CN113897331A - 一种诱导人源动脉内皮细胞的分化方法 - Google Patents
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Abstract
本发明提供了一种诱导人源动脉内皮细胞的分化方法,属于生物技术领域。所述分化方法为:使用内皮细胞培养基对内皮祖细胞进行贴壁培养0‑1天;使用添加血管内皮生长因子和去甲肾上腺素的内皮细胞基础培养基对所述内皮祖细胞进行连续培养1‑3代,得到所述诱导人源动脉内皮细胞。本发明制备的动脉内皮细胞在表型和功能上与成体动脉内皮细胞一致,而且其实验周期较短,成本低,易操作。该发明可为体外血管疾病研究、血管疾病治疗和药物开发,以及内皮化组织工程血管的构建提供特异动脉内皮细胞来源。
Description
技术领域
本发明属于生物技术领域,尤其是指一种诱导人源动脉内皮细胞的分化方法。
背景技术
动脉血管病变包括动脉粥样硬化、动脉狭窄、动脉瘤和动脉夹层等,常会引发心肌梗死和脑埂塞等重大疾病。目前针对动脉血管类疾病的治疗包括药物处理、介入治疗和血管旁路移植等。供患者移植的血管多为自体静脉血管和非内皮化的人造血管,易形成血栓和再堵塞。人原代血管内皮细胞分离困难、体外培养扩增能力有限、存在去分化等问题,严重限制了人类血管疾病的病理机制研究和治疗策略研究。人多能干细胞(humanpluripotent stem cells,hPSCs)包括诱导多能干细胞(induced pluripotent stemcells,iPSCs)和胚胎干细胞(embryonic stem cells,ESCs),可在体外无限扩增,并分化为内皮细胞(endothelial cells,ECs),为血管类疾病的细胞治疗、药物开发和相容性更好的内皮化人工生物血管开发提供了无限人源内皮细胞资源。现阶段,科学家已开发出hPSCs向ECs分化的方法,所获得细胞表达内皮细胞标志物CD31和CD144等。但这种hPSCs来源的内皮细胞(hPSC-derived ECs,hPSC-ECs)是一种不成熟(表达内皮祖细胞标志物CD34)的多类型细胞群体,同时具有动脉内皮细胞(artery endothelial cells,AECs)和静脉内皮细胞(vein endothelial cells,VECs)特征,难以获得成熟的、高纯度的AECs或VECs。血管内皮的不成熟性和异质性会影响细胞治疗中移植细胞与宿主的整合和功能匹配,药物评估敏感性和内皮化人工血管的顺应性等,不利于临床转化。
现有技术的主要问题为:1)细胞成熟度低,表达内皮祖细胞标志物CD34;2)细胞异质性高,同时具有部分动脉内皮细胞(artery endothelial cells,AECs)和静脉内皮细胞(vein endothelial cells,VECs)特征,不利于多能干细胞来源内皮细胞(hPSC-derivedECs,hPSC-ECs)的临床转化和市场开发。
目前已经有一些研究试图将hPSCs诱导为特异的AECs亚型,主要通过改变物理微环境和添加生物活性因子的方法。改变物理微环境包括在分化过程中模拟血流剪切应力和调节氧气含量(缺氧)来诱导hPSCs向AECs分化,此类方法目前诱导效率低,方法不成熟。添加生物活性因子的方法主要通过向培养基中添加促血管生长因子EGF、VEGF和bFGF等(Sriram et al.Stem Cell Research&Therapy(2015)6:261),或同时添加促血管生长因子(VEGF、bFGF)和化学小分子(SB431542、RESV和L690)等(www.pnas.org/cgi/doi/10.1073/pnas.1702295114)。此类方法添加因子较多,操作复杂,诱导效率不稳定。
