间充质干细胞定向分化及其在肝脏疾病应用中的启示

王汉裕 刘拥军

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间充质干细胞定向分化及其在肝脏疾病应用中的启示

王汉裕 刘拥军★

间充质干细胞向肝细胞诱导分化成功后，分化后的肝细胞样细胞应用于肝病成为了研究热点。不仅未分化的间充质干细胞能治疗肝病，其来源的分化后的肝细胞样细胞亦能有效治疗肝病。因此，有必要对间充质干细胞分化前后的细胞治疗效果进行比较评价。本文从诱导分化培养方案、鉴定指标及相关细胞生物学功能进行述评。

间充质干细胞；定向分化；细胞治疗；肝病

干细胞研究的发展促进了应用干细胞治疗肝病的研究，丰富了肝病的治疗手段，尤其是肝硬化及肝功能衰竭的治疗。基于干细胞的分化潜能，利用干细胞，尤其是间充质干细胞（mesenchymal stem cells，MSCs），向肝细胞进行诱导分化培养，可获得肝细胞样细胞（hepatocyte-like cells，HLCs），并应用于肝病的治疗。不少研究开始比较MSCs向肝细胞分化前后用于肝病治疗。我们发现MSCs经过向肝细胞分化诱导培养后，丢失了高分泌基质金属蛋白酶（matrix metalloproteinases，MMPs）和肝细胞生长因子（hepatocyte growth factor，HGF）的能力。我们及其他实验均发现分化后的HLCs治疗肝病的效果不如未分化的MSCs。本文结合本实验的研究，综合目前相关研究文献，对MSCs向肝细胞定向分化及其在肝病中的应用进行简要述评。

1 分化诱导培养方案的选择依据

2001年小鼠胚胎干细胞（embryonic stem cells，ESCs）[1]和2002年多潜能成体祖细胞（multipotent adult progenitor cells，MAPCs）[2]在体外向HLCs诱导分化培养获得成功后，2004年MSCs在体外向HLCs分化诱导培养也获得成功[3]。小鼠ESCs向HLCs的诱导分化方案中，ESCs在不含白血病抑制因子（leukemia inhibitory factor，LIF）的培养基中培养5天，继而接种于I型胶原包被的培养皿中培养4天（基础培养基为含20% 胎牛血清和300μM硫代甘油的IMDM，依次添加酸性成纤维细胞生长因子（acidic fibroblast growth factor，aFGF，100 ng/mL）、HGF（20 ng/mL）；在诱导培养的第15天，添加抑瘤素M（oncostatin M，OSM，10 ng/mL）、地塞米松（dexamethasone，DXM，10-7M）、ITS混合物（5 mg/mL胰岛素、5 mg/mL转铁蛋白、5μm/mL亚硒酸）培养3天。经过18天的诱导分化培养，ESCs成功分化为具有部分肝细胞功能的HLCs。基于ESCs和MAPCs的诱导方案，改进的MSCs诱导方案采用了两步法，首先用含肝HGF（20 ng/mL）、碱性成纤维细胞生长因子（basic fi broblast growth factor，bFGF，10 ng/mL）和尼克酰胺（0.61 g/L）的IMDM培养基培养1周；更换为诱导成熟培养基，即含OSM（20 ng/mL）、地塞米松（1×10-6mol/L）和 ITS 混合物（25 g/L胰岛素、25 g/L转铁蛋白、25 mg/L亚硒酸钠）的IMDM，继续培养3周。经过4周的诱导分化培养，细胞形态从梭型转变为典型的肝细胞样椭圆形，并能检测到细胞色素P450（cytochrome P450，CYP）的2B6（CYP 2B6）亚型。随后文献中出现的培养分化方案都在此方案上加于变化，比如预先在I型[4]或IV型胶原[5]包被的培养皿上培养2天，再继续往下分化培养；把MSCs接种在Matrigel包被的纳米聚酰胺纤维上进行分化培养[6]。

在干细胞向HLCs分化培养过程中，除了HGF外，目前的分化培养方案中均需要添加bFGF[1～5]。我们简化了上述的分化方案，MSCs向HLCs分化诱导培养依然获得成功。我们的诱导方案也是采取两步法，在第一阶段采用10-8mol/L的地塞米松、50 ng/mL的HGF、10 ng/mL的表皮生长因子（epidermal growth factor，EGF）、1% ITS的DMEM-F12（高糖）培养基诱导培养2周；第二阶段，使用含2%的FBS、20 ng/mL的OSM、10-6mol/L的DXM、1% ITS的DMEM-F12培养基诱导培养4周。考虑到第二阶段的培养时间延长，添加低浓度的FBS以维持细胞的活性。经过6周的诱导培养后，细胞逐渐变成圆形（图1），并具有成熟肝细胞的功能，比如合成糖元（图2）、分泌尿素（图3）等。这说明MSCs向HLCs分化的过程中，bFGF不是必需的。bFGF是胚胎干细胞培养基中维持ESCs未分化状态的一个重要组成因子[7]。最近也有研究发现bFGF联合骨形态发生蛋白4（bone morphogenetic protein-4，BMP-4）促进了ESCs向成骨细胞和成软骨细胞分化[8]。

