CN114085805B - 冬眠动物干细胞分化为肝细胞的方法、利用该肝细胞筛选人体外细胞保护的代谢产物的方法 - Google Patents
冬眠动物干细胞分化为肝细胞的方法、利用该肝细胞筛选人体外细胞保护的代谢产物的方法 Download PDFInfo
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Abstract
本发明公开了冬眠动物多能干细胞分化为肝细胞的方法，该方法采用ActivinA和CHIR99021分别激活TGFβ和Wnt信号通路，将多能干细胞诱导分化成内胚层细胞,随后以肝细胞生长因子使其向肝样细胞分化，最后以含制瘤素M及地塞米松的培养液促进其成熟。该方法能够有效缩短多能干细胞分化成肝样细胞的时间，提高诱导分化效率，并能够实现冻存复苏，可持续传代，为研究冬眠模式动物冷适应‑复温、缺氧‑复氧等实验模型中的代谢调控机制提供了工具基础。本发明还提供一种利用上述肝细胞筛选为体外冷保存人体细胞提供保护的代谢产物的方法。
Description
技术领域
本发明涉及生物学技术领域。特别地，本发明涉及一种利用冬眠动物干细胞分化诱导为成熟的肝样细胞的方法；以及，利用得到的成熟的肝样细胞为工具研究体外冷保存复温损伤的机制，寻找具有保护人体外细胞保存作用的代谢产物的方法。
背景技术
冬眠模式动物因其非凡的生理特点，如能大幅度进行代谢调控和生命体征调控、抵抗缺血和低温的能力而闻名。在自然冬眠期间动物处于蛰伏期与阵觉期的交替循环；在蛰伏期，动物的核心温度急剧下降，伴随着心跳、呼吸和血流等关键生命体征降至正常时期的1-10％并可维持数天；在阵觉期，动物的核心温度迅速回复到37摄氏度，心跳、呼吸和血流等关键生命体征也迅速恢复正常水平，并维持数小时后，将再次进入蛰伏期。面对这些大幅和快速的生理变化，未发现冬眠动物会出现任何器官损伤。在冬眠季节之外，冬眠动物能够承受医源性损伤，如缺血/再灌注损伤(IRI)和能量剥夺，而人体的IRI会导致器官衰竭，如器官移植和心肌梗死。结合这些特点，冬眠动物是一个最佳的自然模型，可用于研究应用于器官移植等条件下的新型保存技术。然而冬眠动物资源短缺，冬眠相关的细胞和分子机制研究总体进展十分缓慢，其瓶颈在于缺乏强大便捷的体外研究工具。
发明内容
为了解决上述现有技术存在的问题，本发明目的之一在于提供一种冬眠动物干细胞分化为肝细胞的方法，该方法能够在较短时间内将干细胞分化成肝样细胞，诱导分化效率高，分化成熟的肝样细胞能实现冻存复苏，可持续传代，为研究冬眠模式动物冷适应-复温、缺氧-复氧等实验模型中的代谢调控机制提供了工具保障。
为实现上述目的，本发明采用如下技术方案：
冬眠动物干细胞分化为肝细胞的方法，其包括以下步骤：
S01、分化前处理：将冬眠动物的多能干细胞消化成单细胞后接种于包被Geltrex胶的培养皿中，置于无菌培养箱中培养；
S02、内胚层分化：待分化前处理的培养皿中细胞生长汇合度达到10％～20％，采用第一诱导分化培养基进行培养，1天后采用第二诱导分化培养基进行培养，再过1天后采用第三诱导分化培养基培养1～2天；所述第一诱导分化培养基为添加了Activin A、CHIR9901、B27的RPMI-1640培养基，所述第二诱导分化培养基为添加了Activin A、KSR的RPMI-1640培养基，所述第三诱导分化培养基为添加了Activin A、KSR的RPMI-1640培养基，且第三诱导分化培养基中添加的KSR的浓度高于第二诱导分化培养基中KSR的浓度；
S03、肝祖细胞分化：采用第四诱导分化培养基进行培养，所述第四诱导分化培养基为添加了HGF、FBS、谷氨酰胺、非必需氨基酸的Advanced F12培养基；
