CN113278686A - 一种研究dna甲基化调控重编程过程的方法 - Google Patents
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Abstract
本发明涉及一种研究DNA甲基化调控重编程过程的方法，包括以下步骤：步骤（1）、在山中因子Oct4、Sox2、Klf4及c‑Myc诱导的基础上添加DNA甲基化酶抑制剂5’aza，按照山中伸弥提供的诱导方法对体细胞进行诱导，在不同诱导天数观察细胞形态；步骤（2）、当观察到出现细胞克隆时收集细胞进行荧光定量检测、间接免疫荧光检测、碱性磷酸酶染色、染色体核型分析和edu增殖检测；步骤（3）、确定5’aza的效果后，将四因子和5’aza分别进行组合处理体细胞进行诱导，检测ips克隆的形成，确定最有效的组合形式。通过本发明，通过山中因子和DNA甲基化抑制剂5’aza的不同组合诱导，探明DNA甲基化在体细胞重编程诱导中的作用。
Description
技术领域
本发明涉及一种研究DNA甲基化调控重编程过程的方法，属于生物技术领域。
背景技术
细胞重编程是逆分化的一种过程，通过体细胞核移植、转录因子诱导、细胞融合、细胞质孵育等手段，可以将分化完全的体细胞重编程为全能性细胞。体细胞核移植通过收集体细胞核注入去核的卵母细胞中，将融合后的细胞进行体外培养至胚胎进而植入母体，获得克隆动物。这种方法切实有效且能真实产生后代，但其在一定程度上违反了伦理道德，无法在人类上利用。细胞融合手段主要在植物育种和制备单克隆抗体时应用广泛，而在动物中仍涉及伦理道德问题。而自2006年山中伸弥发现体细胞导入四个转录因子（Oct4，Sox2，Klf4及c-Myc），可重编程为诱导性多能干细胞后，转录因子诱导技术不断发展。其无需使用卵细胞和胚胎，而仅适用人体其他细胞即可获得多能干细胞的优势免除了伦理争议，因此广受关注。而其仅从转录因子的角度研究了体细胞重编程的因素，大量调控因子尚未被挖掘。研究表明，添加组蛋白去乙酰化酶抑制剂VPA后，显著提高重编程过程，仍有相当多的表观遗传因子有待挖掘。
发明内容
本发明的目的就是针对上述现有问题，DNA甲基化作为表观遗传修饰的一种，广泛存在于各个生命过程中。在体细胞中多能性基因往往处于高甲基化状态，表达抑制以维持体细胞稳定，而多能干细胞中DNA甲基化水平大多处于低甲基化状态，多能性基因活跃。因此DNA甲基化无疑决定着体细胞重编程的命运，然而目前尚未有报道系统地研究DNA甲基化对体细胞重编程的调控，仍需进一步研究。通过本发明，提供一种研究DNA甲基化调控重编程过程的方法。
本发明的技术方案是：一种研究DNA甲基化调控重编程过程的方法，其特征是，包括以下步骤：
步骤（1）、在山中因子Oct4、Sox2、Klf4及c-Myc诱导的基础上添加DNA甲基化酶抑制剂5’aza，按照山中伸弥提供的诱导方法对体细胞进行诱导，在不同诱导天数观察细胞形态；
步骤（2）、当观察到出现细胞克隆时收集细胞进行荧光定量检测、间接免疫荧光检测、碱性磷酸酶染色、染色体核型分析和edu增殖检测；
步骤（3）、确定5’aza的效果后，将四因子和5’aza分别进行组合处理体细胞进行诱导，按步骤（2）中方法检测ips克隆的形成，确定最有效的组合形式；
在此基础上，单独使用5’aza进行诱导，按步骤（2）中方法检测ips克隆形成，进一步确定DNA甲基化抑制剂的单独诱导作用；从而有效探明DNA甲基化在重编程过程中的作用。
步骤（2）中，荧光定量检测的方法为：收集克隆样细胞，添加Trizol 裂解液，进行RNA提取，反转录为cDNA后，实时荧光定量PCR检测Oct4、Sox2、Klf4及c-Myc的表达，同时检测多能性基因的表达以及体细胞标记基因的表达；
间接免疫荧光检测方法为：去除细胞培养孔板中的培养基，添加4%的多聚甲醛对细胞固定30min，PBS清洗后，0.1%的Triton 透膜处理15min，离心清洗后，封闭2h，加入Oct4，Sox2，Klf4，c-Myc，全能性标记蛋白和体细胞标记抗体，孵育过夜，清洗后，加入二抗孵育2h，清洗后加入DAPI孵育10min，荧光倒置显微镜下观察标记蛋白的表达；
