中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (19): 3544-3549.doi: 10.3969/j.issn.1673-8225.2010.19.025

• 干细胞基础实验 basic experiments of stem cells • 上一篇    下一篇

胚胎肝脏原始细胞表型及生物学特性

余强锋，周剑寅，尹震宇，王效民   

  1. 厦门大学附属中山医院肝胆外科(福建医科大学教学医院)，福建省厦门市 361004
  • 出版日期:2010-05-07 发布日期:2010-05-07
  • 通讯作者: 王效民，博士，教授，主任医师，厦门大学附属中山医院肝胆外科(福建医科大学教学医院)，福建省厦门市 361004 wxm@xmzsh.com
  • 作者简介:余强锋，男，1982年生，福建省龙岩市人，汉族，2009年福建医科大学毕业，硕士，医师，主要从事胚胎肝干细胞的基础研究。 fengwolf@126.com
  • 基金资助:

    福建省自然科学基金资助项目(2006J0122)。

Phenotypes and biological properties of embryonic hepatic progenitor cells

Yu Qiang-feng, Zhou Jian-yin, Yin Zhen-yu, Wang Xiao-min   

  1. Department　of　Hepatobiliary　Surgery, Zhongshan Hospital Affiliated to Xiamen University, Xiamen 361004, Fujian Province, China
  • Online:2010-05-07 Published:2010-05-07
  • Contact: Wang Xiao-min, Doctor, Professor, Chief physician, Department　of　Hepatobiliary　Surgery, Zhongshan Hospital Affiliated to Xiamen University, Xiamen 361004, Fujian Province, China wxm@xmzsh.com
  • About author:Yu Qiang-feng, Master, Physician, Department　of　Hepatobiliary　Surgery, Zhongshan Hospital Affiliated to Xiamen University, Xiamen 361004, Fujian Province, China fengwolf@126.com
  • Supported by:

    the Natural Science Foundation of Fujian Province, No. 2006J0122*

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摘要：

背景：目前肝干细胞尚无特异性标志物，从胚胎中建立具有干细胞样特性的肝脏原始细胞群是鉴定及研究肝干细胞的一个可行方法。

目的：观察胚胎肝原始细胞表型及生物学特性，寻找肝原始/干细胞候选细胞群。

方法：酶消化法分离、培养孕13.5 d的BALB/C小鼠胚胎肝原始细胞，免疫细胞化学染色及免疫荧光染色、Western blot、RT-PCR法测定AFP/Albumin/CK19/c-Met/E-cadherin等肝干细胞表面标记，以及CD45、CD14、SMA等非肝干细胞标记的表达；相差显微镜及透射电镜观察细胞形态结构；流式细胞仪测定细胞周期分布时相；表皮生长因子加二甲基亚砜诱导分化成熟后行PAS糖原染色。

结果与结论：实验成功分离得到纯度较高的干细胞，原代细胞多呈集落样生长，小圆形。细胞周期测定提示细胞多数处于静止期，符合干细胞特点。透射电镜观察细胞核浆比例大，细胞器不成熟，呈现原始细胞特点。流式细胞周期测定结果S期占10.8%，G2/M期占10.6%，无异常DNA含量。PAS染色后阳性对照组肝癌细胞的糖原主要表达在靠近一侧的核周边胞质内，呈强阳性；而胚胎肝前体细胞诱导组，核浆比例大，部分细胞胞质内可见较弱的糖原表达，充满胞质。提示实验初步建立表达AFP+Albumin+CK19+c-Met+E-cadherin+标志的集落样生长的肝原始细胞群，其表达多种干细胞表型，具有未成熟肝干细胞特点，可能作为肝干细胞候选细胞。

关键词: 肝干细胞, 表型, 分离, 鉴定, 胚胎

Abstract:

BACKGROUND: There are no specific markers of hepatic stem cells (HSCs), so to establish a cluster of hepatic progenitor cells from embryo is a feasible method for studying HSCs.

OBJECTIVE: To investigate phenotypes and biological properties and to search for candidate cell of embryonic HSCs. 

METHODS: 13.5 day postcoitum (dpc) embryonic hepatic progenitor cells were isolated from BALB/C mice by enzyme digestion method. Immunocytochemical staining, immunofluorescence staining, Western blot assay and reverse transcription-polymerase chain reaction were applied to identify HSC phenotypes of AFP/Albumin/CK19/c-Met/E-cadherin, and other cell markers of CD45, CD14 and SMA. Phase-contrast microscope and transmission electron microscope were used to observe the cell morphology. Flow cytometry was used to determine the cell cycle disposition. Epidermal growth factor + dimethyl sulphoxide induced cell differentiation, and PAS staining for glycogen was performed.

RESULTS AND CONCLUSION: Stem cells with a high purity could be obtained. Primary cultured cells presented colony-like structure and small round shape. Cell cycle detection has indicated that a majority of cells entered stationary phase, which was accorded to stem cell characteristics. Transmission electron microscope has demonstrated big karyoplasmic ratio, immature organelle, showing properties of initial cells. Flow cytometry results exhibited that 10.8% of cell cycle disposition in S phase and 10.6% was in G2/M phase, without abnormal DNA content. Following PAS staining, glycogen in hepatoma carcinoma cells mainly expressed near to unilateral cytoplasm surrounding nuclei, showing strong positive expression in the positive control group. Big karyoplasmic ratio and weak expression of glycogen in cytoplasm were visible in the embryonic hepatic progenitor cell group. Hepatic progenitor cell clusters expressing AFP+Albumin+CK19+c-Met+E-cadherin+ were established, which expressed some phenotypes and immature characteristics of stem cells. They may be chosen as candidate cells of HSCs.

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