Construction Of GAcDNA Antisense Eukaryotic Expression Vector And Its Effects On The Biological Activity Of Gastric Carcinoma Cells

Objectives: To study whether antisense GAcDNA can inverse malignancy phenotype of the gastric carcinoma cells by blocking the glutaminase gene with antisense nucleotide techniques in order to explore a new approach for the gene therapy of tumor and to establish the foundation of following research and clinical application.Methods: At first, the antisense eukaryotic expression vector contenting the segment of GAcDNA, named pcDNA3.0/GA, was constructed by standard gene recombinant techniques. Then transfect it into gastric carcinoma cells (SGC-7901) by liposomes. This transfected cells were named SGC-7901GA after filtration and identification. At last, to study the changes of the biological activity of tansfected gastric carcinoma cells, we depicted the cells growth curves of with MTT method and compared the differences of apoptosis with terminal deoxynucleotidyl transferase-mediated nick end labeling technique (TUNEL) and proliferating cell nuclear antigen (PCNA) with immunohistochemistry method between transfected cells and untransfected cells.Results:1. The antisense Eukaryotic expression vector pcDNA3.0/GA contenting the segment of GAcDNA was constructed successfully, which was confirmed by enzymes cutting and gene sequencing.2. Through transfection by liposomes, GAcDNA segment was integrated into the genome of SGC-7901 cells, which was confirmed by PCR. The transfected cells, which can live after selecting with G418, were named SGC-7901GA.3.The growth speed of SGC-7901GA is significant slower than control group bydepicting the cells growth curves with MTT method(p<0.05).4. The e group(p<0.01).4. The expression of PCNA in SGC-7901GA is significant lower than control5.The apoptosis index of SGC-7901GA is significant higher than control group(p<0.01).Conclusions: Through transfecting antisense eukaryotic expression vector contenting the segment of GAcDNA into gastric carcinoma cells, we found the proliferation ability of tumor cells is decreased, the apoptosis index is increased and the level of tumor's malignancy is decreased. Blocking the gene of glutaminase with antisense nucleotide techniques may be a new way for the gene therapy of tumor.