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

中国组织工程研究与临床康复第 15 卷 第 3 期 2011–01–15 出版Vol.15, No.3Journal of Clinical Rehabilitative Tissue Engineering Research January 15, 2011C3A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3Biological features of C3A hepatocytes versus L-02 immortalized hepatocytes under hypothermic stored conditionLi An-quan1, Li Qing-yong1,2, Zhang Qing-hua1, Jiang Zhi-xin1, Sha Hang1, Gao De-lu1, Gao Yi3AbstractBACKGROUND: 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. Li AQ, Li QY, Zhang QH, Jiang ZX, Sha H, Gao DL, Gao Y. Biological features of C3A hepatocytes versus L-02 immortalized hepatocytes under hypothermic stored condition. Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu. 2011;15(3):473-477. [ ]1The 305 Hospital of Chinese PLA, Beijing 100017, China; 2 Postgraduate College of Southern Medical University, Guangzhou 510515, Guangdong Province, China; 3 Second Department of Hepatobiliary Surgery, Zhujiang Hospital Affiliated to Southern Medical University, Guangzhou 510253, Guangdong Province, China Li An-quan, Associate chief physician, the 305 Hospital of Chinese PLA, Beijing 100017, China phdlqy@ Correspondence to: Zhang Qing-hua, Doctor, Professor, Doctoral supervisor, the 305 Hospital of Chinese PLA, Beijing 100017, China mdlqy@yahoo. 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* Received: 2010-10-25 Accepted: 2010-11-29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 doi:10.3969/j.issn.1673-8225.2011.03.023 李安全，李庆勇，张清华，蒋知新，沙杭，高德禄，高毅.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[J].中国组织工程研究与临床康复，2011，15(3):473-477. [ ]细胞库是BALSS普遍推广的基础[3]。

C3A细胞是 0 引言 生 物 人 工 肝 (bioartificial liver support system, BALSS)为肝功能衰竭的治疗开辟了新 的途径[1]。