CN111073843A - 一种肝样细胞成熟与扩增的方法 - Google Patents
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Abstract
本发明涉及一种肝样细胞成熟与扩增的方法,步骤包括:步骤S1:干细胞在体外使用诱导液进行培养诱导间充质干细胞分化为肝样细胞;步骤S2:将肝样细胞通过脾脏定点注射到FAH基因缺陷大鼠肝脏载体;步骤S3:刺激FAH基因缺陷大鼠肝脏载体自体肝细胞凋亡,促进肝样细胞成熟、增殖;步骤S4:分离与纯化得到人原代肝细胞。本发明在体外诱导人间充质干细胞形成肝样细胞,通过脾脏定点注射到FAH基因缺陷大鼠肝脏中,利用FAH基因缺陷大鼠肝脏内环境,使肝样细胞高效地分化为成熟的人肝细胞并实现肝细胞的快速扩增。从而解决了肝细胞体外再生的方法得到的肝细胞不成熟、规模小、成本高等缺点。
Description
【技术领域】
本发明涉及生物医学技术领域,涉及细胞改造技术领域,尤其涉及肝样细胞成熟与扩增的方法。
【背景技术】
人原代肝细胞是指从人肝组织取出后立即培养的肝细胞,可用于基础生命科学研究、药物研发、肝细胞移植以及肝脏器官3D打印,市场容量可达千亿。然而,肝细胞来源问题一直制约其广泛应用。一方面由于文化因素,我国的公民离世后的器官捐献率极低;另一方面,我国关于器官捐献的法规尚不完善,这都导致人原代肝细胞极为缺乏。
目前,解决人原代肝细胞来源问题的技术手段主要包括:1)肝细胞体外再生,即利用胚胎干细胞、诱导多能干细胞或者间充质干细胞在体外利用不同的诱导因子及诱导步骤分化成肝样细胞;2)肝细胞体内扩增,即将人原代肝细胞接种到免疫缺陷、自体肝细胞诱导凋亡的小鼠模型中,长出人鼠嵌合肝。肝细胞体外再生的方法得到的肝细胞为肝样细胞,表达部分肝细胞标志基因,仅具备肝细胞部分生理功能,与人原代肝细胞还有较大差距。另外,肝细胞体外再生的方法具有操作要求高、规模小、成本高等缺点,在实际应用中不具有可行性。肝细胞(小鼠)体内扩增的方法可得到一定数量的成熟人原代肝细胞,但无法实现个性化定制,且肝细胞数量有限。
【发明内容】
本发明要解决的技术问题是提供一种肝样细胞成熟与扩增的方法。
本发明采用如下技术方案:
本发明提供了一种肝样细胞成熟与扩增的方法,具体步骤包括:
步骤S1:干细胞在体外使用相应的诱导液进行培养诱导干细胞分化为肝样细胞;
步骤S2:将肝样细胞通过脾脏定点注射到FAH基因缺陷大鼠肝脏载体;
步骤S3:刺激FAH基因缺陷大鼠肝脏载体自体肝细胞凋亡,促进肝样细胞成熟、增殖;
步骤S4:分离与纯化得到人原代肝细胞。
进一步的,所述干细胞包括骨髓来源的间充质干细胞、胚胎干细胞或诱导多能干细胞。
进一步的,所述骨髓来源的间充质干细胞体外诱导间充质干细胞分化为肝样细胞具体步骤为:在细胞融合度为85%时,加入肝细胞诱导液,每3天换1次液,诱导培养2周后,向肝细胞诱导液加入30ng/ml的humanOncostatinM,继续诱导1周后经免疫荧光鉴定白蛋白表达;
肝细胞诱导液包括:IMDM培养液、100units/mL青霉素、100mg/mL链霉素、25mMHepesBuffer Solution、1倍体积的ITS+1LiquidMedia Supplement、20μg/mldexamethasone 和20ng/mlHGF。
进一步的,所述胚胎干细胞培养体系中加RPMI基础培养基补充1mM L- 谷氨酰胺、0.5%v/v胎牛血清、100ng/ml激活素-A、25ng/ml Wnt3a,诱导培养2天;RPMI基础培养基补充1mM L-谷氨酰胺、0.5%v/v胎牛血清、100ng/ml 激活素-A、继续诱导培养2天;接着培养基换成HCM基础培养基,添加20ng/ml 重组人骨形态发生蛋白、30ng/ml成纤维细胞生长因子-4,继续诱导培养6天; HCM基础培养基补加20ng/ml肝细胞生长因子,诱导培养5天;HCM基础培养基补加10μg/ml human OncostatinM,100nM地塞米松,诱导培养15天。
