Adipose-derived mesenchymal stem cells from hepatitis B virus infection patients differentiate into hepatocyte-like cells in vitro

doi:10.3969/j.issn.2095-4344.2017.25.008

Abstract

Abstract:

BACKGROUND: It has been reported that human adipose-derived mesenchymal stem cells can be induced to differentiate into hepatocyte-like cells with the function of albumin synthesis and urea secretion in vitro.
OBJECTIVE: To investigate the potential of adipose-derived mesenchymal stem cells differentiating into hepatocyte-like cells in vitro.
METHODS: Adipose-derived mesenchymal stem cells were isolated from the subcutaneous fat of hepatitis B virus infection patients by collagenase digestion and adherent method. Adipose-derived mesenchymal stem cells
were induced by three-phase induction method and observed morphologically. The expression levels of alpha-fetoprotein, albumin and cytokeratin 18 were detected by immunohistochemical staining and glycogen synthesis function was detected by glycogen staining method.
RESULTS AND CONCLUSION: Most of adipose-derived mesenchymal stem cells induced by three-phase induction method were differentiated into hepatocyte-like cells with polygonal morphology. Immunohistochemistry staining results showed that hepatocyte-like cells expressed alpha-fetoprotein, albumin and cytokeratin 18, and the expression levels of albumin and cytokeratin 18 increased with the culture time. The induced cells had the function of glycogen synthesis and were positive for periodic acid Schiff staining. These results showed that the subcutaneous adipose-derived mesenchymal stem cells could be induced into functional hepatocyte-like cells in hepatitis B virus infection patients.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Mesenchymal Stem Cells, Subcutaneous Fat, Cell Differentiation, Tissue Engineering

CLC Number:

R394.2

Wang Yan-hui, Wang Zhi-ru, Sun Yan, Zhao Wen-jing, Yang Shu-yuan, Zhao Shu-li. Adipose-derived mesenchymal stem cells from hepatitis B virus infection patients differentiate into hepatocyte-like cells in vitro[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(25): 3983-3988.

Figures/Tables 2

2.1 hADSCs的分离培养 Ⅳ型胶原酶消化脂肪组织获得的细胞培养48 h后，镜下观察，贴壁细胞大多数呈梭形，少数细胞呈多角形，培养7-10 d单层生长细胞达80%-90%融合，呈旋涡状排列。10例供者的hADSCs全部培养成功，成功率为100%，原代培养时间为(8.3±1.2) d。传代培养的hADSCs贴壁较快，呈梭形的成纤维细胞样细胞，研究分离获得的10例hADSCs在体外连续传代培养15代无衰老征象，生长状态良好，见图1。 2.2 hADSCs细胞的生长曲线体外培养的hADSCs生长曲线均呈“S”形，3代和5代hADSCs细胞的生长曲线基本相同，细胞增殖速度快，传代周期较短，培养三四天进入对数生长期，第7天进入平台期，见图2。 2.3 hADSCs表面抗原测定流式细胞仪检测结果显示，5代hADSCs高表达间充质干细胞的特征性表面抗原，(93.5±2.9)%的细胞表达CD44，100%的细胞表达CD29，(90.1±3.6)%的细胞表达CD105，仅有(1.4±0.7)%的细胞表达造血干细胞的特征性表面标志CD34，见图3。 2.4 hADSCs体外诱导分化为脂肪细胞和成骨细胞实验结果显示，在成脂诱导培养第3周，实验组大部分细胞油红O染色阳性，镜下观察细胞胞浆内有橘红色脂肪滴，对照组细胞油红O染色阴性；成骨诱导第2周，实验组大部分细胞碱性磷酸酶染色阳性，镜下观察细胞胞浆内可见有灰黑色沉淀，对照组细胞碱性磷酸酶染色阴性，见图4。 2.5 hADSCs体外诱导分化为有功能的肝样细胞 hADSCs经三阶段诱导培养后，大部分细胞呈多角形，类肝细胞形态，细胞形态变化与文献报道相同[24]。免疫组织化学染色结果显示，实验组诱导培养后hADSCs白蛋白、细胞角蛋白18和甲胎蛋白染色阳性，镜下观察细胞胞浆内可见黄棕色沉淀，且细胞白蛋白和细胞角蛋白18的表达随着诱导培养时间的延长逐渐增加；对照组细胞白蛋白、细胞角蛋白18和甲胎蛋白染色阴性，见图5。过碘酸雪夫染色染色结果显示，对照组细胞过碘酸雪夫染色阴性；实验组诱导培养后hADSCs过碘酸雪夫染色阳性，镜下观察细胞胞浆内可见紫色沉淀，见图5。结果提示hADSCs体外可以诱导分化为具有糖原合成功能的肝样细胞。"

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