现有技术中存在的技术问题,常规hPSCs分化获得的ECs成熟度低(表达内皮祖细胞标志物CD34),具有细胞异质性(同时具有AECs和VECs特征),异质性的程度仍然不明确,需要进一步向AECs进行诱导分化。现有hPSCs向AECs直接定向分化的方法操作复杂、成本高、效率低、稳定性较差。
多能干细胞来源内皮细胞可为血管疾病研究、药物开发和内皮化组织工程血管提供无限内皮细胞来源。但现有多能干细胞分化内皮细胞具有明显不成熟性和异质性,难以获得高纯度、高成熟度动脉内皮细胞,阻碍了其临床转化和市场化开发。
发明内容
为解决上述技术问题,本发明提供了一种诱导人源动脉内皮细胞的分化方法。
一种诱导人源动脉内皮细胞的分化方法,包括以下步骤:
(1)使用内皮细胞培养基对内皮祖细胞进行贴壁培养0-1天;
(2)使用添加血管内皮生长因子(VEGF)和去甲肾上腺素(NE)的内皮细胞基础培养基对所述内皮祖细胞进行连续培养1-3代,得到所述人源动脉内皮细胞。
在本发明的一个实施例中,步骤(1)中,所述内皮祖细胞选自原代分离的内皮祖细胞或CD34阳性的内皮祖细胞。
在本发明的一个实施例中,所述CD34阳性的内皮祖细胞的分化方法为:
(1)使用PSCeasy、mTeSRTM1或TeSRTM-E8TM干细胞培养基对人多能干细胞进行维持培养,对细胞密度达到80%以上的人多能干细胞进行解离传代培养;
(2)使用添加3-6μM CHIR99021的CDM3培养基对步骤(1)中所述人多能干细胞培养;
(3)使用添加25-50ng/mL碱性成纤维细胞生长因子(bFGF)的CDM3培养基对步骤(2)中所得细胞培养;
(4)使用添加25-50ng/mL血管内皮生长因子(VEGF)和10-25ng/mL骨形态发生蛋白4的CDM3培养基对步骤(3)中所得细胞培养;
(5)将步骤(4)中所得细胞进行分离纯化得到所述CD34阳性的内皮祖细胞。
在本发明的一个实施例中,步骤(1)中,所述人多能干细胞选自人胚胎干细胞或诱导多能干细胞。
在本发明的一个实施例中,步骤(1)中,所述CD34阳性的内皮祖细胞的纯化方法包括CD34抗体流式分选或磁珠法。
在本发明的一个实施例中,步骤(2)中,所述内皮细胞基础培养基选自ECM基础培养基、EGM2基础培养基或DMEM/DF12基础培养基。
在本发明的一个实施例中,步骤(2)中,所述血管内皮生长因子在内皮细胞基础培养基中的浓度为50-100ng/μL。
在本发明的一个实施例中,步骤(2)中,所述去甲肾上腺素(NE)在内皮细胞基础培养基中的浓度为10nM-1μM。
本发明所述的人多能干细胞(hPSCs)为包括人胚胎干细胞(hESCs)和诱导多能干细胞(hiPSCs)。在本发明中,可以使用可商购获得的hiPSCs,也可以使用自行对人源体细胞例如成纤维细胞重编程获得的hiPSCs。为伦理学考虑,可以在本发明中使用的人胚胎干细胞,是可商购获得的胚胎干细胞的成熟细胞系,或者是从未经体内发育的受精14天以内的人类胚胎分离或获取的胚胎干细胞。需要特别指出,本发明中的人多能干细胞(hPSCs),无论人胚胎干细胞(hESCs)还是人源诱导性多能干细胞(hiPSCs),均不能够发育成完整个体。
本发明的上述技术方案相比现有技术具有以下优点:
本发明通过采用血管内皮生长因子(VEGF)和去甲肾上腺素(NE)多能干细胞来源内皮细胞,可获得90%以上纯度的动脉内皮细胞,制备的动脉内皮细胞在表型和功能上与成体动脉内皮细胞一致,而且其实验周期较短,成本低,易操作。该发明可为体外血管疾病研究、血管疾病治疗和药物开发,以及内皮化组织工程血管的构建提供特异动脉内皮细胞来源。