进一步的,所述诱导多能干细胞体外诱导分化为肝样细胞具体步骤为: CDM-PVA培养基(250mL DMEM-F12,250mL IMDM,0.5g聚乙烯醇,5ml 浓缩脂类,20μL硫代甘油,25μg/mL转铁蛋白,10μg/L重组人胰岛素, 100units/mL青霉素,100mg/mL链霉素),补加10ng/mL激活素-A和12ng/mL 碱性成纤维细胞生长因子,培养2天;CDM-PVA培养基,补加100ng/mL激活素-A,100ng/mL碱性成纤维细胞生长因子,10ng/mL重组人骨形态发生蛋白4,10μMLY294002,3μM CHIR99021,培养24小时;CDM-PVA培养基,补加100ng/mL激活素-A,100ng/mL碱性成纤维细胞生长因子,10ng/mL重组人骨形态发生蛋白4,10μM LY294002,培养24小时;RPMI-B27培养基 (490mL RPMI 1640,补加10mL B27,5mL NEAA,100units/mL青霉素,100mg/mL链霉素),补加100ng/mL激活素-A,100ng/mL碱性成纤维细胞生长因子,诱导培养24小时;RPMI-B27培养基,补加50ng/mL激活素-A,诱导培养3天;RPMI-B27培养基,补加20ng/mL重组人骨形态发生蛋白4,10ng/mL成纤维细胞生长因子-10,诱导培养3天;Hepatocyte Basal Medium,补加50ng/ml HGF,30ng/ml human OncostatinM,诱导培养3天。
进一步的,所述FAH基因缺陷大鼠脾脏载体通过受精卵显微注射下使用 CRISPCas9基因敲除方法获得。
进一步的,所述FAH基因缺陷大鼠肝脏载体的获得方法具体是通过将诱导分化完成的肝样细胞通过胰酶消化、离心、0.9%生理盐水重悬,制成细胞密度为1×107活细胞/500μl的肝样细胞悬液,从脾尖利用1ml注射器,将肝样细胞悬液缓慢注射进入脾脏,肝样细胞随血液流动慢慢进入肝脏载体。
进一步的,所述刺激FAH基因缺陷大鼠肝脏载体自体肝细胞凋亡,促进肝样细胞成熟、增殖是去掉FAH基因缺陷大鼠维持药物NTBC,加入免疫抑制剂混合物和抗感染药物进行培养,并且每隔7天收集一次样本置于-80℃保存待检测。
进一步的,所述步骤4具体是:利用4℃预冷的灌注溶液I原位灌注大鼠肝脏约20min,直至肝组织中的血液被冲洗干净;利用37℃预热的灌注溶液II灌注30min,直至肝组织失去弹性;将肝组织转移到盛有足量肝细胞洗液的培养皿中,用无菌镊子扯破肝组织,得到肝细胞悬液;肝细胞悬液经过70μm细胞筛,以去除组织团块;细胞悬液转移至50ml离心管中,50×g,4℃,离心5min;去掉上清,肝细胞洗液重悬细胞沉淀,重复离心2次;铺板培养液重悬肝细胞,利用台盼蓝拒染法检测细胞活力与活细胞数;细胞悬液以(1.5-2.5)×105活细胞/cm2的接种密度,接种到胶原包被的培养板或培养皿中,摇匀,在37℃、5%CO2培养箱中培养4-6h后,将铺板培养液换成肝细胞维持培养液。
进一步的,所述灌注溶液I为D-Hank’s溶液中含有乙二醇双(2-氨基乙醚)四乙酸、半胱氨酸的混合液,pH值为7.4,0.22μm滤膜过滤,4℃保存;灌注溶液II为D-Hank’s溶液中含有5mmol/l氯化钙、0.1g/l六水氯化镁、0.3g/lⅣ型胶原酶、1.0g/l II型分离酶、50mg/l核酸酶的混合液,pH值为7.4,0.22μm滤膜过滤,4℃保存;所述肝细胞洗液为William’smediumE培养基里加入2mmol/l谷氨酰胺、100units/mL 青霉素、100mg/mL链霉素,2~5%m/v牛血清白蛋白;
所述肝细胞铺板培养液是在William’smediumE培养基里加入1%v/v的ITS+premix、 2mmol/l谷氨酰胺、10μg/l表皮生长因子、18mg/l氢化可的松、40μg/l地塞米松、100units/mL 青霉素、100mg/mL链霉素和5%v/vFBS;
所述肝细胞维持培养液,是在William’s medium E培养基里加入1%v/v的ITS+premix、2mmol/l谷氨酰胺、10μg/l表皮生长因子、18mg/l氢化可的松、 40μg/l地塞米松、100units/mL青霉素、100mg/mL链霉素和2%v/v DMSO。
进一步的,还需要还鉴定人原代肝细胞功能,具体的步骤:
将刺激FAH基因缺陷大鼠肝脏载体自体肝细胞凋亡得到的血清样本用 ELISA检测人白蛋白、大鼠白蛋白的分泌;
利用免疫荧光检测步骤4得到的细胞培养物中FAH蛋白阳性率;
将步骤4得到的细胞培养物和步骤1得到的肝样细胞,利用RT-PCR鉴定人肝细胞标志基因表达。
与现有技术相比,本发明的有益效果在于:
1、本发明可以通过干细胞诱导形成肝样细胞,干细胞来源广泛包括胚胎干细胞、诱导多能干细胞或间充质干细胞,实现人原代肝细胞的个性化订制。
2、本发明将肝样细胞定点注射到大鼠脾脏,通过血流带动肝样细胞到达肝脏,相较于肝门静脉,脾脏定点注射可达到注射细胞量大、成功率高。
3、本发明在体外诱导人间充质干细胞形成肝样细胞,通过脾脏定点注射到FAH基因缺陷大鼠肝脏中,利用FAH基因缺陷大鼠肝脏内环境即自体肝细胞逐步凋亡,使肝样细胞高效地分化为成熟的人肝细胞并实现肝细胞的快速扩增。从而解决了肝细胞体外再生的方法得到的肝细胞不成熟、规模小、成本高等缺点,以及针对肝细胞(小鼠)体内扩增的方法无法实现个性化定制且肝细胞数量有限的问题。
【附图说明】
图1为免疫荧光检测肝样细胞中白蛋白的表达;
图2为免疫荧光检测肝样细胞中细胞核;
图3为移植大鼠血浆中大鼠白蛋白、人白蛋白水平;
图4为RT-PCR检测人肝细胞标记基因与大鼠肝细胞作为检测的阴性对照;
图5为免疫荧光检人肝细胞的FAH染色;
图6为免疫荧光检人肝细胞的细胞核染色;
图7为免疫荧光检人肝细胞的细胞核与FAH染色重叠图。
【具体实施方式】
本发明目的的实现、功能特点及优点将结合实施例,做进一步说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
本发明提供了一种肝样细胞成熟与扩增的方法,具体步骤包括:
步骤S1:干细胞在体外使用相应的诱导液进行培养诱导干细胞分化为肝样细胞;
步骤S2:将肝样细胞通过脾脏定点注射到FAH基因缺陷大鼠肝脏载体;
步骤S3:刺激FAH基因缺陷大鼠肝脏载体自体肝细胞凋亡,促进肝样细胞成熟、增殖;
步骤S4:分离与纯化得到人原代肝细胞。
这里所述干细胞包括骨髓来源的间充质干细胞、胚胎干细胞或诱导多能干细胞等等。
一实施例中,所述骨髓来源的间充质干细胞体外诱导间充质干细胞分化为肝样细胞具体步骤为:在细胞融合度为85%时,加入肝细胞诱导液,每3天换1次液,诱导培养2周后,向肝细胞诱导液加入30ng/ml的human Oncostatin M,继续诱导1周后经免疫荧光鉴定白蛋白表达;
肝细胞诱导液包括:IMDM培养液、100units/mL青霉素、100mg/mL链霉素、25mMHepes Buffer Solution、1倍体积的ITS+1LiquidMedia Supplement、20μg/mldexamethasone 和20ng/ml HGF。
具体的是,骨髓来源的间充质干细胞,细胞融合度为85%时,加入肝细胞诱导液,每 3天换1次液,肝细胞诱导液成分为:IMDM培养液(购买至美国Gibco公司),补加100units/mL青霉素(购买至美国Gibco公司)、100mg/mL链霉素(购买至美国Gibco公司),25mM Hepes Buffer Solution(购买至美国Gibco公司),1倍体积的ITS+1LiquidMediaSupplement(购买至美国Sigma公司),20μg/ml dexamethasone(购买至美国Sigma公司),20ng/ml HGF(购买至美国cellsciences公司)。诱导培养2周后,向肝细胞诱导液加入30ng/ml 的human Oncostatin M(购买至美国cellsciences公司),继续诱导1周后经免疫荧光等鉴定白蛋白表达。白蛋白表达阳性则确定肝样细胞诱导成功,如图1、图2所示。
一实施例中,所述胚胎干细胞体外诱导分化为肝样细胞具体步骤为:RPMI 基础培养基(购买至美国Gibco公司)补充1mM L-谷氨酰胺(购买至美国Gibco公司), 0.5%v/v胎牛血清(购买至美国Gibco公司),100ng/ml激活素-A(购买至美国R&D System 公司),25ng/ml Wnt3a,诱导培养2天;RPMI补充1mM L-谷氨酰胺,0.5%v/v胎牛血清,100ng/ml激活素-A,诱导培养2天;HCM基础培养基(购买至美国Gibco公司)补加20ng/ml 重组人骨形态发生蛋白2,30ng/ml成纤维细胞生长因子-4,诱导培养6天;HCM基础培养基补加20ng/ml肝细胞生长因子,诱导培养5天;HCM基础培养基补加10μg/ml human OncostatinM,100nM地塞米松,诱导培养15天。
