修回日期: 2015-05-26
接受日期: 2015-08-04
在线出版日期: 2015-11-18
全能干细胞主要包括胚胎干细胞(embryonic stem cell, ESC)和自体诱导多能干细胞(induced pluripotent stem cell, iPSC)两类, 其在肝脏方向的分化及移植应用, 可为肝细胞替代治疗提供新的细胞来源, 有望成为终末期肝病的有效辅助疗法. 近年干细胞技术在肝脏领域研究进展迅速, 如iPSC可在保留全能分化性的同时大大减轻ESC应用面临的伦理及免疫排斥问题, 同时干细胞分化水平也逐渐从肝系细胞的单细胞状态提升到肝脏组织的高度. 这些进展一方面推动了肝功能替代治疗技术进步; 另一方面也促进了肝脏器官体外发育基础理论的丰富和更新.
核心提示: 近些年干细胞技术在肝脏领域研究进展迅速, 全能干细胞可为肝细胞替代治疗提供新的细胞来源, 有望成为终末期肝病的有效辅助疗法, 同时干细胞分化水平也逐渐从肝系细胞的单细胞状态提升到肝脏组织的高度, 从而促进了肝功能替代治疗技术的进步.
引文著录: 吉斐, 胡安斌. 全能干细胞在肝脏方向分化研究的进展. 世界华人消化杂志 2015; 23(32): 5101-5106
Revised: May 26, 2015
Accepted: August 4, 2015
Published online: November 18, 2015
Pluripotent stem cell mainly contain two types: embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC). Their hepatic differentiation and application in transplantation may make them serve as new seed cells for replacement therapy and become an effective adjunctive therapy for end-stage hepatic diseases. Recently, great progress has been made in the research of stem cell technology. For example, iPSCs can maintain pluripotency, and the application of iPSCs can avoid the ethical issues associated with the use of ESCs. The research of differentiation of stem cells has greatly shifted from differentiation into hepatic single-cell lineage to differentiation into liver tissues. All of these can improve the development of replacement therapy, and update the basic knowledge of ectogenesis of the liver.
- Citation: Ji F, Hu AB. Hepatic differentiation of pluripotent stem cells. Shijie Huaren Xiaohua Zazhi 2015; 23(32): 5101-5106
- URL: https://www.wjgnet.com/1009-3079/full/v23/i32/5101.htm
- DOI: https://dx.doi.org/10.11569/wcjd.v23.i32.5101
病毒性肝炎和药物等原因导致的肝功能衰竭等终末期肝病, 肝脏移植是其唯一有效疗法[1-4]. 目前全球约有5亿肝病患者需肝功能替代治疗, 大部分患者由于供肝严重不足等原因而无法施行肝移植[5-9], 急需肝细胞移植和生物人工肝等肝功能过渡替代治疗[10-12]. 因此, 肝细胞和肝脏组织等肝功能替代物也急需新的来源. 胚胎干细胞(embryonic stem cell, ESC)是来源于囊胚内细胞团的全能干细胞, 具备全能分化性和无限增殖能力[13-15], 很早即体现了向肝细胞分化的能力, 但分化移植应用面临的伦理及排斥问题仍未解决. 自体诱导多能干细胞(induced pluripotent stem cell, iPSC)是将自体成体细胞导入重编程基因, 使细胞重编程并去分化达到ESC分化水平的干细胞技术[16], 可在具备全能分化性和向肝细胞分化的同时, 大大减轻ESC应用所面临的伦理和排斥问题[17,18]. 同时, 就分化技术和水平而言, ESC和iPSC在肝脏方向的分化, 也从单细胞状态逐渐向肝脏组织水平进展提高, 从而体现了干细胞本身和其分化技术水平的进步, 本文就这方面的进展做一分析和评价.