本发明关键在于发现了VEGF和去甲肾上腺素促进动脉内皮细胞分化的方法。用VEGF和去甲肾上腺素处理纯化后的内皮祖细胞,连续处理2代之后进行检测,动脉内皮细胞诱导的效率达到了90%以上,所获取的动脉内皮细胞具有正常动脉内皮细胞所具有的功能。VEGF是常用的诱导动脉内皮细胞特征的细胞因子,本发明基于去甲肾上腺素可通过激活ERK信号通路促进鸡胚中动脉内皮细胞迁移的这一现象,提出利用去甲肾上腺素与VEGF联合处理诱导人源动脉内皮细胞分化,发现相较于VEGF单独处理,可使动脉内皮细胞分化效率提高15%以上,很大的提高了动脉内皮细胞的诱导效率,而且其实验周期较短,成本低,易操作。为体外建模血管疾病和建立组织工程血管提供特异亚型的内皮细胞来源。
本发明采用的是人源动脉内皮细胞的,与动物源动脉内皮细胞相比能更好反映人动脉内皮细胞的特征,用于人类疾病模拟、药物开发和治疗。
附图说明
为了使本发明的内容更容易被清楚的理解,下面根据本发明的具体实施例并结合附图,对本发明作进一步详细的说明,其中
图1是本发明H1-EFNB2mCherrry/-多能干细胞分化内皮细胞明场和免疫荧光照片。
图2是本发明中对照组和实验组(VEGF和NE联合处理)细胞的免疫荧光检测,红色为mCherry阳性,代表动脉标志分子EFNB2高表达。
图3是本发明中对照组和实验组(VEGF和NE联合处理)细胞的流式细胞分析,以评估mCherry阳性细胞的动脉内皮细胞比例。
图4是本发明所获动脉内皮细胞(AECs)中动脉标志分子(CXCR4、NRP1和DLL4)的表达水平分析,对照细胞为静脉内皮细胞(VECs)(*表示p<0.05)。
图5是本发明所获动脉内皮细胞(AECs)中一氧化氮(NO)释放水平,成人原代动脉内皮细胞(HAEC)和脐静脉内皮细胞(HUVEC)分别作为阳性对照和阴性对照(*表示p<0.05,ns表示差异不显著)。
图6是本发明所获动脉内皮细胞的血管形成能力检测,A为动脉内皮细胞体外小管形成实验,B为动脉内皮细胞移植到小鼠皮下后的形成血管能力检测。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
实施例1:
多能干细胞来源内皮细胞分化和CD34阳性内皮祖细胞分选
本发明实施例所用多能干细胞为H1-EFNB2mCherrry/-多能干细胞系,干细胞系分化为动脉内皮细胞后,在动脉内皮细胞标志基因EFNB2的启动子驱动下,表达mCherry荧光报告基因,使动脉内皮细胞发红色荧光,便于鉴定动脉内皮细胞。具体实施过程如下:
H1-EFNB2mCherrry/-多能干细胞在基质胶包被的皿中生长至90%融合度时,将培养基更换为由RPMI1640(Thermo Fisher,美国)、牛血清白蛋白(BSA,Thermo Fisher,美国)和L-抗坏血酸(Sigma-Aldrich,美国)组成的CDM3培养基,并添加6μM CHIR99021处理2天;随后更换含有25ng/mL碱性成纤维细胞生长因子(bFGF)的CDM3培养1天;采用含有50ng/mL血管内皮生长因子(VEGF)和25ng/mL骨形态发生蛋白4(BMP4)的CDM3继续培养3天。第6天,在显微镜下可看到网状分布的内皮细胞,其中部分细胞在荧光显微镜下发红色荧光(见图1)。
内皮细胞分化第6天,采用人CD34抗体磁珠(Miltenyi,德国)进行分选,获得CD34阳性的内皮祖细胞。
实施例2:
CD34阳性内皮祖细胞向动脉内皮细胞定向诱导
本实施例采用本发明的方法,利用血管内皮生长因子(VEGF)和去甲肾上腺素(NE)联合处理H1-EFNB2mCherrry/-多能干细胞分化的CD34阳性内皮祖细胞,并评估所制备动脉内皮细胞的表型和功能特征,具体实施过程如下:
H1-EFNB2mCherrry/-多能干细胞分化的CD34阳性内皮祖细胞接种于0.1%明胶包被的培养皿中,在内皮细胞培养基ECM(ScienCell,美国)中贴壁培养1天。
第2天,将培养基更换为ECM基础培养基,并添加血管内皮生长因子(VEGF,终浓度50ng/mL)和去甲肾上腺素(NE,终浓度1μM)进行联合处理,在此条件下联系培养2代,以获得动脉内皮细胞。