图1 诱导分化前后的细胞形态Figure 1 Cells morphology changes after hepatic differentiation

图2 糖原染色（PAS法）Figure 2 Glycogen detection by PAS staining

图3 随着诱导分化培养时间的延长，尿素分泌逐渐增多Figure 3 The differentiated cells synthesized urea in a timedependent manner

2 分化诱导的鉴定标记及其意义

不管大鼠[9]、小鼠[10]，还是人类[3]，各种来源的（骨髓[11]、脐带、脐带血[12]或脂肪[11,13,14]）MSCs均能分化为HLCs，为将来肝病的细胞治疗解决了细胞来源的技术难题。但是，目前没有提出一个特异的指标来明确干细胞向HLCs分化成功与否，也没有明确的鉴定指标来评价分化效率。因此，在相关文献中，均采用多指标评价系统。常用的鉴定标记有白蛋白（albumin）、甲胎蛋白（α-fetoprotein，AFP）、细胞角蛋白（cytokeratine，CK）、肝细胞核因子（hepatocyte nuclear factor，HNF）、细胞色素（cytochrome，CYP）P450、酪氨酸转氨酶（tyrosine-aminotransferase）、色氨酸2，3双加氧酶（tryptophan 2，3-dioxygenase）等，体现具有肝细胞功能的鉴定方法有过碘酸-希夫（Periodic Acid-Schiff，PAS）染色、Dil-Ac-LDL吸收、尿素合成等。另外，部分上述指标还存在亚型，比如细胞角蛋白包括CK7[6]、CK18[5]、CK19[2]，HNF包括HNF-1α[2]、HNF-3β[2]、HNF-4α[5]。然而，尚未有某一篇文献全部采用上述指标。有研究发现诱导分化早期出现的指标有HNF-3β、CK19、AFP，而CK18、albumin、HNF-1α、CYP则表达于诱导分化晚期[2]。经过4周的诱导，HLCs表达 CYP 2B6，而肝细胞核因子4（HNF-4）在诱导6周后才表达[3]。

不少文献提到MSC在诱导分化之前没有表达ALB、AFP、CK18[5,15,16]、CK19[15]。亦有文献报道，经过诱导分化培养后的细胞只有70%表达白蛋白[5]。BM-MSCs低表达白蛋白、CK18、色氨酸2，3双加氧酶，但不表达 AFP[3]。但是，我们发现未经过诱导分化的UC-MSCs本身高表达白蛋白、CK19和AFP（图4）。这些数据证明了白蛋白、色氨酸2，3双加氧酶、CK18、CK19和AFP不能作为骨髓和脐带来源的MSCs在向肝细胞诱导分化的鉴定指标。分泌尿素是肝细胞活性的一个特性，但是肾小管上皮细胞也分泌尿素。CYP虽然首先在肝细胞中发现，但也在其他细胞中表达[18]。即使是肝细胞相对特异性指标的CYP，其不同亚型的活性亦存在种属特异性，如大鼠的CYP 2B1[19]、小鼠的CYP 2B9和CYP 2B13[20,21]、人的CYP 2B6[2]。尽管肝细胞具有LDL吸收的功能，但其他细胞也有这样的特性[22]。综合目前的文献报道，只有肝细胞能合成和存储糖原[2]。在包被了Matrigel上培养的MAPCs分化为HLCs（同时表达白蛋白、CK18、HNF-3β）的分化率能达到 91%[2]。经过6周的诱导，大约 50%的分化细胞具有存储糖原的功能[3]。

3 诱导前后的细胞生物学相关研究

自从建立了干细胞向HLCs分化诱导的成熟方案，不少研究者随即对这些HLCs生物学功能和特性进行深入的研究。由于ESCs具有高度的致瘤性，在体外进行诱导分化时，很容易混杂未能充分分化的ESCs[23,24]，这就给临床使用带来高风险，限制其临床应用。和ESCs相比，成体干细胞不具有明显潜在的成瘤性，没有伦理问题的困扰，非常适合于临床的应用[25]。

图4 免疫荧光方法检测间充质干细胞表达的白蛋白、细胞角蛋白19、甲胎蛋白Figure 4 Expressions of albumin, CK19 and AFP from MSCs by immuno fl uorescence microscopy