ESC最初从小鼠囊胚中的内细胞团分离建系, 具有分化为所有组织类型和细胞的潜能. 肝细胞属内胚层消化上皮细胞, 其分化需内皮细胞、中胚层细胞和间质细胞的诱导. 这种协同诱导方式可在ESC体外首先发育形成的拟胚体(embryoid body, EB)阶段实现, EB是ESC体外重演胚胎发育部分过程的产物, 类似体内囊胚阶段并包含三胚层细胞. 早期, Hamazaki等[19]将ESC体外培养5 d形成的EB贴壁培养于培养板中, 再模拟体内肝脏发育所需的生长因子, 依次加入成纤维细胞生长因子(fibroblast growth factor, FGF)、肝细胞生长因子(hepatocyte growth factor, HGF)和制瘤素(oncostatin, OSM)等并分别作为早、中、晚期诱导剂, 连续诱导9 d后分析结果, 发现实验组检测到6-磷酸葡萄糖(glucose-6-phosphate, G6P)和酪氨酸氨基转移酶(tyrosineaminotransferase, TAT)两种成熟肝细胞酶的基因表达, 白蛋白(albumin, ALB)的表达量也提高9.5倍, 进一步检测表明培养系统中分化出现了成熟肝细胞. 在此阶段, 其他研究者包括本课题组也通过EB阶段定向分化获得肝细胞和胆管细胞等, 在我们的肝细胞分离和再培养系统中, 我们将肝细胞和胆管细胞的分化效率提高到72.6%[20]和7.4%[21], 为细胞修复和肝脏组织工程学提供了丰富的细胞来源.
后来, Ishii等[22]研究了EB阶段对ESC向肝细胞分化的影响, 发现联合激活素A(activin A)诱导分化时, 不经过EB阶段的ESC分化效果更好, 还发现Activin A和Wnt3a一起可以加快限定性内胚层的形成并能诱导更多类肝细胞的产生[23,24]. FGF和骨形态发生蛋白(bone morphogenetic protein, BMPs)在肝诱导过程中有着重要意义, 两者联合使用可有效的促进ALB的表达[25,26], 同时可以观察到肝性标志物上皮细胞黏附分子(epithelial cell adhesion molecule, EpCAM)、细胞角蛋白(cytokeratin, CK)7、CK19表达阳性的细胞成簇出现[27].
2007-11, 日本和美国科学家[28,29]分别宣布发现利用反转录病毒将Oct4、Sox2、Klf4和c-myc基因导入人体皮肤细胞, 转化得到与ESC功能相同的全能干细胞, 称为iPSC. iPSC具有和ESC类似的功能, 细胞处于原始状态, 分化能力强且具有无限增殖能力, 并避开了ESC研究中面临的伦理和法律问题[30]. 同时, 因为来源于自体细胞, 移植应用亦可避免免疫排斥问题. 由于以上优点, iPSC出现后迅速成为干细胞分化及移植研究中的新兴力量[30].
同ESC类似, iPSC可在某些化学物质或者诱导因子作用下向肝细胞分化. 诱导过程一般分三个步骤[31-35], 首先Activin A、Wnt3a或Foxa2可将iPSC转化为限定性内胚层细胞, 并促进其转化为肝前体细胞, 随后, 肝细胞特异生长因子FGFs、BMPs以及HGF可使其向肝系细胞分化, OSM和地塞米松(dexamethasone, DXM)使其分化为成熟的类肝细胞. 利用RT-PCR或者免疫荧光方法可检测到诱导后的类肝细胞表达肝性标志物ALB、甲胎蛋白(α-fetoprotein, AFP)、CK8/18/19、肝细胞功能蛋白细胞色素P450 1A2(cytochrome P450 1A2, CYP1A2)以及转甲状腺素蛋白(transthyretin, TTR)并且具有尿素合成和糖原储备能力[18,36]. Ogawa等[37]研究发现在培养基中加入环磷酸腺苷(cyclic adenosine monophosphate, cAMP)能够有效促进iPSC向类肝细胞分化和成熟, Ratziu等[38]将ESC和iPSC与小鼠胚胎成纤维细胞(Swiss 3T3细胞)进行3D共培养, 发现其分泌的Ⅰ型胶原能够显著促进类肝细胞的成熟. 细胞呈多边或立方形肝细胞形态, 出现双核或多核细胞核, 胞浆丰富呈索状排列, 电镜下还可看到丰富的线粒体、过氧化物酶体、脂质体、完整的高尔基体以及微绒毛和囊泡, 肝细胞侧面还有与毛细胆管分布相一致的连接复合体. 最后, 将分化的肝细胞移植体内, 发现肝细胞可以整合到受体肝组织内并进一步发挥成熟肝细胞功能[39].