培养2代后,对所获动脉内皮细胞进行诱导效率分析、细胞特征鉴定和功能检测。检测方法和结果分析如下:
动脉内皮细胞的诱导效率检测。在荧光显微镜下,实验组内皮细胞中的红色荧光(mCherry)强度和密度明显高于对照组细胞(见图2);流式细胞分析显示,EFNB2-mCherry阳性的动脉内皮细胞比例达到90%以上,显著高于对照组(见图3)。
动脉内皮细胞的标志基因检测。采用qPCR技术评估了纯化后动脉内皮细胞标志基因(CXCR4、NRP1和DLL4)的表达水平,发现上述标志物在本发明所制备的mCherry阳性动脉内皮细胞(AECs)中的表达显著高于人静脉内皮细胞(VECs)(见图4,*表示p<0.05)。
动脉内皮细胞的功能检测。一氧化氮(NO)高产出是动脉内皮细胞区别于静脉内皮细胞的一个标志。本实施例比较了mCherry阳性动脉内皮细胞(AECs)、原代分离的人成体动脉内皮细胞(HAEC)和人脐静脉内皮细胞(HUVEC)中NO的释放量,发现本发明所制备的动脉内皮细胞中NO的释放量和人成体动脉内皮细胞(HAEC)相当,明显高于HUVEC(见图5,*表示p<0.05,ns表示差异不显著)。此外,本实施例利用体外小管形成实验和体内细胞移植实验证实,本发明所获动脉内皮细胞在体外和体内均具有血管形成能力(见图6)。
显然,上述实施例仅仅是为清楚地说明所作的举例,并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (10)
1.一种诱导人源动脉内皮细胞的分化方法,其特征在于,包括以下步骤:
(1)使用内皮细胞培养基对内皮祖细胞进行贴壁培养0-1天;
(2)使用添加血管内皮生长因子和去甲肾上腺素的内皮细胞基础培养基对所述内皮祖细胞进行连续培养1-3代,得到所述人源动脉内皮细胞。
2.根据权利要求1所述的分化方法,其特征在于,步骤(1)中,所述内皮祖细胞选自原代分离的内皮祖细胞或CD34阳性的内皮祖细胞。
3.根据权利要求2所述的分化方法,其特征在于,所述CD34阳性的内皮祖细胞的分化方法为:
(1)使用PSCeasy、mTeSRTM1或TeSRTM-E8TM干细胞培养基对人多能干细胞进行维持培养,对细胞密度达到80%以上的人多能干细胞进行解离传代培养;
(2)使用添加CHIR99021的CDM3培养基对步骤(1)中所述人多能干细胞培养;
(3)使用添加碱性成纤维细胞生长因子的CDM3培养基对步骤(2)中所得细胞培养;
(4)使用添加血管内皮生长因子和骨形态发生蛋白4的CDM3培养基对步骤(3)中所得细胞培养;
(5)将步骤(4)中所得细胞进行分离纯化得到所述CD34阳性的内皮祖细胞。
4.根据权利要求3所述的分化方法,其特征在于,所述CHIR99021的浓度为3-6μM。
5.根据权利要求3所述的分化方法,其特征在于,所述血管内皮生长因子浓度为25-50ng/mL;所述骨形态发生蛋白4的浓度为10-25ng/mL。
6.根据权利要求3所述的分化方法,其特征在于,步骤(1)中,所述人多能干细胞选自人胚胎干细胞或诱导多能干细胞。
7.根据权利要求3所述的分化方法,其特征在于,步骤(5)中,所述CD34阳性的内皮祖细胞的纯化方法包括CD34抗体流式分选或磁珠法。
8.根据权利要求1所述的分化方法,其特征在于,步骤(2)中,所述内皮细胞基础培养基选自ECM基础培养基、EGM2基础培养基或DMEM/DF12基础培养基。
9.根据权利要求1所述的分化方法,其特征在于,步骤(2)中,所述血管内皮生长因子在内皮细胞基础培养基中的浓度为50-100ng/μL。
10.根据权利要求1所述的分化方法,其特征在于,步骤(2)中,所述去甲肾上腺素在内皮细胞基础培养基中的浓度为10nM-1μM。
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