碱性磷酸酶染色方法为：吸去培养基,PBS清洗干净后加入ALP固定液，固定3 min，PBS清洗后，加入ALP孵育液，避光15-20 min，PBS清洗；加入核固红染色或甲基绿染色3-5min，PBS清洗后进行镜检，观察克隆细胞能否呈现碱性磷酸酶活性；
染色体核型分析方法为：将细胞克隆按5×106的细胞接种在100mm口径培养皿中，至对数期，加入0.2ml的10μmol/L秋水仙酰胺，培养4h后，弃去培养基，加入胰蛋白酶孵育3min，加入10%FBS的DMEM终止消化，离心后，加入40mmol/L KCl和25mmol/L枸橼酸钠的低渗液重悬，静置后，加入等体积新鲜配制的冰冷的1:3醋酸甲醇固定液，静置10min，立新10min后，醋酸甲醇溶液重悬；用巴氏吸管吸一滴细胞悬液，从3米处滴在用冰块透凉的载玻片中；烘干后进行吉姆萨染色，5min后冲掉染液，观察染色体核型；
edu增殖检测方法为：取对数生长的细胞克隆，每孔1×105细胞接种于96孔板中，持续培养；每孔加入50μM Edu培养基孵育2小时，弃培养基，PBS清洗2次，加入4%多聚甲醛室温孵育30min，弃固定液；加入甘氨酸孵育5min，清洗后加入0.5% TritonX-100的PBS孵育10min；清洗后加入1× Apollo®染色反应液，避光孵育10min，弃反应液，加入0.5%TritonX-10的PBS清洗2-3次，甲醇清洗1-2次，Hoechest 33342反应液孵育30min，PBS清洗后，进行观察。
本发明方法先进科学，通过本发明，本研究操作简单方便，实验思路清晰，可行性较高，具有很强的适用性和延展性。通过山中因子和DNA甲基化抑制剂5’aza的不同组合诱导，探明DNA甲基化在体细胞重编程诱导中的作用。本研究应用广泛，在不同物种的体细胞中均可适用。
具体实施方法
下面结合具体实施例对本发明进一步进行描述。但本发明的保护范围并不仅限于此：
1.DNA甲基化抑制剂联合山中因子诱导重编程；
将逆转录病毒包裹的Oct4，Sox2，Klf4及c-Myc过表达载体联合DNA甲基化酶抑制剂5’aza对体细胞进行诱导，在不同诱导天数观察细胞状态，同时进行拍照记录。
2.ips细胞的鉴定；
当观察到出现克隆样细胞时，收集细胞进行鉴定。
荧光定量检测：收集克隆样细胞，添加Trizol 裂解液，进行RNA提取，反转录为cDNA后，实时荧光定量PCR检测Oct4，Sox2，Klf4及c-Myc的表达，同时检测多能性基因的表达以及体细胞标记基因的表达。
间接免疫荧光：去除细胞培养孔板中的培养基，添加4%的多聚甲醛对细胞固定30min，PBS清洗后，0.1%的Triton 透膜处理15min，离心清洗后，封闭2h，加入Oct4，Sox2，Klf4，c-Myc，全能性标记蛋白和体细胞标记抗体，孵育过夜，清洗后，加入二抗孵育2h，清洗后加入DAPI孵育10min，荧光倒置显微镜下观察标记蛋白的表达。
碱性磷酸酶染色：吸去培养基,PBS清洗干净后加入ALP固定液，固定3 min，PBS清洗后。加入ALP孵育液，避光15-20 min，PBS清洗。加入核固红染色或甲基绿染色3-5min，PBS清洗后进行镜检。观察克隆细胞能否呈现碱性磷酸酶活性。
染色体核型分析：将细胞克隆按5×106的细胞接种在100mm口径培养皿中，至对数期，加入0.2ml的10μmol/L秋水仙酰胺，培养4h后，弃去培养基，加入胰蛋白酶孵育3min，加入10%FBS的DMEM终止消化，离心后，加入40mmol/L KCl和25mmol/L枸橼酸钠的低渗液重悬，静置后，加入等体积新鲜配制的冰冷的1:3醋酸甲醇固定液，静置10min，立新10min后，醋酸甲醇溶液重悬。用巴氏吸管吸一滴细胞悬液，从约3米处滴在用冰块透凉的载玻片中。烘干后进行吉姆萨染色，5min后冲掉染液，观察染色体核型。
Edu增殖检测：取对数生长的细胞克隆，每孔1×105细胞接种于96孔板中，持续培养。每孔加入50μM Edu培养基孵育2小时，弃培养基，PBS清洗2次，加入4%多聚甲醛室温孵育30min，弃固定液。加入甘氨酸孵育5min，清洗后加入0.5% TritonX-100的PBS孵育10min。清洗后加入1× Apollo®染色反应液，避光孵育10min，弃反应液，加入入0.5% TritonX-10的PBS清洗2-3次，甲醇清洗1-2次，Hoechest 33342反应液孵育30min，PBS清洗后，进行观察。
