中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (3): 473-477.doi: 10.3969/j.issn.1673-8225.2011.03.023

• 生物材料基础实验 basic experiments of biomaterials • 上一篇    下一篇

C3A细胞与L-02永生化肝细胞低温保存条件下的生物学特性比较

李安全1，李庆勇1,2，张清华1，蒋知新1，沙  杭1，高德禄1，高  毅3   

  1. 1解放军第三○五医院, 北京市  100017
    2南方医科大学研究生院, 广东省广州市   501515
    3南方医科大学珠江医院肝胆二科, 广东省广州市   510253
  • 收稿日期:2010-10-25 修回日期:2010-11-29 出版日期:2011-01-15 发布日期:2011-01-15
  • 通讯作者: 张清华，博士，教授，博士生导师，解放军第三○五医院老年病中心，北京市100017 mdlqy@yahoo.com.cn
  • 作者简介:李安全，男，1954年生，辽宁省营口市人，汉族，1988年北京医科大学毕业，副主任医师，主要从事老年病研究。 phdlqy@126.com
  • 基金资助:

    国家高技术研究发展计划（863计划）基金资助(2006AA02A141)；全军医学科研“十一五”计划专项基金资助项目（08Z017）。

Biological features of C3A hepatocytes versus L-02 immortalized hepatocytes under hypothermic stored condition

Li An-quan1, Li Qing-yong1,2, Zhang Qing-hua1, Jiang Zhi-xin1, Sha Hang1, Gao De-lu1, Gao Yi3   

  1. 1The 305 Hospital of Chinese PLA, Beijing   100017, China
    2Postgraduate College of Southern Medical University, Guangzhou   510515, Guangdong Province, China
    3Second Department of Hepatobiliary Surgery, Zhujiang Hospital Affiliated to Southern Medical University, Guangzhou   510253, Guangdong Province, China
  • Received:2010-10-25 Revised:2010-11-29 Online:2011-01-15 Published:2011-01-15
  • Contact: Zhang Qing-hua, Doctor, Professor, Doctoral supervisor, the 305 Hospital of Chinese PLA, Beijing 100017, China mdlqy@yahoo.com.cn
  • About author:Li An-quan, Associate chief physician, the 305 Hospital of Chinese PLA, Beijing 100017, China phdlqy@126.com
  • Supported by:

    the National High Technology Research and Development Program of China (863 Program), No.2006AA02A141*; the Special Fund of the Eleventh Five-year Plan for Military Medical Project, No. 08Z017*

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摘要：

背景：获得大量功能良好的肝细胞是生物人工肝的核心。探索出一种可靠的肝细胞低温保存方法进而构建一个肝细胞库是目前生物人工肝研究的热点。
目的：比较用UW液在4 ℃条件下保存已经进入Ⅲ期临床试验的C3A细胞与国内构建的永生化肝细胞株L-02细胞的生物学特性。
方法：贴壁培养C3A与L-02细胞，胰酶消化，制备成细胞悬液，UW液保存。4 ℃低温保存0，24，48及72 h后，采用流式细胞术分别测定细胞存活率与凋亡率，测定谷草转氨酶与乳酸脱氢酶释放、尿素合成功能及白蛋白分泌功能。
结果与结论：随低温保存时间延长，C3A与L-02细胞存活率呈下降的趋势，但C3A细胞的存活率明显高于L-02细胞(P < 0.01)；细胞凋亡率呈上升趋势，但48 h后C3A细胞同L-02细胞无差异(P  > 0.05)。谷草转氨酶及乳酸脱氢酶释放呈现上升的趋势，但C3A细胞明显低于L-02细胞(P  < 0.01)。白蛋白分泌功能呈下降的趋势，但C3A细胞明显优于L-02细胞(P  < 0.01)。尿素合成功能呈下降的趋势，但是L-02细胞明显优于C3A细胞(P  < 0.01)。结果提示，UW液4 ℃保存C3A细胞与L-02细胞时间不易超过48 h。以C3A细胞为材料的人工肝可能更适用于肝功能衰竭合并低白蛋白血症，以L-02细胞为材料的人工肝更适用于肝功能衰竭合并肝性脑病。

关键词: 生物人工肝, 低温保存, 永生化肝细胞, C3A细胞, L-02细胞

Abstract:

BACKGROUND: A large number of functional liver cells is the core of bioartificial liver. It is the existing research focus of bioartificial liver to explore a reliable method of cryopreservation of liver cells and to construct liver cell bank.
OBJECTIVE: To compare the biological characteristics of C3A hepatocytes that has entered clinical Ⅲ stage test and L-02 immortalized hepatocytes stored in UW solution at 4 ℃.
METHODS: The C3A hepatocytes and L-02 hepatocytes were adherent cultured, digested by 0.25% trypsinization, prepared into cell suspension, and stored in UW solution. At 0, 24, 48, 72 hours of hypothermic storage (4 ℃), the cell viability rate and cell apoptosis rate were measured using flow cytometry. Lactate dehydrogenase and aspartate aminotransferase release, the ability of hepatocytes to synthesize urea and secrete albumin were also determined.
RESULTS: The cell viability of C3A hepatocytes and L-02 hepatocytes was degraded following the time, but the cell viability of C3A hepatocytes was higher than L-02 hepatocytes (P < 0.01). Cell apoptosis was increased, but there was no difference between C3A hepatocytes and L-02 hepatocytes after 48 hours (P > 0.05). The lactate dehydrogenase and aspartate aminotransferase release were ascended, but the release of C3A hepatocytes was lower than L-02 hepatocytes (P < 0.01). The ability of hepatocytes to secrete albumin was decreased, but the ability of C3A hepatocytes was better than L-02 hepatocytes   (P < 0.01). The ability of hepatocytes to synthesize urea was also decreased, however, the ability of L-02 hepatocytes was better than C3A hepatocytes (P < 0.01). Results demonstrated that, the time of the C3A hepatocytes and L-02 hepatocytes which are hypothermicaly stored (4 ℃) in UW solution should not exceed 48 hours. The artificial liver prepared by C3A hepatocytes is more suitable for the liver function failure combined low albuminaemia, and artificial liver prepared by L-02 hepatocytes fits the liver function failure combined hepatic encephalopathy.

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