永生化人肝细胞系的建立和生物学特性研究  

Establishment and biological characteristics of immortalized human hepatocyte cell line

作　　者：张然星[1] 李宓[2] 张文健[3] 顾锋[4] 门秀丽[3] 叶丽亚[3] 房青[3] 徐梅[3] 高福云[3] 娄晋宁[3] 

机构地区：[1]北京协和医学院研究生院,100730 [2]中山大学附属第五医院肾内科,珠海519000 [3]中日友好医院临床医学研究所,北京100029 [4]中国医学科学院北京协和医院内分泌科,100730

出　　处：《中国医药生物技术》2008年第1期11-18,共8页Chinese Medicinal Biotechnology

基　　金：国家高技术发展研究计划(863计划)(2006AA02Z482)

摘　　要：目的建立永生化人肝细胞系(IHCL),评价其作为制备生物人工肝细胞材料的可行性。方法利用基因转移技术对原代培养的人肝细胞转染永生化基因猴病毒40大T抗原和人端粒酶逆转录酶,建立IHCL。以噻唑蓝法测定IHCL细胞生长曲线;流式细胞仪分析细胞增殖周期;裸鼠皮肤和肝脏局部接种观察细胞致瘤性;RT-PCR检测肝细胞相关基因mRNA表达;蛋白质印迹法检测肝功能相关蛋白的表达。应用Cytodex 3微载体培养IHCL,分析细胞的生长状态和细胞密度。将IHCL细胞分别与5例重症肝功能衰竭患者的血清共培养,培养前后检测血清中胆红素和尿素氮水平,评价IHCL的解毒功能。结果所建立的IHCL具有原代肝细胞的形态,并具有较好的增殖能力,细胞已经传代超过80次。IHCL细胞以DNA二倍体整数倍方式增殖,细胞倍增周期为38h。裸鼠接种IHCL部位均未见肿瘤生长。RT-PCR检测显示IHCL细胞表达α1-抗胰蛋白酶、尿苷二磷酸葡萄糖醛酸基转移酶、谷胺酰胺合成酶和谷胱甘肽硫转移酶的mRNA;蛋白质印迹分析证实IHCL细胞表达这4种蛋白。IHCL细胞可以在微载体上贴附生长,细胞密度可以达到2.86×106个/ml。将IHCL细胞与重症肝功能衰竭患者血清共培养12、24和48h后,血清中总胆红素由(346±94)μmol/L分别下降至(330±97)、(315±97)和(295±100)μmol/L,直接胆红素由(243±70)μmol/L下降至(218±76)、(198±79)和(184±75)μmol/L,24、48h与共培养前比较差异均有统计学意义(P<0.05);而尿素氮进行性地升高,由(6.6±0.9)μmol/L分别升高至(9.0±0.9)、(9.9±1.1)和(12.5±1.6)μmol/L,3个时间点与共培养前比较差异均有统计学意义(P<0.01)。结论所建立的IHCL细胞系具有正常人肝细胞的基本生物学特性和主要的功能特性,具备成为生物人工肝细胞材料的可行性。Objective To establish an immortalized human hepatocyte cell line （IHCL） and evaluate the feasibility of producing artificial liver cells by using the cell line. Methods Primary human hepatocytes were transfected with simian virus 40 large tumor （SV40T） antigen and human telomerase reverse transcriptase （hTERT） to establish IHCL. The proliferation curve of IHCL was analyzed by MTT assay, and the cell cycle was analyzed by flow cytometry. The IHCL was inoculated locally at the skin and liver of nude mice to evaluate the tumorigenesis of the cells. The mRNA expression of hepatocyte-specific genes was detected by RT-PCR, and the expressions of the proteins associated with liver function were determined by Western blotting. The IHCL was cultured on microcarriers, Cytodex 3, and the proliferation and density of the cells were analyzed. The IHCL was co-cultured with sera from 5 patients with severe liver failure, the serum levels of bilirubin and urea nitrogen were determined to evaluate the therapeutic effect of IHCL.Results The established IHCL maintained the morphology of primary hepatocyte with a high proliferative ability and has been passaged for more than 80 times. The IHCL was diploid, and the doubling time was 38 hours. No tumorigenesis was found at the inoculation site in the nude mice. RT-PCR and Western blotting showed the expression of mRNA and protein of ctl-antitrypsin, UDP-glucuronosyltransferase, glutamine synthetase, and glutathione-S-transferase in the IHCL. The IHCL grew well attaching to microcarrier, reaching a cell density of 2.86 × 10^6/ml. After being co-cultured with the sera obtained from patients with liver failure for 12 h, 24 h and 48 h, the concentration of total bilirubin in the sera decreased from （346±94）μmol/L to （330±97）μmol/L, （315±97） μmol/L, and （295±100）μmol/L respectively, and the concentration of direct bilirubin from （243±70）μmol/L to （218±76）μmol/L,（198±79）μmol/L, and （184±75）μmol/L, the differences between the base

关 键 词：模型 生物学 肝 人工 基因转移技术 肝细胞 肝功能衰竭 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]