MSCs向HLCs诱导分化的培养方案已经得到很好的建立[3]。分化后的HLCs具有向损伤肝脏部位迁移的能力，而且能在体内分泌白蛋白[26～28]。人脂肪来源的MSCs诱导分化为HLCs后，能减轻四氯化碳对裸鼠造成的急性肝损伤程度[29]。这些结果说明来源于干细胞的HLCs合适于治疗肝病，尤其是急性肝衰竭和终末期肝病[30]。分化后的HLCs和未经分化诱导的MSC均有报道其对肝病的治疗作用，例如肝纤维化[31～33]、肝损伤[34,35]、爆发性肝衰竭[27,29,36]和肝再生[26,37,38]。由于MSCs向肝细胞分化前后，对肝病的治疗均有效果，因此，需要对分化前后的细胞进行功能相关比较研究。

动物实验证明，MSCs不仅能迁移到损伤的部位，修复损伤的组织，并使损伤的组织恢复一定的功能[27,39,40]；而且能减少细胞的死亡，并促进内源性再生[41,42]。另外，MSCs也易于体外培养扩增。这些特性使得MSCs成为临床细胞治疗的最佳来源。MSCs修复组织的可能机制在于分泌可溶性细胞因子，这些细胞因子能抑制炎症和免疫反应，也能刺激内源性干细胞的增殖和分化，从而起到组织修复作用[43,44]。体内实验证明未分化的MSCs比分化后的HLCs治疗急性肝衰竭的效果更好[27]，我们的数据也证明了这点。由于未分化的MSCs亦表达高水平的白蛋白，这有益于肝病的治疗。这也说明前期动物实验发现HLCs在肝脏分泌白蛋白的数据是值得商榷的。需要注意的是，发现MSCs输入治疗后有向肝细胞分化的实验研究中，均采用的是免疫缺陷小鼠[15,26]，而MSCs在免疫系统正常小鼠体内存留时间大概为23天[45]。在不同的动物模型中，MSCs能改善肝脏功能，而没有出现细胞融合[27,46]。

HGF是促进肝细胞增殖[47,48]和肝再生[47,49]的最强效细胞因子。HGF刺激肝干/祖细胞的增殖来修复肝损伤[50]。HGF可引起Bcl-xL的强烈表达，阻断Fas及其配体激活后的信号传导，抑制Fas介导的肝细胞凋亡[51]，从而治疗Fas引起的爆发性肝衰竭[52]。HGF除了能诱导肝实质细胞的迅速增殖外，还能刺激胆管上皮细胞增殖，因而影响整个器官的发育。由于HGF有促进肝细胞增殖和抑制凋亡的特性，使得HGF可能是肝病细胞治疗的一个关键因子。我们和其他的研究小组都发现MSCs分泌高水平的HGF[53]。而且，脐带来源的MSCs分泌HGF的量比骨髓来源的MSCs高30倍[53]。 除了HGF，MSCs也分泌高水平的MMPs，这些分泌的MMPs有利于组织修复和肝纤维化的治疗。MSCs分化为HLCs后，其分泌的HGF（图5）和MMPs（表1）都显著降低。也有数据证明MSCs向HLCs分化培养结束后，并不是处于分化终末期的稳定状态，这增加了成瘤性的风险[54]。

图5 细胞分化前后肝细胞生长因子的表达水平变化Figure 5 The change of hepatocyte growth factor expression after hepatic differentiation

表1 细胞分化前后基质金属蛋白酶的分泌水平变化Table 1 Matrix metalloproteinase changes after hepatic differentiation

综上所述，由于没有明确的鉴定标准，也难于评价分化效率。MSCs分化为HLCs后，丢失了分泌HGF和MMPs的能力，而HGF和MMPs均有利于相关肝病的治疗。这些数据说明直接用未分化的MSCs治疗肝病效果好于经过诱导分化培养后的细胞，而且还节约培养成本。

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Mesenchymal stem cells committed differentiation and its application in liver diseases therapy

WANG Hanyu, LIU Yongjun★
(Alliancells Institute of Stem Cells and Translational Regenerative Medicine, Tianjin 300300, China)

The protocols for differentiation of hepatocyte-like cells (HLCs) from mesenchymal stem cells (MSCs) have been well established. Previous datas have shown that MSCs and their derived-HLCs were able to engraft injured liver and alleviate injuries. It is necessary to elvaluate the cells therapeutic effects before and after MSCs differentiation. We will make a brief comment on the induction and differentiation culture protocol, hepatic markers and related functions of cell biology.

Mesenchymal stem cells; Committed differentiation; Cytotherapy; Liver diseases

国家自然科学基金（30872618）

和泽干细胞转化再生医学研究中心，天津 300300

★通讯作者：刘拥军，E-mail: andyliuliu2001@yahoo.com.cn