另一实施例中,所述诱导多能干细胞体外诱导分化为肝样细胞具体步骤为: CDM-PVA培养基(250mLDMEM-F12,250mL IMDM,0.5g聚乙烯醇(购买至美国Sigma 公司),5ml浓缩脂类(购买至美国Gibco公司),20μL硫代甘油(购买至美国Sigma 公司),25μg/mL转铁蛋白(购买至美国Roche公司),10μg/L重组人胰岛素(购买至美国Roche公司),100units/mL青霉素,100mg/mL链霉素),补加10ng/mL激活素-A 和12ng/mL碱性成纤维细胞生长因子,培养2天;CDM-PVA培养基,补加100ng/mL激活素-A,100ng/mL碱性成纤维细胞生长因子,10ng/mL重组人骨形态发生蛋白4,10μM LY294002,3μM CHIR99021,培养24小时;CDM-PVA培养基,补加100ng/mL激活素-A, 100ng/mL碱性成纤维细胞生长因子,10ng/mL重组人骨形态发生蛋白4,10μM LY294002,培养24小时;RPMI-B27培养基(490mLRPMI 1640,补加10mLB27(购买至美国Gibco 公司),5mLNEAA(购买至美国Gibco公司),100units/mL青霉素,100mg/mL链霉素),补加100ng/mL激活素-A,100ng/mL碱性成纤维细胞生长因子,诱导培养24小时;RPMI-B27 培养基,补加50ng/mL激活素-A,诱导培养3天;RPMI-B27培养基,补加20ng/mL重组人骨形态发生蛋白4,10ng/mL成纤维细胞生长因子-10,诱导培养3天;Hepatocyte BasalMedium(购买至美国Lonza公司),补加50ng/ml HGF,30ng/ml human OncostatinM,诱导培养3天。
进一步的,所述FAH基因缺陷大鼠通过受精卵显微注射下使用CRISP Cas9基因敲除方法获得。所述FAH基因缺陷大鼠肝脏载体的获得方法具体是通过将诱导分化完成的肝样细胞通过胰酶消化、离心、0.9%生理盐水重悬,制成细胞密度为1×107活细胞/500μl的肝样细胞悬液,从脾尖利用1ml注射器,将肝样细胞悬液缓慢注射进入脾脏。
进一步的,所述刺激FAH基因缺陷大鼠肝脏载体自体肝细胞凋亡,促进肝样细胞成熟、增殖是去掉FAH基因缺陷大鼠维持药物NTBC,加入免疫抑制剂混合物和抗感染药物进行培养,并且每隔7天收集一次样本置于-80℃保存待检测。
进一步的,所述步骤4具体是:利用4℃预冷的灌注溶液I原位灌注大鼠肝脏约20min,直至肝组织中的血液被冲洗干净;利用37℃预热的灌注溶液II灌注30min,直至肝组织失去弹性;将肝组织转移到盛有足量肝细胞洗液的培养皿中,用无菌镊子扯破肝组织,得到肝细胞悬液;肝细胞悬液经过70μm细胞筛,以去除组织团块;细胞悬液转移至50ml离心管中,50×g,4℃,离心5min;去掉上清,肝细胞洗液重悬细胞沉淀,重复离心2次;铺板培养液重悬肝细胞,利用台盼蓝拒染法检测细胞活力与活细胞数;细胞悬液以(1.5-2.5)×105活细胞/cm2的接种密度,接种到胶原包被的培养板或培养皿中,摇匀,在37℃、5%CO2培养箱中培养4-6h后,将铺板培养液换成肝细胞维持培养液。
进一步的,所述灌注溶液I为D-Hank’s溶液中含有乙二醇双(2-氨基乙醚)四乙酸、半胱氨酸的混合液,pH值为7.4,0.22μm滤膜过滤,4℃保存;灌注溶液II为D-Hank’s溶液中含有5mmol/l氯化钙、0.1g/l六水氯化镁、0.3g/lⅣ型胶原酶、1.0g/l II型分离酶、50mg/l核酸酶的混合液,pH值为7.4,0.22μm滤膜过滤, 4℃保存;所述肝细胞洗液为William’smedium E培养基里加入2mmol/l谷氨酰胺、 100units/mL青霉素、100mg/mL链霉素,2~5%m/v牛血清白蛋白;
所述肝细胞铺板培养液是在William’s mediumE培养基里加入1%v/v的ITS+premix、 2mmol/l谷氨酰胺、10μg/l表皮生长因子、18mg/l氢化可的松、40μg/l地塞米松、100units/mL 青霉素、100mg/mL链霉素和5%v/vFBS;
所述肝细胞维持培养液,是在William’s medium E培养基里加入1%v/v的 ITS+premix、2mmol/l谷氨酰胺、10μg/l表皮生长因子、18mg/l氢化可的松、40μg/l地塞米松、100units/mL青霉素、100mg/mL链霉素和2%v/vDMSO。
进一步的,还需要还鉴定人原代肝细胞功能,具体的步骤:
将刺激FAH基因缺陷大鼠肝脏载体自体肝细胞凋亡得到的样本用ELISA检测人白蛋白、大鼠白蛋白的分泌;
利用免疫荧光检测步骤4得到的细胞培养物中FAH蛋白阳性率;
将步骤4得到的细胞培养物和步骤1得到的肝样细胞,利用RT-PCR鉴定人肝细胞标志基因表达。
具体的实验步骤:
步骤1:体外诱导间充质干细胞分化为肝样细胞
骨髓来源的间充质干细胞,细胞融合度为85%时,加入肝细胞诱导液,每3天换1次液,肝细胞诱导液成分为:IMDM培养液(购买至美国Gibco公司),补加100units/mL青霉素(购买至美国Gibco公司)、100mg/mL链霉素(购买至美国Gibco公司),25mMHepes BufferSolution(购买至美国Gibco公司),1倍体积的ITS+1LiquidMedia Supplement(购买至美国Sigma公司),20μg/ml dexamethasone(购买至美国Sigma公司),20ng/ml HGF (购买至美国cellsciences公司)。诱导培养2周后,向肝细胞诱导液加入30ng/ml的human Oncostatin M(购买至美国cellsciences公司),继续诱导1周后经免疫荧光鉴定白蛋白表达。白蛋白表达阳性则确定肝样细胞诱导成功,如图1、图2所示。
步骤2:将肝样细胞通过脾脏定点注射到FAH基因缺陷大鼠肝脏
通过CRISP Cas9基因敲除方法以及受精卵显微注射,获得的FAH(延胡索酰乙酰乙酸水解酶)缺失的大鼠,FAH基因缺失大鼠在饮水中加入5.4mg/L 2-(2-nitro-4-trifluoro-methylbenzoyl)-1,3-cyclo-hexanedione(NTBC),以维持大鼠的生命。从手术前3天起,每天向大鼠食物中加入一定量的免疫抑制剂混合物(4mg/kg tacrolimus, 20mg/kg mycophenolate mofetil,4mg/kg prednisone);手术前1天,从饮用水中去掉生命维持药物NTBC;移植当天,将诱导分化完成的肝样细胞通过胰酶消化、离心、0.9%生理盐水重悬,制成细胞密度为1×107活细胞/500μl的肝样细胞悬液;经戊巴比妥钠麻醉的 FAH基因缺陷大鼠放置于超净工作台内,腹部去毛、碘伏消毒,经过手术解剖暴露脾脏,从脾尖利用1ml注射器(26gauge针头),将肝样细胞悬液缓慢注射进入脾脏,确保注射无泄露与脾脏无出血,最后手术缝合,伤口处涂碘伏。
步骤3:刺激FAH基因缺陷大鼠自体肝细胞凋亡、肝样细胞成熟与增殖
将步骤2得到的动物,放入SPF级的独立通风系统,每天向大鼠的食用面包中加入免疫抑制剂混合物(4mg/kg tacrolimus,20mg/kg mycophenolate mofetil,4mg/kgprednisone),向饮用水中加入抗感染药物(25mg/kg头孢克肟,5mg/kg ganciclovir),加入经高压的灭菌的垫料与食物;术后每隔7天收集一次大鼠血清,-80℃保存待检测;
FAH基因缺失大鼠,去除药物NTBC后,大鼠自体肝细胞发生凋亡,为肝样细胞成熟与增殖提供微环境。
利用免疫抑制剂(4mg/kg tacrolimus,20mg/kg mycophenolate mofetil,4mg/kg以及抗感染药物(25mg/kg头孢克肟,5mg/kg ganciclovir)可以达到免疫抑制效果,同时抵抗大鼠可能的感染,使大鼠得以生存。
步骤4:分离人原代肝细胞
术后第8周,戊巴比妥钠麻醉的大鼠转移到生物安全柜中,解剖暴露大鼠肝门静脉,利用4℃预冷的灌注溶液I原位灌注大鼠肝脏约20min,直至肝组织中的血液被冲洗干净,再利用37℃预热的灌注溶液II灌注30min,直至肝组织失去弹性;将肝组织转移到盛有足量肝细胞洗液的培养皿中,用无菌镊子扯破肝组织,得到肝细胞悬液;肝细胞悬液经过70μm细胞筛,以去除组织团块;细胞悬液转移至50ml离心管中,50×g,4℃,离心5min;去掉上清,肝细胞洗液重悬细胞沉淀,重复离心2次;铺板培养液重悬肝细胞,利用台盼蓝拒染法检测细胞活力与活细胞数;细胞悬液以(1.5-2.5)×105活细胞/cm2的接种密度,接种到胶原包被的培养板或培养皿中,摇匀,在37℃、5%CO2培养箱中培养4-6h后,将铺板培养液换成肝细胞维持培养液。
