中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (18): 3301-3304.doi: 10.3969/j.issn.1673-8225.2012.18.016

• 器官移植基础实验 basic experiments of organ transplantation • 上一篇    下一篇

一种简易的人原代肝细胞分离培养方法***☆

吴青松1，李治国1，刘广波1，高  毅1, 2   

  1. 1南方医科大学珠江医院肝胆外科，广东省广州市 510282；2南方医科大学再生医学研究所，广东省广州市  510282
  • 收稿日期:2011-12-08 修回日期:2011-12-18 出版日期:2012-04-29 发布日期:2012-04-29
  • 通讯作者: 高毅，教授，博士生导师，南方医科大学珠江医院肝胆二科，广东省广州市 510282 gaoyi6146@163.com
  • 作者简介:吴青松☆，男，1971年生，湖南省常宁市人，汉族，南方医科大学在读博士，主任医师，主要从事肝胆外科临床、生物人工肝细胞材料研究。 nanhuaren@yahoo.com.cn
  • 基金资助:

    国家高技术研究发展计划(863计划)项目(2006AA02A141)；广东省科技计划项目(2007A032100005)；广东省自然科学基金项目(07300180)。

A simple method for human primary hepatocytes separation  

Wu Qing-song1, Li Zhi-guo1, Liu Guang-bo1, Gao Yi1, 2   

  1. 1Department of Hepatobiliary Surgery, Zhujiang Hospital of Southern Medical University, Guangzhou  510282, Guangdong Province, China; 2Institute of Regeneration Medicine, Southern Medicine University, Guangzhou  510282, Guangdong Province, China
  • Received:2011-12-08 Revised:2011-12-18 Online:2012-04-29 Published:2012-04-29
  • Contact: Gao Yi, Professor, Doctoral supervisor, Department of Hepatobiliary Surgery, Zhujiang Hospital of Southern Medical University, Guangzhou 510282, Guangdong Province, China; Institute of Regeneration Medicine, Southern Medical University, Guangzhou 510282, Guangdong Province, China gaoyi6146@163.com
  • About author:Wu Qing-song☆, Studying for doctorate, Chief physician, Department of Hepatobiliary Surgery, Zhujiang Hospital of Southern Medical University, Guangzhou 510282, Guangdong Province, China nanhuaren@yahoo.com.cn
  • Supported by:

    National High Technology Research and Development Program (863 Program), No.2006AA02A141*; Guangdong Science and Technology Program, No. 2007A032100005*; Guangdong Natural Science Foundation, No. 2007A032100005*

PDF

569

摘要：

背景：采用原位两步灌流法分离肝细胞数量多，活性高，但灌流装置价格昂贵，操作环节多，技术要求高，所需时间长，易污染，限制了在一般实验室及人原代肝细胞在医学中的应用。
目的：建立人原代肝细胞体外分离培养方法。
方法：采用多点穿刺灌注方法将预温38 ℃的前灌流液及Ⅱ型胶原酶溶液灌依次灌入人肝脏组织中，经消化及离心后获得人原代肝细胞；倒置显微镜和电镜观察细胞形态特征，并鉴定。
结果与结论：经多点穿刺灌注法消化分离后可获得约5×105个肝细胞，分离获得的肝细胞活率达90%；细胞培养24 h后呈铺路石样生长；电镜示肝细胞呈典型形态特征：细胞核大，胞质少；培养的肝细胞 PAS糖原染色阳性，白蛋白表达阳性。说明多点穿刺灌注法分离人原代肝细胞简单易行，肝细胞产量高、活力好、纯度高，分离培养的肝细胞具有特异性生物学功能表达，适宜在一般条件实验室推广应用。
 

关键词: 原代肝细胞, 分离, 培养, Ⅱ型胶原酶, 灌注法 ,  

Abstract:

BACKGROUND: The two-step in situ perfusion for liver cells isolation was high in quantity and activity, but the perfusion device was expensive, complicate in operational steps, high in technique requirement, long time to operate and easily to pollute. And the human primary liver cells are limited to use in the general laboratory and in medical applications.
OBJECTIVE: To establish the separate and culture methods for human primary hepatocytes in vitro.
METHODS: The liver tissue was perfused with pre-perfusate solution at 38 ℃ and Ⅱ collagenase solution by using multi-point puncture perfusion technique, and then the human primary hepatocytes were obtained after digestion and centrifugation; Hepatocyte morphology was observed by inverted microscope and electron microscope and the function was identified.
RESULTS AND CONCLUSION: The amount of human primary hepatocytes was 5×105 and the viability was 90% after digested and separated by multi-point puncture perfusion. Electron microscope showed hepatocytes were cobblestone-like growth, a typical large nucleus and little cytoplasm after cultured for 24 hours. Glycogen PAS staining and albumin expression of cultured hepatocytes were positive. The multi-point puncture perfusion hepatocytes technique was simple and easy to operate, with advantages of high yield, good viability and high purity and the isolated and cultured hepatocytes have specific biological functions, so it would be applied widely in ordinary laboratory.

中图分类号: