的种子细胞，具有广阔的应用前景。
本发明相对于现有技术，具有如下的优点及效果：
本发明证实钠钙交换体1启动子联合丹参酚酸B可有效诱导iPSCs心肌定向分化。丹参酚酸B可有效促进NCX1表达，同时促进心肌标志物α肌动蛋白(α-actin)、心肌肌钙蛋白T(cTnT)表达显著提高。且iPSCs形态可见心肌细胞样梭形改变。
附图说明
图1是NCX1慢病毒转染诱导性多能干细胞后NCX1表达情况；其中，A：EGFP；B：Oct4；C：NCX1；D：Merged。
图2是钠钙交换体1启动子联合丹参酚酸B诱导iPSCs分化后形态改变；其中，A：对照组；B：丹参酚酸B(1μM)；C：丹参酚酸B(10μM)；D：丹参酚酸B(100μM)；E：NCX1组；F：NCX1+丹参酚酸B(1μM)；G：NCX1+丹参酚酸B(10μM)；H：NCX1+丹参酚酸B(100μM)。
图3是钠钙交换体1启动子联合丹参酚酸B诱导iPSCs分化后心肌特异性标志物表达情况。
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2钠钙交换体1启动子制备
1)慢病毒Neo-Promoter_419bp>Luciferase:IRES:DsRed_Express2构建和鉴定：
引物设计：根据Genebank中钠钙交换体1启动子基因的cDNA序列，设计钠钙交换体1启动子引物序列为：
上游引物：5′-GCAACAGACATACAAACTAAAGAAT-3′；
下游引物：5′-GGAGCAACATAGTTAAGAATACC-3′。
PCR扩增：在上述引物的作用下，以钠钙交换体1启动子的cDNA为模板进行扩增，电泳检测PCR产物，切胶回收纯化目的条带。将载体Neo-Promoter_419bp>Luciferase:IRES:DsRed_Express2与扩增钠钙交换体1启动子在内切酶作用下重组，取2μL重组反应液转化50μL感受态细胞，新霉素筛选重组阳性克隆并扩增，菌液行PCR扩增验证，挑取构建正确的菌落培养过夜，提取质粒DNA，构建正确标记。
2)慢病毒的包装和浓缩：
将慢病毒包装质粒Vira Power TM Lentiviral Packaging Mix和载体质粒pLV[Exp]-Neo-Promoter_419bp>Luciferase:IRES:DsRed_Express2以1：1的摩尔比例混合，在Lipofectamine2000的作用下共转染293T细胞，置于37℃、体积分数5％CO2的培养箱中培养。12h后换液，加入新鲜293T细胞培养液。72h后观察有强红色色荧光及细胞融合现象时收集培养上清，4℃、4500r/min离心15min，将病毒上清液用0.45μm滤膜过滤以去除细胞碎片，4℃、50 000×g高速离心90min。用RNase-free L-DMEM悬浮病毒颗粒沉淀，-80℃冻存备用。同法构建只含红色色荧光蛋白的空病毒，标记pLV[Exp]-Neo-Luciferase:IRES:DsRed_Express2。
3)慢病毒的滴度测定：接种293T细胞于6孔板，每孔接种2×105个细胞，37℃培养过夜；第2天将慢病毒溶液用无血清DMEM(不含抗生素)按1×10-2，1×10-3，1×10-4，1×10-5，1×10-6，1×10-7进行系列稀释，将其分别加入到相应的孔中，加入聚酰胺(Polybrene)终浓度至8mg/L，置37℃、体积分数5％CO2培养箱培养。12h后全量换液，加入新鲜的293T细胞培养液。第4天观察荧光表达情况，荧光细胞数随稀释倍数增加而减少，计数出表达荧光的细胞个数，将得到的数值乘以相应的稀释倍数就得到病毒原液的滴度数。
4)pLV[Exp]-Neo-Promoter_419bp>Luciferase:IRES:DsRed_Express2包装iPSCs：将对数生长期诱导性多能干细胞(iPSCs)消化离心后，以分化培养基重悬成2×107L-1细胞悬液，分化培养基为含体积分数1％非必需氨基酸、1％谷氨酰胺、0.25％β-巯基乙醇，体积为15％胎牛血清的DMEM。细胞悬液以20μL/滴接种到直径10cm的培养皿上，培养皿内加少许PBS。悬滴48h后将形成的拟胚体悬浮到含新鲜分化培养液的培养皿中，将病毒pLV[Exp]-Neo-Promoter_419bp>Luciferase:IRES:DsRed_Express2和对照病毒转染诱导性多能干细胞的拟胚体。
