胚胎干细胞向肝实质细胞体外定向诱导分化过程中的肝卵圆细胞
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国家重点基础研究发展规划(973)项目(2001CB510101),国家自然科学基金重点项目(30230350),国家自然科学基金项目(60278014)和广东省自然科学基金项目(36704).


Hepatic Oval Cells Obtained In The Course of Directional Induction and Differentiation of Mouse Embryonic Stem Cells Into Hepatocytes in Vitro
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This work was supported by grants from National Basic Research Program of China (2001CB510101), The National Natural Science Foundation of China (30230350,60278014) and The Natural Sciences Founation of Guangdong Province (36704).

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    摘要:

    如果肝脏严重受损致使肝细胞大部分坏死,或由于某些原因 ( 肝毒性物质、致癌物质的作用 ) 抑制残存肝细胞增殖时,肝内前体/干细胞———肝卵圆细胞便被激活并分化生成肝细胞和胆管细胞等以参与肝修复 . 基于此理论,人们建立了啮齿类动物肝卵圆细胞诱导实验模型 . 但显然上述模型不适用于人类,所以有必要开发一种适用于人类的、高效的肝卵圆细胞的新诱导模型 . 选用小鼠胚胎干细胞,转成拟胚体分化 3 天后分组,诱导组添加肝细胞生长因子 (HGF) 、表皮生长因子 (EGF) 作定向诱导分化 . 其间用免疫细胞化学 (ICC) 检测肝卵圆细胞标志物 A6 等的表达,用流式细胞仪筛选肝卵圆细胞并行 RT-PCR 、透射电镜检测 . 所筛选的肝卵圆细胞进一步体外培养并进行 ICC 和 RT-PCR ,检测其分化生成成熟的肝细胞和胆管细胞的能力 . 研究证实胚胎干细胞体外定向诱导生成肝实质细胞的过程中,存在着有双向分化能力的肝卵圆细胞这个中间分化阶段 . 诱导组肝卵圆细胞分化率均显著地高于对照组,最高时可达 6.11% 左右 . HGF 和 EGF 能显著性诱导胚胎干细胞源性卵圆细胞的生成 . 流式细胞仪筛选 Sca-1+/CD34+ 细胞占总细胞数的 4.59% ,其中 A6 阳性肝卵圆细胞占 90.81% 左右 . 使用流式细胞仪可获得高富集的 A6+/Sca-1+/CD34+ 肝卵圆细胞 . 提供了一种可适用于人类的肝卵圆细胞的新诱导模型 .

    Abstract:

    When either massive damage is inflicted on the liver or liver regeneration after damage is compromised by a variety of toxins and carcinogens, the hepatocyte progenitor/stem cells which called “oval cells” is activated and differentiate into a variety of cell lineages including hepatocytes and biliary epitheliums. Those models used in the study of rodents' oval cells are obviously not suitable for the study on human hepatic oval cells. A new hepatic oval cells produced model suitable for human need be developed. Mouse embryonic stem cells (ES cells) were cultured and induced to develop into embryonic bodies. The induced group and the control group were set up then. Hepatocyte growth factor (HGF) and epidermal growth factor (EGF) was added to the culture medium of the induced group. The markers such as A6 antigen, which is expressed by hepatic oval cell, are detected by means of immunocytochemistry. As the descendants of mouse ES cells, hepatic oval cells sorted by FACS were detected by RT-PCR and transmission electron microscopy. The hepatic oval cells sorted by FACS were further cultured and tested the ability of bipotential differentiation by ICC and RT-PCR. It was firstly confirmed that the midterm phase of hepatic oval cells which are bipotential does occur during the course of the directional induction and differentiation of mouse ES cells into hepatic parenchyma cells. The differentiation ratios of hepatic oval cells from the induced group are significantly higher than that from the control group, and the maximal ratio from the induced group could be about 6.11%. HGF and EGF could promote the proliferation of hepatic oval cells derived from ES cells. About 4.59% of the cells sorted by FACS are Sca-1+/CD34+, and about 90.81% of the Sca-1+/CD34+ cells are A6 positive. Highly purified A6+/ Sca-1+/CD34+ hepatic oval cells derived from ES cells could be obtained by FACS. A new hepatic oval cells produced model suitable for human was developed.

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银东智,蔡继业,郑启昌,肇静娴,刘美莉,戴 云,谢秋玲,洪 岸.胚胎干细胞向肝实质细胞体外定向诱导分化过程中的肝卵圆细胞[J].生物化学与生物物理进展,2005,32(10):959-968

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