3.确定DNA甲基化对细胞重编程的调控作用；
将四因子分别与5’aza进行组合诱导体细胞，按当观察到出现细胞克隆时收集细胞进行荧光定量检测、间接免疫荧光检测、碱性磷酸酶染色、染色体核型分析和edu增殖检测的方法鉴定获得的细胞克隆，确定最有效的组合形式。在此基础上，单独使用5’aza进行细胞诱导，利用当观察到出现细胞克隆时收集细胞进行荧光定量检测、间接免疫荧光检测、碱性磷酸酶染色、染色体核型分析和edu增殖检测的方法进行细胞鉴定，以此确定DNA甲基化对细胞重编程的调控作用。
Claims (2)
1.一种研究DNA甲基化调控重编程过程的方法，其特征是，包括以下步骤：
步骤（1）、在山中因子Oct4、Sox2、Klf4及c-Myc诱导的基础上添加DNA甲基化酶抑制剂5’aza，按照山中伸弥提供的诱导方法对体细胞进行诱导，在不同诱导天数观察细胞形态；
步骤（2）、当观察到出现细胞克隆时收集细胞进行荧光定量检测、间接免疫荧光检测、碱性磷酸酶染色、染色体核型分析和edu增殖检测；
步骤（3）、确定5’aza的效果后，将四因子和5’aza分别进行组合处理体细胞进行诱导，按步骤（2）中方法检测ips克隆的形成，确定最有效的组合形式；
在此基础上，单独使用5’aza进行诱导，按步骤（2）中方法检测ips克隆形成，进一步确定DNA甲基化抑制剂的单独诱导作用；从而有效探明DNA甲基化在重编程过程中的作用。
2.根据权利要求1所述的一种研究DNA甲基化调控重编程过程的方法，其特征是，步骤（2）中，荧光定量检测的方法为：收集克隆样细胞，添加Trizol 裂解液，进行RNA提取，反转录为cDNA后，实时荧光定量PCR检测Oct4、Sox2、Klf4及c-Myc的表达，同时检测多能性基因的表达以及体细胞标记基因的表达；
间接免疫荧光检测方法为：去除细胞培养孔板中的培养基，添加4%的多聚甲醛对细胞固定30min，PBS清洗后，0.1%的Triton 透膜处理15min，离心清洗后，封闭2h，加入Oct4，Sox2，Klf4，c-Myc，全能性标记蛋白和体细胞标记抗体，孵育过夜，清洗后，加入二抗孵育2h，清洗后加入DAPI孵育10min，荧光倒置显微镜下观察标记蛋白的表达；
碱性磷酸酶染色方法为：吸去培养基,PBS清洗干净后加入ALP固定液，固定3 min，PBS清洗后，加入ALP孵育液，避光15-20 min，PBS清洗；加入核固红染色或甲基绿染色3-5min，PBS清洗后进行镜检，观察克隆细胞能否呈现碱性磷酸酶活性；
染色体核型分析方法为：将细胞克隆按5×106的细胞接种在100mm口径培养皿中，至对数期，加入0.2ml的10μmol/L秋水仙酰胺，培养4h后，弃去培养基，加入胰蛋白酶孵育3min，加入10%FBS的DMEM终止消化，离心后，加入40mmol/L KCl和25mmol/L枸橼酸钠的低渗液重悬，静置后，加入等体积新鲜配制的冰冷的1:3醋酸甲醇固定液，静置10min，立新10min后，醋酸甲醇溶液重悬；用巴氏吸管吸一滴细胞悬液，从3米处滴在用冰块透凉的载玻片中；烘干后进行吉姆萨染色，5min后冲掉染液，观察染色体核型；
edu增殖检测方法为：取对数生长的细胞克隆，每孔1×105细胞接种于96孔板中，持续培养；每孔加入50μM Edu培养基孵育2小时，弃培养基，PBS清洗2次，加入4%多聚甲醛室温孵育30min，弃固定液；加入甘氨酸孵育5min，清洗后加入0.5% TritonX-100的PBS孵育10min；清洗后加入1× Apollo®染色反应液，避光孵育10min，弃反应液，加入0.5% TritonX-10的PBS清洗2-3次，甲醇清洗1-2次，Hoechest 33342反应液孵育30min，PBS清洗后，进行观察。
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| CN106754729A (zh) * | 2016-12-31 | 2017-05-31 | 内蒙古赛科星家畜种业与繁育生物技术研究院有限公司 | 利用Xist Tale抑制性转录因子制备诱导性多能干细胞的方法 |
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