S04、肝样细胞成熟：采用第五诱导分化培养基进行培养，所述第五诱导分化培养基为添加了Oncostatin M、dexamethasone的Lonza培养基，得到成熟肝细胞。
一种实施方式，步骤S01中，采用Accutase酶消化成单细胞，细胞接种密度为5×103cells/cm2。
一种实施方式，步骤S02中，第一诱导分化培养基中Activin A浓度为100ng/ml，CHIR9901浓度为3uM；第二诱导分化培养基中Activin A浓度为100ng/ml，KSR浓度为0.5％；第三诱导分化培养基中Activin A浓度为100ng/ml，KSR浓度为5％。
一种实施方式，步骤S03中，第四诱导分化培养基中HGF浓度为100ng/ml、FBS浓度为8％、谷氨酰胺浓度为1％、非必需氨基酸浓度为1％。
一种实施方式，步骤S03中，细胞进行隔天换液，培养7天。
一种实施方式，步骤S04中，三天换液一次，培养5天，获得成熟肝细胞。
一种实施方式，步骤S04获得成熟肝细胞采用Lonza培养基添加2％FBS进行传代冻存。
本发明的另一目的是利用上述获得的成熟肝细胞来研究体外冷保存-复温、缺氧-复氧等应激实验造成细胞损伤的机制，提供一种利用上述肝细胞筛选为人体细胞体外保存提供保护的代谢产物的方法。以体外冷保存-复温实验为例，该方法，包括以下步骤：
取所述冬眠动物的成熟肝细胞进行冷冻保存，然后定性定量测量所有代谢物；接着进行复温，再定性定量测量所有代谢物；
取人体肝样细胞进行冷冻保存，然后定性定量测量所有代谢物；接着进行复温，再定性定量测量所有代谢物；
找出冬眠动物的成熟肝细胞在冷冻保存阶段和复温阶段含量具有显著差异的代谢物标记为显著差异代谢物，并对比所述显著差异代谢物在人体肝样细胞的冷冻保存阶段和复温阶段含量有无显著差异，如该代谢物在人体肝样细胞的冷冻保存或复温阶段无显著差异，则标记所述显著差异代谢物为特殊代谢物；
将所述特殊代谢物加入细胞培养基中，采用添加了所述特殊代谢物的细胞培养基冷保存人肝样细胞，评估保存效果，若细胞的存活率高，则该特殊代谢物为人体外细胞保护的代谢产物。
一种实施方式，冷冻保存的温度为4℃，复温为37℃。
一种实施方式，评估保存效果的方法为：以营养培养基冷保存人肝样细胞为对照组，以添加了所述特殊代谢物的营养培养基冷保存人肝样细胞为实验组，进行24h以上冷保存，比较对照组和实验组的细胞存活率，若实验组的细胞存活率高于对照组，则说明所述特殊代谢物为人体外细胞保护的代谢产物。
与现有技术相比，本发明的有益效果是：
1、本发明的冬眠动物干细胞分化为肝细胞的方法能够有效缩短干细胞分化成肝样细胞的时间，提高诱导分化效率，只需要2周的时间就可以将干细胞分化成成熟的肝样细胞，并能够实现冻存复苏，可持续传代，为冬眠模式动物冷适应-复温、缺氧-复氧等应激条件下保护细胞免受损伤的代谢调控机制研究提供了工具基础。
2、基于本发明的分化方法获得了冬眠动物的成熟肝细胞，提供了通过冬眠动物的成熟肝细胞来研究、寻找能够为人体细胞体外保存提供保护的代谢产物的方法，促进人体器官移植保存技术的进步、减轻缺血再灌注损伤有极大的意义。
附图说明
图1是本发明冬眠动物干细胞分化为肝细胞的技术线路图；
图2是本发明肝细胞分化过程中各阶段细胞在光学显微镜下的形态；
图3是本发明肝细胞分化过程中各阶段的特异性基因及蛋白检测图；
图4是本发明分化肝细胞的功能验证实验结果图；
图5是本发明十三条纹地松鼠肝样细胞的差异性代谢产物图；
图6是本发明人肝样细胞差异性代谢产物图；
图7是本发明的对照组和实验组的人肝样细胞的存活率图；
图8为本发明的对照组和实验组的人肝样细胞在冷冻-复温过程中线粒体膜电位图；
图9为本发明的对照组和实验组的人肝样细胞在冷冻-复温过程中线粒体活性氧自由基(ROS)的变化图；
图10为本发明大鼠肝脏冷保存-复温过程的组织形态和酶学指标变化图；
图11为本发明大鼠肝脏冷保存-复温过程凋亡细胞变化图。
具体实施方式
下面通过具体实施例子和附图对本发明做进一步详细说明。
实施例1
请参见图1，图1为本实施方式的冬眠动物干细胞分化为肝细胞的技术线路图。图1显示了本实施方式的肝细胞分化方案，是采用Activin A和CHIR99021分别激活TGFβ和Wnt信号通路，将多能干细胞诱导分化成内胚层细胞,肝细胞生长因子(HGF)使其向肝样细胞分化，最后以含制瘤素M(oncostatin M)及地塞米松(dexamethasone)的培养液促进其成熟。具体方法如下：
一种冬眠动物干细胞分化为肝细胞的方法，包括以下步骤：
S01、分化前处理：
在分化前一天，将十三条纹地松鼠的诱导性多能干细胞用accutase酶消化成单细胞，然后按照细胞按密度5×103cells/cm2接种于提前包被Geltrex胶的培养皿中，置于37℃，5％CO2的无菌培养箱中培养过夜。
S02、内胚层分化：
第一阶段：待分化前处理的培养皿中细胞生长汇合度达到10％～20％，采用第一诱导分化培养基进行培养，时间为24h。所述第一诱导分化培养基为添加了Activin A、CHIR9901、B27的RPMI-1640培养基，Activin A浓度为100ng/ml，CHIR9901浓度为3uM。
第二阶段：采用第二诱导分化培养基进行培养24h，所述第二诱导分化培养基为添加了Activin A、KSR的RPMI-1640培养基，Activin A浓度为100ng/ml，KSR浓度为0.5％。
第三阶段：采用第三诱导分化培养基培养1～2天，所述第三诱导分化培养基为添加了Activin A、KSR的RPMI-1640培养基，Activin A浓度为100ng/ml，KSR浓度为5％。
S03、肝祖细胞分化：
采用第四诱导分化培养基进行培养，所述第四诱导分化培养基为添加了HGF、FBS、谷氨酰胺、非必需氨基酸的Advanced F12培养基，HGF浓度为100ng/ml、FBS浓度为8％、谷氨酰胺浓度为1％、非必需氨基酸浓度为1％，隔天换液维持7天。
S04、肝样细胞成熟：
采用第五诱导分化培养基进行培养，所述第五诱导分化培养基为添加了Oncostatin M、dexamethasone的Lonza培养基，oncostatin M浓度为20ng/ml，dexamethasone浓度为100nM，三天换液一次，培养5天，即可获得成熟的肝细胞。
以上分化肝细胞的各个阶段过程中，细胞的形态如图2所示。图2为光学显微镜下分化过程中各个阶段的细胞形态。由图2可以看出，诱导分化肝样细胞具有类似肝细胞的形态结构，呈多面体形；核大而圆,居中,常染色质丰富,部分有双核或多倍体核结构。
采用逆转录-聚合酶链反应(RT-PCR)及免疫荧光检测分化各个阶段细胞特异性基因和蛋白表达水平，结果如图3所示，均为强阳性。
为了验证本实施方式分化诱导的肝细胞功能，图4A，4B分别通过western blot蛋白电泳及流式细胞术检测肝样细胞的标志性蛋白。采用过碘酸.雪夫反应(PAS)试验和吲哚氰绿(ICG)摄取试验分别检测分化细胞是否具备糖原合成和ICG摄取释放等肝细胞功能。采用BODIPY脂质代谢检测肝样细胞脂滴水平。图中显示结果均为强阳性。
由以上验证实验可以证明，本实施方式冬眠动物干细胞分化为肝细胞的方法，采用冬眠模式生物十三条纹地松鼠的诱导性多能干细胞成功分化出了可持续传代冻存的冬眠物种肝样细胞，提供了强大便捷的体外研究工具。为直观地探索冬眠物种冷适应-复温、缺氧-复氧等应激条件下保护细胞免受损伤的分子机制及代谢调控机制等的研究提供了基础。
实施例2
一种利用肝细胞筛选人体外细胞保护的代谢产物的方法，该方法包括以下步骤：