所述灌注溶液I为D-Hank’s溶液中含有2mmol/l乙二醇双(2-氨基乙醚)四乙酸、5mM半胱氨酸的混合液,pH值为7.4,0.22μm滤膜过滤,4℃保存;所述灌注溶液II为 D-Hank’s溶液中含有5mmol/l氯化钙、0.1g/l六水氯化镁、0.3g/lⅣ型胶原酶、1.0g/l II 型分离酶、50mg/l核酸酶的混合液,pH值为7.4,0.22μm滤膜过滤,4℃保存;所述肝细胞洗液为William’s medium E培养基里加入2mmol/l谷氨酰胺、100units/mL青霉素、 100mg/mL链霉素,2~5%m/v牛血清白蛋白;所述肝细胞铺板培养液是在William’s medium E培养基里加入1%v/v的ITS+premix、2mmol/l谷氨酰胺、10μg/l表皮生长因子、18mg/l 氢化可的松、40μg/l地塞米松、100units/mL青霉素、100mg/mL链霉素和5%v/v FBS;所述肝细胞维持培养液,是在William’s medium E培养基里加入1%v/v的ITS+premix、 2mmol/l谷氨酰胺、10μg/l表皮生长因子、18mg/l氢化可的松、40μg/l地塞米松、100units/mL 青霉素、100mg/mL链霉素和2%v/v DMSO。
所述胶原包被的培养板或培养皿的制备方法为:将172μL冰醋酸加入到100mL去离子水中,再加入终浓度为50μg/mL的Ⅳ型鼠尾胶原,无菌条件下,用0.22μm滤膜过滤除菌后,得到胶原工作液,4℃保持待用;将该胶原工作液以5μg/cm2的包被量加入到需要包被的培养板或培养皿中,常温孵育1h后,将胶原工作液吸出,自然晾干后封口,4℃保藏。上述胶原包被的培养板或培养皿使用前,需用PBS清洗一次,以去除残留的冰醋酸。
步骤5:鉴定人原代肝细胞功能
将步骤3得到的大鼠血清利用ELISA检测人白蛋白、大鼠白蛋白的分泌,结果如图3所示;利用免疫荧光检测步骤4得到的细胞培养物中FAH蛋白阳性率,结果如图4所示。将步骤4得到的细胞培养物和步骤1得到的肝样细胞,利用RT-PCR鉴定人肝细胞标志基因表达,结果如图5、图6、图7所示。
具体的鉴定方法与结果分析:
1)商品化人白蛋白、大鼠白蛋白ELISA试剂盒购自美国Bethyl公司。ELISA检测培养上清中白蛋白的含量的具体方法为:分别设空白孔、标准孔、待测样品孔,空白孔加入新鲜培养液50μl,余孔分别加入标准品或待测样品,每个标准品或待测样品重复3孔,轻轻摇匀,酶标板覆膜后37℃反应45分钟;弃去液体,洗涤5次,拍干后每孔分别加入 HRP标记的抗白蛋白二抗100μl,37℃孵育1小时;弃去孔内液体,拍干,洗板5次,拍干;每孔加入显色液100μl,37℃避光显色15分钟;每孔加入终止液100μl,用酶标仪在450纳米波长下测量各孔OD值。最后根据标准孔确立的标准曲线计算出样品中白蛋白的含量。
结果分析:移植后,人白蛋白分泌持续增加,大鼠白蛋白持续下降,到移植后第8周,人白蛋白水平可达20mg/ml,显著高于大鼠白蛋白水平6mg/ml,说明在移植后第8周人肝细胞在大鼠肝脏中占主要。
2)RT-PCR鉴定人肝细胞标志基因表达:
RT-PCR鉴定人肝细胞标志基因表达标记基因:细胞用Trizol reagent裂解保存(用量为1ml每孔六孔板),保存于-80℃不超过1个月;加入0.2ml氯仿,剧烈震荡,静置2min后,4℃条件下12000×g离心15min;将上层水相转移至新的无RNase的EP管,加入0.5 ml异丙醇,颠倒混匀,静置10min后,4℃条件下12000×g离心10min;此时可见白色沉淀,弃上清,加入1ml无RNaes的75%乙醇(可保持于-20℃至少1年)剧烈震荡后,4℃条件下7500×g离心5min;弃上清,常温干燥5~10min;加入20μl无RNaes水,55-60℃干热10~15min,以使RNA溶解充分;将RNA放于冰上,用分光光度计测定RNA浓度;利用Takara的反转录试剂盒进行样品的反转录,合成cDNA;利用TAKARA的PCR Master Mix配制反应体系,短暂离心收集到管底;体系如下:
在PCR仪上进行RNA的半定量检测,程序如下:
人肝细胞标志基因的特异性检测引物包括:
结果分析:人间充质干细胞不表达人肝细胞标志基因,包括人白蛋白、人肝细胞核因子4α、人细胞色素P4503A4;经过诱导,人间充质干细胞分化成肝样细胞,较低水平表达人肝细胞标志基因;经过大鼠FAH基因缺陷模型成熟与扩增后,人-鼠嵌合肝细胞(即混合肝细胞)高水平表达人肝细胞标志基因。大鼠肝细胞的人肝细胞标志基因全都阴性,
