转基因肝星状细胞株的建立及生物学特性

Establishment of a transgenic hepatic stellate cell strain and study on its biological behaviors

目的 建立稳定表达肝细胞生长因子 (HGF)的肝星状细胞株CFSC/HGF。方法 构建携带hgf基因全长序列的重组逆转录病毒pMSCV HGF ,反复感染肝星状细胞系CFSC建立转基因肝星状细胞株。对建立的CFSC/HGF用逆转录 聚合酶链反应 (RT PCR )和酶联免疫吸附试验 (ELISA)分别从基因和蛋白水平进行鉴定 ,并通过免疫细胞化学检测、细胞增殖活性测定 ,以及对人肝癌细胞HepG2的扩散试验等对其生物学特性进行初步研究。结果 构建了重组逆转录病毒质粒 pMSCV HGF及包装细胞株PT/HGF ,建立并筛选出HGF表达水平分别为0 .15、2 .2 3、5 .48、9.76μg/L的 4个细胞株CFSC/HGF ,细胞表达结蛋白和α 平滑肌肌动蛋白 ;随HGF表达水平不同 ,CFSC/HGF细胞增殖活性显示出明显的差别 ;CFSC/HGF培养上清可使HepG2细胞扩散 ,生长受抑。结论 建立了稳定表达hHGF的肝星状细胞株CFSC/HGF ,并对其生物学行为进行了初步鉴定。

Objective To establish a rat hepatic stellate cell strain CFSC/HGF expressing hHGF stably,and investigate its biological character.Methods HGF gene was amplified from plasmid pcDNA3 HGF by PCR technique and subcloned to retroviral vector pMSCVneo.The highly virus producing cell line PT/HGF was screened out following transferring the recombined plasmid into packaging cell line PT67 with lipofectamine,G418 selecting and NIH3T3 cells viral titer determining.After repeatedly infecting CFSCs with the culture supernatant,the gene transferred cells were selectively cultured with G418 and identified by RT PCR and ELISA.Immuno cytochemistry,MTT and the human hepatoma cell HepG2 dispersing test were carried out.Results The recombined retrovirus pMSCV HGF and its packaging cell line PT/HGF were constructed after the HGF segment (2?200 bp) was amplified with PCR and proved encoding HGF gene by sequence determination.After being infected with the supernatant of PT/HGF for 3 times,CFSCs with HGF gene transferred were selectively cultured with 700 mg/L G418 and the positive clones obtained in 14 days.The 4 cell lines,CFSC/HGF (1 4),which expressed HGF stably were selected out and identified by RT PCR and ELISA.Immuno cytochemistry proved that the new cell strain expressed desmin and α SMA.MTT indicated that the cellular proliferation rate varied with the HGF expressing rate.HepG2 cells could be scattered with the supernatant of the CFSC/HGF.Conclusion A new rat hepatic stellate cell strain,CFSC/HGF,which could express hHGF gene stably was established successfully,and the primary study on its biological behaviors has been made.