构建特异高表达人谷氨酰胺合成酶基因的肝细胞

Construction of hepatocytes with specific high-expression of human glutamine synthetase

目的建立高表达人谷氨酰胺合成酶(hGS)基因的人肝细胞系。方法克隆hGS基因及人甲胎蛋白转录调控元件(AFP TREs),构建真核表达载体pLNChGS及插入人AFP TREs的载体pLNAFhGS;经包装产生重组逆转录病毒并感染HepG2细胞,得到阳性细胞克隆HepG2/ pLNChGS和HepG2/pLNAFhGS;进行细胞增殖、hGS mRNA半定量及酶活性检测。结果HepG2/ pLNChGS、HepG2/pLNAFhGS细胞的hGS mRNA表达分别为8.306±0.336和21.358±1.716,均显著高于对照细胞(P<0.01),且两者之间比较差异有统计学意义(P<0.01);HepG2/pLNChGS、HepG2/pLNAFhGS细胞的hGS酶活性分别为3.279±0.328和4.557±0.253,均显著高于对照细胞(P<0.05),两者之间比较差异也有统计学意义(P<0.05)。结论成功构建高分泌hGS的人肝细胞系,为进一步降氨毒功能的研究打下基础。

Objective To construct hepatocytes with specific high-expression of human glutamine synthetase （hGS）. Methods hGS gene and AFP transcriptional regulatory elements （TREs） were cloned by designed primers. A eukaryotic expression vector, pLNChGS, was constructed by enzyme digestion and ligation. Another vector, pLNCAFhGS, was constructed by inserting human AFP TREs into vector pLNChGS. PA317 cells were transfected with pLNCX, pLNChGS and pLNCAFhGS and further produced recombinant retrovirus, which infected HepG2 cells respectively. HepG2/pLNCX, HepG2/pLNChGS and HepG2/pLNAFhGS cells were obtained. The proliferation of these cells was determined, and the expression of hGS mRNA and enzymatic activity were detected. Results The expression of hGS mRNA in HepG2/ pLNChGS cells （ 8. 306 ± 0. 336 ） and HepG2/pLNAFhGS cells （ 21. 358 ± 1. 716 ） was much stronger than in the controls significantly （ P 〈 0.01 ）, and that in HepG2/pLNAFhGS cells was stronger than in HepG2/pLNChGS cells obviously （ P 〈 0. 01 ）. The hGS enzymatic activity of HepG2/pLNChGS cells （3.279 ± 0.328） and HepG2/pLNAFhGS cells （4.557 ± 0.253 ） was higher （ P 〈 0.05 ） than in controis, and that in HepG2/pLNAFhGS cells was also higher than in HepG2/pLNChGS cells （ P 〈 0.05 ）. Conclusion The hepatocytes with specific high-expression of hGS is successfully established, swhich lays foundation for further experiment on ammonia metabolism.