目前, ESC向肝系细胞的诱导分化已经取得很大进展, 但分化细胞多呈单细胞状态而不能形成肝组织, 国外Ogawa等[37]曾报道ESC分化为肝细胞组织化现象, Tsutsui等[40]检测到小鼠ESC分化为肝组织, 发现分化的肝组织不仅能够表达Cyp2b5、Cyp2b10和Cyp3a11等基因, 还能表达肝特异性的Cyp7a1, 从而证明了肝组织的存在. 另外, 我们研究[41]结果显示ESC分化形成的肝脏组织除了肝细胞和胆管上皮细胞出现外, 还有内皮细胞和血管生成标志物血管内皮生长因子受体1(vascular endothelial growth factor receptor 1, VEGFR-1)、CD31/血小板内皮细胞黏附分子1(platelet endothelial cell adhesion molecule 1, PECAM-1)的表达, 并出现了许多脉管网状结构[42], 其间分布着ALB阳性的类肝系细胞和CK19阳性的胆管上皮细胞. 从功能学和形态学上均提示, 分化系统中形成了包含肝细胞、胆管上皮细胞、内皮细胞和血管网在内的类肝脏组织结构. 我们进一步研究[42]表明上皮型钙黏蛋白(E-cadherin, E-cad)通过促进细胞黏附和降低细胞分化增殖中的上皮间质转化(epithelial-mesenchymal transition, EMT)水平, 而使ESC在向肝方向分化时保持三维立体结构, 从而有利于肝脏组织的形成.
在早期肝脏分化过程中, 来自前肠内胚层的肝特异性细胞分化形成肝芽需要内胚层上皮细胞、间质细胞以及内皮细胞的相互作用, 因此, 采用共培养的方式能模拟发育进程中的细胞的相互作用以及影响. 研究[42]显示ESC或分化的ESC与肝脏非实质细胞、胎肝细胞共培养, 对于ESC向肝细胞分化具有重要意义. 但目前共培养如何影响分化进程和细胞表型, 仍然不很清楚. Hui等[43]与Takebe等[44,45]将人iPSC与间质细胞、脐静脉内皮细胞、间充质干细胞共培养, 48 h后分化成立体结构的细胞簇, PCR检测示肝特异性基因表达, 并检测到间质细胞相关的FGF和BMP信号通路表达上调, 表明间质细胞相关的旁分泌对肝芽的形成非常重要. 进一步将体外培养形成的肝芽植入裸鼠体内, 移植后48 h即可发现肝芽新生血管与受体血管相连并有血流灌注, 新生血管在形态和密度上跟成熟肝脏血管相似, 功能性血管形成后进一步刺激肝芽的成熟从而形成肝脏组织. 显微镜下可看到肝索状结构, 并表达细胞间紧密连接蛋白1(zona occludens 1, ZO1)、ALB、CK8/18、ASGR1(asialoglycoprotein receptor 1)以及Ⅳ型胶原. 将该类肝组织移植后, 血中ALB浓度较前期报道的ALB浓度高5-10倍, 细胞色素P450功能活性增强, 并显著改善了小鼠肝损伤模型的预后.
各种原因所致的肝功能不全与肝功能衰竭严重威胁着人类健康, 肝细胞移植及生物人工肝在肝病治疗中取得了一定研究进展. 目前以干细胞为基础的肝病治疗主要包括肝脏基因表达或蛋白分泌异常的疾病, 包括遗传疾病、代谢缺陷性疾病、凝血障碍疾病. 有学者[46]将ESC经过诱导分化, 再与肝非实质细胞进一步共培养, 经过ALB启动子驱动荧光蛋白表达并进行富集, 将种入该细胞的肝支持设备整体移植入受损的肝脏组织中, 明显改善了肝衰竭小鼠的存活率. 另有报道[47]将能够在AFP启动子作用下表达Hyg/EGFP的转基因ESC经过诱导, 分化成表达AFP的内胚叶细胞并移植入肝衰竭小鼠, 可以明显改善动物的短期存活率.
