Three kinds of engineered hepatic tissues constructed using rat bone marrow mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2014.41.004

Abstract

Abstract:

BACKGROUND: Engineered hepatic tissue is considered a promising strategy for healing acute liver failure. But, there are series of hindrances in the construction of engineered hepatic tissues, including acquisition of vital hepatocytes, choice of scaffolds and culture system, and nutrition supply.

OBJECTIVE: To construct three kinds of engineered hepatic tissues in hope to screen the optimal one for transplantation in acute liver failure.

METHODS: After purification, amplification, bone marrow mesenchymal stem cells were induced to differentiate into hepatocyte-like cells which were co-cultured with acellular amniotic membrane, DACRON PATCH cardiovascular surgical patch, biological surgical patch, respectively to construct three kinds of engineered hepatic tissues. After 3 days of culture, morphological and functional detections were performed.

RESULTS AND CONCLUSION: Rat bone marrow mesenchymal stem cells with higher purity were successfully harvested by using density gradient centrifugation and adherent methods, and then the cells were differentiated into hepatocyte-like cells. In the three kinds of engineered hepatic tissues, hepatocyte-like cells were found to be combined with the biological surgical patch to the maximum extent, and their combination exhibited stronger ability of urea synthesis and albumin secretion, which provides experimental basis for treatment of acute liver failure.

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

全文链接：

Key words: bone marrow, mesenchymal stem cells, liver failure, acute, liver, artificial

CLC Number:

R394.2

Yu Jian-xiong, Yuan Jing, Xu Rui-yun. Three kinds of engineered hepatic tissues constructed using rat bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(41): 6578-6584.

Figures/Tables 2

2.1 SD大鼠骨髓间充质干细胞的分离、纯化、扩增及标记在倒置相差显微镜下，可观察有核细胞在培养瓶底散在分布；3 d后首次换液，可见有少量贴壁细胞；5-9 d后细胞成集落式生长，呈明显的长梭形；10-13 d后原代细胞融合成单层排列有明显方向性，细胞排列成漩涡状、网状、辐射状。细胞经传代培养后，细胞生长较快，4-7 d即可汇合成单层，原代和传代骨髓间充质干细胞形态及生长特点无明显改变。流式细胞仪分析第3代骨髓间充质干细胞的表型，结果显示，骨髓间充质干细胞具有较高的均一性，流式细胞仪检测表型为CD29(99.8%)、CD44(62%)阳性，而CD34(1.8%)、CD45(2.5%)为阴性，也不表达CD80(6.8%)和CD86(2.1%)两种重要的共刺激分子，见图1。共聚焦显微镜下观察可见全部细胞标记后胞膜均显红色荧光，证实CM-DiI标记阳性率为100%，见图2。 2.2 骨髓间充质干细胞的转分化骨髓间充质干细胞转分化组中，可见细胞由梭形向三角形或椭圆形或圆形转变；细胞体积增大，并且相互集聚，形成小梁状排列，类似肝小叶结构；诱导3周后，细胞小梁样排列逐渐模糊，细胞发生凋亡。转分化细胞在第14天行免疫荧光检测，发现肝细胞标记物白蛋白和甲胎蛋白均为阳性表达，其中红色为CM-DiI标记荧光，绿色为肝细胞标记物的FITC染色荧光；而对照组细胞共聚焦显微镜下观察肝细胞标记物白蛋白和甲胎蛋白为阴性(图3)。RT-PCR结果证实了转分化过程，诱导组细胞在不同时间段白蛋白和甲胎蛋白的mRNA均有阳性表达，其中在第7天出现少量白蛋白的mRNA表达，随诱导时间的延长，其表达量逐渐增强，21 d时呈最强表达；甲胎蛋白的mRNA在14 d时表达量最强，21 d时表达量有所降低。而未诱导组白蛋白、甲胎蛋白的mRNA未见明显表达。表明转分化的肝样细胞是介于胚肝细胞和成熟肝细胞之间的尚未完全终末化的类肝样细胞。 2.3 人工肝组织支架的透射电镜观察透射电镜下可见支架1板层纤维排列规则，相邻板层间纤维走向相互垂直；透射电镜下可见支架3由相互交叉排列的纤维状结构组成，部分胶原纤维组织出现降解现象(图4)；而支架2因材料的特殊性在透射电镜下无法观察。 2.4 人工肝组织的构建及评估细胞在支架1和2的表面生长，其中在支架1生长良好，细胞数量较多，细胞与基质的黏附性好；而在支架2上细胞数量很少，形态欠佳且与支架黏附疏松。相比而言，在支架3中，细胞不仅在表层，而且在内层中顺纤维基质排列生长，呈多角形或圆形，形成多层细胞结构(图5)。尿素合成实验和白蛋白分泌实验也证实人工肝组织3具有更强的功能，与人工肝组织1和人工肝组织2相比差异有显著性意义(P < 0.05及P < 0.01)，为最佳人工肝组织(图6)。"

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