Hepatoma Membrane Anti Antigen Hab18g/cd147, Antisense Rna Expression Vector And Its Inhibition Of Tumor Invasion Of The Preliminary Studies

HAblSG is the correspondent membrane-antigen of HAbl8 which is a hepatoma monoclonal antibody with high specificity and affinity purified by our own lab. We used HAbl 8 to select cDNA library of Human Hepatocarcinoma Cell and obtained a 1.7kb-long cDNA sequence of HAblSG. Open reading frame of HAbl 8G is homologous with CD147. Molecular CD147. namely EMMPRIN(Excellular Matrix Metal loproteinase Inducer), can stimulate fibroblasts to produce Matrix Metalloproteinase(MMPs).MMPs,is closely related to invasion of tumor because it can digest the excellular matrix.We want to construct antisense RNA expression vector of CD 147 to inhibit the expression of CD 147 and MMPs,then to explore its function of inhibiting invasion of tumor.Three steps are included in the experiments process. 1.Constructing the antisense RNA expression vector of HAbl8G/CD147. 2. Transfecting HAM8G/CD147 antisense RNA vector to HHCC, selecting the positive clone.3.Observing its function of inhibiting invasion of hepatoma. We cut the pBluescript sk(+)HAb 18Concluding full length cDNA of HAbl 8G ) with two enzymes and obtain 1.7kb HAblSG cDNA,then, ligased the cDNA reversely to mammalian expression vector PCI-neo cut by two similiar enzymes too.After that,we transfected the antisense RNA expression vector to hepatoma strain HHCC which express HAblSG highly. After selecting by G418, picking out single clone by method of limited dillution ,enlargly cultuing,we identified theclones by immuno-histochemical SP method , cytoflowing method and observed states of protein expression being inhibited.Gelatinase Zymography was used to study the function of HAblSG in aspect of inducing secretion of type IV collagenase;culture chamber is used to test the function of inhibiting invasion of hepatoma cells. The result shows the vector is constructed successfully;expression of HAblSG is blocked efficiently;secretion of type IV collagenase reduced;invasion of hepatoma cells are lowered.Conclusion: we inhibit the expression of HAblSG through molecullar level ;and .because of this,its capability of stimulating human fibroblasts to secret MMPs is lowered;at last, the infitration and metastasis of hepatoma cells was indirectly inhibited.