3)免疫荧光检测人FAH蛋白的具体方法是:以12孔板的1孔为例,细胞培养至2 个月时,吸去旧培养液,用常温PBS洗1次,立即用冰冷的4%多聚甲醛1mL(具体配方为:称取4g多聚甲醛,置于三角烧瓶中,加入80mL去离子水,放入37℃恒温水浴箱,每隔1-2h摇晃混匀,16-24h后多聚甲醛会完全溶解,补充去离子水至100mL,调节pH值至7.2),常温固定10-15min;用PBS在水平摇床上洗3次,每次5min;为增强抗体与细胞内蛋白的结合,使用0.5mL含0.25%v/v Triton100的PBS溶液常温处理45min;用PBS 在水平摇床上洗3次,每次5min;封闭,加入适量封闭液(含有5%v/v山羊正常血清, 2%m/v BSA的PBS溶液),常温水平摇床孵育至少1h;利用含3%v/v山羊正常血清的 PBS溶液稀释抗体,抗FAH兔多抗(购自武汉三鹰公司)稀释比例为1:500,4℃摇床孵育过夜;再用含3%山羊正常血清的PBS溶液在水平摇床上洗3次,每次10min,以去除非特异结合的抗体;二抗为山羊抗兔lgG偶联DyLight-488(购自美国Thermo Fisher Scientific Inc公司),以1:1000稀释到含3%山羊正常血清的PBS溶液,于暗处避光常温孵育1h,此后操作一律避光;再用含3%山羊正常血清的PBS溶液在水平摇床上洗2次,每次10min,第三次清洗时加入1:1000的DAPI(购自瑞士Roche公司)用于细胞核染色, 常温孵育10min;再清洗一次以清洗掉残留的DAPI,10-20min;加入适量含3%山羊正常血清的PBS溶液防止在观察时细胞因干燥变形。最后,利用Leica DFC425C荧光显微镜(购自德国Leica公司)观察荧光信号。
以上仅为本发明的优选实施例,并非因此限制本发明的专利范围,凡是利用本发明说明书内容所作的等效结构或等效流程变换,或直接或间接运用在其他相关的技术领域,均同理包括在本发明的专利保护范围内。
Claims (11)
1.一种肝样细胞成熟与扩增的方法,其特征在于,具体步骤包括:
步骤S1:干细胞在体外使用相应的诱导液进行培养诱导干细胞分化为肝样细胞;
步骤S2:将肝样细胞通过脾脏定点注射到FAH基因缺陷大鼠肝脏载体;
步骤S3:刺激FAH基因缺陷大鼠肝脏载体自体肝细胞凋亡,促进肝样细胞成熟、增殖;
步骤S4:分离与纯化得到人原代肝细胞。
2.根据权利要求1所述肝样细胞成熟与扩增的方法,其特征在于:所述干细胞包括骨髓来源的间充质干细胞、胚胎干细胞或诱导多能干细胞。
3.根据权利要求2所述肝样细胞成熟与扩增的方法,其特征在于,所述骨髓来源的间充质干细胞体外诱导间充质干细胞分化为肝样细胞具体步骤为:在细胞融合度为85%时,加入肝细胞诱导液,每3天换1次液,诱导培养2周后,向肝细胞诱导液加入30ng/ml的humanOncostatin M,继续诱导1周后经免疫荧光鉴定白蛋白表达;
肝细胞诱导液包括:IMDM培养液、青霉素、链霉素、Hepes Buffer Solution、ITS+1Liquid Media Supplement、dexamethasone和HGF。
4.根据权利要求2所述肝样细胞成熟与扩增的方法,其特征在于,所述胚胎干细胞体外诱导间充质干细胞分化为肝样细胞具体步骤为:胚胎干细胞培养体系中加RPMI基础培养基补充1mM L-谷氨酰胺、0.5%v/v胎牛血清、100ng/ml激活素-A、25ng/ml Wnt3a,诱导培养2天;RPMI基础培养基补充1mM L-谷氨酰胺、0.5%v/v胎牛血清、100ng/ml激活素-A、继续诱导培养2天;接着培养基换成HCM基础培养基,添加20ng/ml重组人骨形态发生蛋白、30ng/ml成纤维细胞生长因子-4,继续诱导培养6天;HCM基础培养基补加20ng/ml肝细胞生长因子,诱导培养5天;HCM基础培养基补加10μg/ml human Oncostatin M,100nM地塞米松,诱导培养15天。