处理分组如下：A：对照组；B：丹参酚酸B(1μM)；C：丹参酚酸B(10μM)D：丹参酚酸B(100μM)；E：NCX1组；F：NCX1+丹参酚酸B(1μM)；G：NCX1+丹参酚酸B(10μM)；H：NCX1+丹参酚酸B(100μM)。比较各组之间心肌细胞的诱导效率，每天更换一次培养基。病毒感染24～48h后，使用新霉素进行筛选。第10天后将悬浮的拟胚体接种到0.1％明胶铺板的培养瓶中，用上述分化培养液继续培养，在倒置显微镜下每日观察其生长情况，且收集经诱导分化处理的细胞后续实验使用。
实施例3细胞免疫荧光以及形态学鉴定
诱导培养20d后，PBS洗涤3次，以4％多聚甲醛固定，PBS冲洗，DAPI复染细胞核10min，树脂胶封片固定。荧光显微镜以及倒置显微镜下下观察，每孔观察10个视野共30个视野。
NCX1慢病毒转染诱导性多能干细胞后NCX1表达情况，如图1所示。结果表明：荧光显微镜定性分析可见NCX1有效表达于iPSCs。
钠钙交换体1启动子联合丹参酚酸B诱导iPSCs分化后形态改变，如图2所示；其中，A：对照组；B：丹参酚酸B(1μM)；C：丹参酚酸B(10μM)；D：丹参酚酸B(100μM)；E：NCX1组；F：NCX1+丹参酚酸B(1μM)；G：NCX1+丹参酚酸B(10μM)；H：NCX1+丹参酚酸B(100μM)。结果表明：相比对照组而言，B～H各组拟胚体均可见边缘不规则，iPSCs如心肌细胞样呈梭形生长。
实施例4心肌特异性标志物(α肌动蛋白(α-actin)、心肌肌钙蛋白T(cTnT))表达情况分析
Western-blot技术：将细胞接种至6孔板，37℃，5％CO2常规条件下培养24h。待细胞稳定后按实施例2中的各分组处理方法处理。提取各组蛋白并进行SDS-PAGE电泳，电泳后转膜用含5％脱脂奶粉的TBST 37摄氏度封闭60min，用相应抗体4℃孵育过夜。TBST漂洗3次后加入相应二抗后37℃孵育60min。然后用TBST漂洗5次后检测。采用图像分析软件进行图像分析，计算条带与β-actin的相对积分光密度值。
钠钙交换体1启动子联合丹参酚酸B诱导iPSCs分化后，心肌特异性标志物表达情况，如图3所示。结果表明：相比对照组而言，其他各组α肌动蛋白(α-actin)和心肌肌钙蛋白T(cTnT)的表达量均有统计学意义(p<0.05)；相比NCX1组而言，不同浓度的丹参酚酸B可有效提高NCX1的心肌分化诱导效率，特别是10～100μM的丹参酚酸B。
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。
SEQUENCE LISTING
<110> 暨南大学
<120> 钠钙交换体1启动子联合丹参酚酸B诱导iPSCs定向心肌分化的方法
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<170> PatentIn version 3.5
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<213> Artificial Sequence
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<223> 上游引物
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Claims (2)
1.一种钠钙交换体1启动子联合丹参酚酸B诱导iPSCs定向心肌分化的方法，其特征在于包括如下步骤：
通过构建NCX1慢病毒颗粒并转染至iPSCs，辅以丹参酚酸B联合诱导；
所述的丹参酚酸B的浓度为100μM。
2.根据权利要求1所述的钠钙交换体1启动子联合丹参酚酸B诱导iPSCs定向心肌分化的方法，其特征在于：
所述的诱导的条件为5% CO2、37℃培养15～20d。
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