Cloning Of Intercellular Adhesion Molecule-1 CDNA And Expression

Objective: To study expression of intercellular adhesion molecule-1 (ICAM-1) on the normal liver tissues and tissues of patients with hepatocellular carcinoma;construct recombinant of ICAM-1 and eukaryotic expression vector ,build human ICAM-1 stable expression cell strain, particular target cells.Recently ICAM-1 has also been shown to be the cellular receptor for the major group of rhinoviruses and subtertian malaria infected red cell.ICAM-1 is expressed on human endothelial cells and a great quantity expressed on inflammatory cells and tumor cells.Increased expressed of ICAM-1 is essential of inflammatory and immune response and contributes to eliminate exotic antigen and tumor cells.But its permanent expression is resulted to chronic inflammatory and develop to tumor.Methods: Immunohistochemical study on the tissues of patients with human hepatocellular carcinoma and normal liver tissues. Frist, Intercellular adhesion molecule-1 cDNA was obtained by reverse transcription PCR of totol RNA extracted from the tissues of patients with human hepatocellular carcinoma.The efficient link was carried out directly between the intercellular adhesion molecule-1 cDNA and the vector by using pGEM-T Easy Vector System.Then the target fragment was subcloned into eukaryotic vector pCDNA3.1hisB to constructeukaryotic expression recombinant pCDNA3.1hisB-ICAM-1.The recombinant pCDNA3.1hisB-ICAM-1 was selected by restriction endonuclease digestion(EcoRI and BamHI) and it was confirmed by DNA sequencing.Last, expression recombinant plasmid was transfected into QSG7701 cell line by using lipofectamine and the stable transfected cell line was selected in medium containing geneticin(G418).The result of reverse transcription PCR of total RNA extracted from the cell line and immunohistochemical stain on the transfected cell line. Result: 1, Immunohistochemistry showed the expression of intercellular adhesion molecule-1 on the HCC tissues with honeycomblike appearance in most cases(87.5%),on the other hand,the normal liver tissues did not or little expression ICAM-1(20%). 2, ICAM-1 cDNA was obtained by RT-PCR extracted from the tissues of patients with human hepatocellular carcinoma .The PCR produce was directly linked up with T vector to be pGEM-ICAM-1;The recombinant digested by restriction endonuclease was linked up with eukaryotic vector ,and the recombinant pCDNA3.1hisB-ICAM-1 was checked up by EcoRI and BamHI digestion and DNA sequencing. 3, The recombinant pCDNA3.1hisB-ICAM-1 transfected QSG7701 cell by lipofectine and the cell was screened with G418 ,the stable expression of ICAM-1 on the transfected cell line,So far,it was accomplished to clone intercellular adhesion molecule-1 and to set a stable transfective cell line.Conclusion: 1, Immunohistochemistry revealed that the hepatocellular carcinoma high expressed ICAM-1 on the cell surface,the normal liver tissue lower expressed; 2, Eukaryotic expression recombinant pCDNA3.1hisB-ICAM-1 was contructed. 3, Stable expression cell strain of human ICAM-1 was biilt.