5.根据权利要求2所述肝样细胞成熟与扩增的方法,其特征在于,所述诱导多能干细胞体外诱导分化为肝样细胞具体步骤为:CDM-PVA培养基(250mL DMEM-F12,250mL IMDM,0.5g聚乙烯醇,5ml浓缩脂类,20μL硫代甘油,25μg/mL转铁蛋白,10μg/L重组人胰岛素,100units/mL青霉素,100mg/mL链霉素),补加10ng/mL激活素-A和12ng/mL碱性成纤维细胞生长因子,培养2天;CDM-PVA培养基,补加100ng/mL激活素-A,100ng/mL碱性成纤维细胞生长因子,10ng/mL重组人骨形态发生蛋白4,10μM LY294002,3μM CHIR99021,培养24小时;CDM-PVA培养基,补加100ng/mL激活素-A,100ng/mL碱性成纤维细胞生长因子,10ng/mL重组人骨形态发生蛋白4,10μM LY294002,培养24小时;RPMI-B27培养基(490mL RPMI 1640,补加10mL B27,5mL NEAA,100units/mL青霉素,100mg/mL链霉素),补加100ng/mL激活素-A,100ng/mL碱性成纤维细胞生长因子,诱导培养24小时;RPMI-B27培养基,补加50ng/mL激活素-A,诱导培养3天;RPMI-B27培养基,补加20ng/mL重组人骨形态发生蛋白4,10ng/mL成纤维细胞生长因子-10,诱导培养3天;Hepatocyte Basal Medium,补加50ng/ml HGF,30ng/mlhuman Oncostatin M,诱导培养3天。
6.根据权利要求1所述肝样细胞成熟与扩增的方法,其特征在于,所述FAH基因缺陷大鼠肝脏载体通过受精卵显微注射下使用CRISP Cas9基因敲除方法获得。
7.根据权利要求6所述肝样细胞成熟与扩增的方法,其特征在于,所述FAH基因缺陷大鼠肝脏载体的获得方法具体是通过将诱导分化完成的肝样细胞通过胰酶消化、离心、0.9%生理盐水重悬,制成细胞密度为1×107活细胞/500μl的肝样细胞悬液,从脾尖利用1ml注射器,将肝样细胞悬液缓慢注射进入脾脏,肝细胞经过血液流动慢慢进入肝脏载体。
8.根据权利要求1所述肝样细胞成熟与扩增的方法,其特征在于,所述刺激FAH基因缺陷大鼠肝脏载体自体肝细胞凋亡,促进肝样细胞成熟、增殖是去掉FAH基因缺陷大鼠肝脏载体维持药物NTBC,加入免疫抑制剂混合物和抗感染药物进行培养,并且每隔7天收集一次样本置于-80℃保存待检测。
9.根据权利要求1所述肝样细胞成熟与扩增的方法,其特征在于,所述步骤4具体是:利用4℃预冷的灌注溶液I原位灌注大鼠肝脏约20min,直至肝组织中的血液被冲洗干净;利用37℃预热的灌注溶液II灌注30min,直至肝组织失去弹性;将肝组织转移到盛有足量肝细胞洗液的培养皿中,用无菌镊子扯破肝组织,得到肝细胞悬液;肝细胞悬液经过70μm细胞筛,以去除组织团块;细胞悬液转移至50ml离心管中,50×g,4℃,离心5min;去掉上清,肝细胞洗液重悬细胞沉淀,重复离心2次;铺板培养液重悬肝细胞,利用台盼蓝拒染法检测细胞活力与活细胞数;细胞悬液以(1.5-2.5)×105活细胞/cm2的接种密度,接种到胶原包被的培养板或培养皿中,摇匀,在37℃、5%CO2培养箱中培养4-6h后,将铺板培养液换成肝细胞维持培养液。
10.根据权利要求9所述肝样细胞成熟与扩增的方法,其特征在于,所述灌注溶液I为D-Hank’s溶液中含有乙二醇双(2-氨基乙醚)四乙酸、半胱氨酸的混合液,pH值为7.4,0.22μm滤膜过滤,4℃保存;灌注溶液II为D-Hank’s溶液中含有氯化钙、六水氯化镁、Ⅳ型胶原酶、II型分离酶、核酸酶的混合液,pH值为7.4,0.22μm滤膜过滤,4℃保存;所述肝细胞洗液为William’s medium E培养基里加入谷氨酰胺、青霉素、链霉素,牛血清白蛋白;
所述肝细胞铺板培养液是在William’s medium E培养基里加入1%v/v的ITS+premix、谷氨酰胺、表皮生长因子、氢化可的松、地塞米松、青霉素、链霉素和5%v/v FBS;
所述肝细胞维持培养液,是在William’s medium E培养基里加入1%v/v的ITS+premix、谷氨酰胺、表皮生长因子、氢化可的松、地塞米松、青霉素、链霉素和2%v/v DMSO。
11.根据权利要求1所述肝样细胞成熟与扩增的方法,其特征在于,还需要还鉴定人原代肝细胞功能,具体的步骤:
将刺激FAH基因缺陷大鼠肝脏载体自体肝细胞凋亡得到的血清样本用ELISA检测人白蛋白、大鼠白蛋白的分泌;
利用免疫荧光检测步骤4得到的细胞培养物中FAH蛋白阳性率;
将步骤4得到的细胞培养物和步骤1得到的肝样细胞,利用RT-PCR鉴定人肝细胞标志基因表达。
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