取所述冬眠动物的成熟肝细胞(实施例1获得的成熟肝细胞)进行冷冻保存，然后定性定量测量所有代谢物；接着进行复温，再定性定量测量所有代谢物；
取人体肝样细胞进行冷冻保存，然后定性定量测量所有代谢物；接着进行复温，再定性定量测量所有代谢物；
找出冬眠动物的成熟肝细胞在冷冻保存阶段和复温阶段含量具有显著差异的代谢物标记为显著差异代谢物，并对比所述显著差异代谢物在人肝样细胞的冷冻保存阶段和复温阶段含量有无显著差异，如该代谢物在人体肝样细胞的冷冻保存或复温阶段无显著差异，则标记所述显著差异代谢物为特殊代谢物；
将所述特殊代谢物加入营养培养基，采用添加了所述特殊代谢物的营养培养基冷保存人肝样细胞，评估保存效果，若细胞的存活率高，则该特殊代谢物为对人体外细胞有保护作用的代谢产物。
一种可选的实施方式，冷冻保存的温度为4℃，复温为37℃。
一种可选的实施方式，评估保存效果的方法为：以营养培养基冷保存人肝样细胞为对照组，以添加了所述特殊代谢物的营养培养基冷保存人肝样细胞为实验组，进行24h以上冷保存，比较对照组和实验组的细胞存活率。
具体地，取实施例1获得的成熟肝细胞，用营养培养基保存，保存条件为4℃、4h，然后定性定量测量代谢产物；接着复温，复温条件为37℃、2h，然后定性定量测量代谢产物，结果如图5所示。
取人体肝样细胞，用营养培养基保存，保存条件为4℃、4h，然后定性定量测量代谢产物；接着复温，复温条件为37℃、2h，然后定性定量测量代谢产物，结果如图6所示。
利用代谢组学对比了冬眠物种(即实施例1的成熟肝细胞)与非冬眠物种(人肝样细胞)的代谢产物，找出差异性代谢产物113种，包括：D-葡萄糖、亚油酸、17α-羟基妊娠烯醇酮、4-羟基-L-苯甘氨酸、1,3-二甲基尿酸、1-(3-甲氧基-4-(磺基)苯基)-1,2-乙二醇、异亮氨酸-缬氨酸、S-磺基-L-半胱氨酸、L-酪氨酸甲酯、甘氨酰-苯丙氨酸、L-缬氨酸-L-甘氨酸、N-甘氨酰-L-亮氨酸、2-甲基丁酰肉碱、5-氨基酮戊酸、β-氨基丙酸、肌氨酸、亮氨酸-酪氨酸、苯丙氨酸-谷氨酸、异亮氨酸-苏氨酸等等。
本发明人发现，5-氨基酮戊酸(5-ALA)在冬眠物种冷适应期间升高，复温期间下降，在人肝样细胞的代谢组5-ALA则无显著差异，因此，将5-ALA标记为显著差异代谢物。
在此基础上发明者在人肝样细胞冷保存过程中添加此代谢产物。采用营养培养基冷保存人肝样细胞为对照组，采用添加不同浓度的5-ALA的营养培养基为实验组，24h、4℃的保存条件后，比较细胞存活率。结果如图7所示，由图7可知，添加了5-ALA的实验组效果好，细胞存活率高，同时能够稳定线粒体膜电位、抑制线粒体ROS的过度生成(如图8，9)。
在本实施例中，所述营养培养基为：HibernateTM-A培养基(货号GIBCOA1247501),10ug/ml转铁蛋白,3ng/ml人表皮生长因子，25.5ug/ml维生素C，10ug/ml胰岛素，10uM氢化可的松，10mg/ml牛血清白蛋白。
为了充分验证5-ALA的在冷保存中能有效减少冷保存-复温引起的线粒体损伤。在此基础上，发明人将其应用在大鼠肝脏冷保存(如图10A所示)，在冷保存48小时复温灌注2小时的大鼠肝脏组织切片HE染色显示，相比于UW液组(UW液是临床用的器官保存液)，UW液+5-ALA组的肝组织形态明显改善(如图10B所示)。如图10C所示，UW液+5-ALA组在灌注2小时期间灌注液的AST，ALT和LDH酶学指标比UW液组显著降低。图11显示了UW液+5-ALA组的肝组织中凋亡细胞减少。
通过本实施例的方法，能够筛选出保护人肝样细胞的代谢产物，促进了器官移植的保存技术的技术，对减轻缺血再灌注损伤有极大的意义。
此外，需要说明的是，使用“第一”、“第二”等词语来限定零部件，仅仅是为了便于对相应零部件进行区别，如没有另行声明，上述词语并没有特殊含义，因此不能理解为对本申请保护范围的限制。