另外, iPSC来源的肝细胞在肝癌细胞株或肝细胞进行药物的筛选研究中也具有重要的作用, 尤其是来源于具有不同细胞色素P450多肽个体的iPSC来源的肝细胞在预测新药物的肝毒性方面具有重要价值[48], 并且疾病特异性iPSC来源的肝细胞可以用来进行新药物的开发和研究[49,50].
ESC和iPSC的无限增殖能力、多向分化潜能以及外源基因高效转染系统的建立为其诱导分化产生肝细胞的研究奠定了坚实的基础, 长远来看, 对肝功能衰竭疾病采用ESC或iPSC分化形成人工肝脏器官以用于器官移植治疗也将成为可能. 另外, 对遗传性肝脏疾病采用干细胞基因介入治疗, 也是最新发展并具有广泛应用前景的肝脏病治疗策略. 因此, 全能干细胞向肝脏方向分化研究已经取得很大进展, 相信也很快将会为肝细胞移植、肝组织工程以及生物人工肝脏器官等治疗手段提供有效的细胞来源. 但是, 该领域研究也存在很多问题, 如不同的诱导体系分化得到肝脏在基因、蛋白表达、成熟程度和功能等方面存在较大的差异, 经过转入外源基因后的iPSC和经过体外培养后, 再输入体内亦会产生排斥反应, 并仍有致畸的可能, 这些都是下一步研究亟需解决的理论和技术难题.
全能干细胞在肝脏方向的分化研究具有广阔的临床应用前景, 但距临床应用还有一定距离, 随着研究水平的提高和深入, 其在肝脏疾病的研究和治疗方面的作用将突显, 尤其对于我国病毒性肝炎高发的状况具有更长远的意义.
肝脏移植是终末期肝病的唯一有效疗法, 但由于供肝的不足, 大部分病患需替代治疗, 肝细胞和肝脏组织等肝功能替代物也急需新的来源, 全能干细胞由于其全能分化性和无限增殖能力的特性而备受关注.
徐迅迪, 教授, 中南大学湘雅二医院肝胆胰外科
全能干细胞向肝脏的分化研究显示分化形成的肝脏组织除了肝细胞和胆管上皮细胞出现外, 还有内皮细胞和血管生成标志物血管内皮生长因子受体1、CD31/血小板内皮细胞黏附分子1的表达, 并出现了许多脉管网状结构, 其间分布着白蛋白(albumin)阳性的类肝系细胞和CK19阳性的胆管上皮细胞.
Hu等报道上皮型钙黏蛋白(E-cad)通过促进细胞黏附和降低细胞分化增殖中的上皮间质转化(epithelial-mesenchymal transition, EMT)水平, 而使胚胎干细胞(embryonic stem cell)在向肝方向分化时保持三维立体结构, 从而有利于肝脏组织的形成.
全能干细胞在肝脏的分化从单细胞状态逐渐向肝脏组织水平进展提高, 体现了干细胞本身和其分化技术水平的进步, 为终末期肝病的治疗提供新思路.
对遗传性肝脏疾病采用干细胞进行基因介入治疗是最新发展起来并具有广泛应用前景的肝脏病治疗策略, 对终末期肝病的治疗采用干细胞分化形成的人工肝脏器官进行器官移植, 这些对于病毒性肝炎高发的中国具有长远意义.
上皮间质转化(EMT): 是指上皮细胞在特定的生理和病理情况下向间质细胞转化的现象.
随着对干细胞包括全能干细胞的认识深入, 对再生医学的发展起到了极大的推动作用. 肝脏作为人体重要的实质脏器, 干细胞在其生理、病理过程中有着广泛而复杂的功能. 本文对全能干细胞在肝脏的分化方面的研究进展以及自己的研究结果进行了简要评述与总结, 提出干细胞的临床应用前景与需要解决的关键问题, 有参考价值, 使用文献较新.
编辑: 郭鹏 电编: 闫晋利
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