对于本领域的技术人员来说，可根据以上描述的技术方案以及构思，做出其它各种相应的改变以及形变，而所有的这些改变以及形变都应该属于本发明权利要求的保护范围之内。
Claims (5)
1.冬眠动物干细胞分化为肝细胞的方法，其特征在于，包括以下步骤：
S01、分化前处理：将冬眠动物的多能干细胞采用Accutase酶消化成单细胞后，按照细胞接种密度为5×103cells/cm2接种于包被Geltrex胶的培养皿中，置于37℃，5％CO2的无菌培养箱中培养；
S02、内胚层分化：待分化前处理的培养皿中细胞生长汇合度达到10％～20％，采用第一诱导分化培养基进行培养，1天后采用第二诱导分化培养基进行培养，再过1天后采用第三诱导分化培养基培养1～2天；所述第一诱导分化培养基为添加了Activin A、CHIR9901、B27的RPMI-1640培养基，所述第二诱导分化培养基为添加了Activin A、KSR的RPMI-1640培养基，所述第三诱导分化培养基为添加了Activin A、KSR的RPMI-1640培养基，且第三诱导分化培养基中添加的KSR的浓度高于第二诱导分化培养基中KSR的浓度；其中，第一诱导分化培养基中Activin A浓度为100ng/ml，CHIR9901浓度为3uM；第二诱导分化培养基中Activin A浓度为100ng/ml，KSR浓度为0.5％；第三诱导分化培养基中Activin A浓度为100ng/ml，KSR浓度为5％；
S03、肝祖细胞分化：采用第四诱导分化培养基进行培养，所述第四诱导分化培养基为添加了HGF、FBS、谷氨酰胺、非必需氨基酸的Advanced F12培养基；其中，第四诱导分化培养基中HGF浓度为100ng/ml、FBS浓度为8％、谷氨酰胺浓度为1％、非必需氨基酸浓度为1％；细胞进行隔天换液，培养7天；
S04、肝样细胞成熟：采用第五诱导分化培养基进行培养，所述第五诱导分化培养基为添加了Oncostatin M、dexamethasone的Lonza培养基，三天换液一次，培养5天，得到成熟肝细胞。
2.根据权利要求1所述冬眠动物干细胞分化为肝细胞的方法，其特征在于，步骤S04获得成熟肝细胞采用Lonza培养基添加2％FBS进行传代冻存。
3.一种利用肝细胞筛选人体外细胞保护的代谢产物的方法，其特征在于，包括以下步骤：
取权利要求1或2任一项获得的冬眠动物的成熟肝细胞进行冷冻保存，然后定性定量测量所有代谢物；接着进行复温，再定性定量测量所有代谢物；
取人体肝样细胞进行冷冻保存，然后定性定量测量所有代谢物；接着进行复温，再定性定量测量所有代谢物；
找出冬眠动物的成熟肝细胞在冷冻保存阶段和复温阶段含量具有显著差异的代谢物标记为显著差异代谢物，并对比所述显著差异代谢物在人体肝样细胞的冷冻保存阶段和复温阶段含量有无显著差异，如该代谢物在人体肝样细胞的冷冻保存或复温阶段无显著差异，则标记所述显著差异代谢物为特殊代谢物；
将所述特殊代谢物加入营养培养基中，采用添加了所述特殊代谢物的营养培养基冷保存人肝样细胞，评估保存效果，若细胞的存活率高，则该特殊代谢物为人体外细胞保护的代谢产物。
4.根据权利要求3所述利用肝细胞筛选保护人体外细胞保护的代谢产物的方法，其特征在于：冷冻保存的温度为4℃，复温为37℃。
5.根据权利要求3所述利用肝细胞筛选人体外细胞保护的代谢产物的方法，其特征在于，评估保存效果的方法为：以营养培养基冷保存人肝样细胞为对照组，以添加了所述特殊代谢物的营养培养基冷保存人肝样细胞为实验组，进行24h以上冷保存，比较对照组和实验组的细胞存活率。
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