| Background and Aims: Gastric cancer is one of the most common malignant tumors in China, but the pathogenesis is unclear. Recent investigations demonstrated that NHEl(Na+-H+ exchanger 1) mRNA expression significantly increased in lots of carcinomas.lt is relative to the occurrence and growth of tumors.The NHEl gene is a ubiquitous, integral membrane protein.lt plays many different physiological roles in mammals, primarily responsible for the regulation of pHi (intracellular pH, pHj). Our earlier studies showed that NHEl gene expression markedly increased in gastric carcinoma than normal gastric mucosa and gastric precancerous lesion.So the NHEl gene could be a good target for antisense gene therapy of gastric cancer.The aim of present study is to explore the effects of the human NHEl antisense gene transefection on the biological behaviours of SGC-7901 human gastric cancer cells,and probe into a new therapeutic strategy for gastric carcinoma.Methods: NHEl antisense gene eukaryotic expression plasmid was constructed using subclone technique and transfected into SGC-7901 gastric carcinoma cells by DOTAP liposome transfection system to directly interfere in the expression of NHEl gene. PCR was used to confirm the transfection. Several items about cells growth were measured, including intracellular pH, cells morphology under light and electrical microscope,cells proliferation capacity with MTT assay, cloning efficiency of SGC-7901 cells in soft agar, cells cycle distribution and apoptotic rates with flowcytometry, tumorigenicity in nude mice .Results : (l)The recombinant from subclone construction was digested and resulted in two fragments as 3.6kb and 5.0kb,the result was consistent with expected recombinant plasmid.(2)PCR showed a specific fragment with length 357bp was amplified from Anti-7901 cells(transfected with NHEl antisense gene) and Zeo-7901 cells(transfected with vector pCDNA3.l), but none from SGC-7901 cells. The results exhibited that the recombinant and the vector pCDNA3.l had integrated into the chromosome of SGC-7901cells respectively. (3) Under observation with light microscope, the appearance of Anti-7901 cells was homologous compared with its parent cells.Meanwhile we could also find that the Anti-7901 cells were usually large with an abundant cytoplasm and decreased nuclear division ,the ratio of nuclei to plasma and atypia.In contrast,the microscopic appearance of SGC-7901 cells and Zeo-7901 cells was heterogenous.Nuclei in these cells were large and hyperchromatic. Nuclear division, the ratio of nuclei to plasma and atypia were increased. Multinucleate giant cells and giant nucleate cells were usually present. Ultrastructure Under transmission electronic microscope revealed that organelles in Anti-7901 cells were abundant compared with its parent cells, the amount of mitochondrion was increased,Golgi apparatus and expanded endoplasmic reticulum was also noted.On the contrary, organelles in SGC-7901 and Zeo-7901 cells were decreased with enlarged nuclei,irregular nuclear membranes and prominent nucleoli.(4) Intracellular pH of Anti-7901 cells(6.77?.05)was significantly lower than that of Zeo-7901 cells and SGC-7901 cells (7.24?.03,7.26?.03,p<0.01).(5) The expression of NHE1 protein in Anti-7901 cells decreased in contrast to Zeo-7901 cells and SGC-7901 cells. (6) Growth curve of Anti-7901 cells slowers than that of Zeo-7901 cell and SGC-7901 cells.Apoptotic rates in Anti-7901 cells was markedly higher than that in Zeo-7901 cells and SGC-7901 cells . Flow cytometric analysis displayed an trifling accumulation of Anti-7901 cells in the GO/GI phase of cell cycle and a slight decreased percentage of cell in S and GaM phase and proliferative index.(7)Cloning efficiency of Anti-7901 cells significantly decreased in soft agar (9?.54%0) compared with Zeo-7901 cells and SGC-7901 cells(40?.53 %o, 38.6?.63%o, P<0.01). No tumorigenicity in nude mice was found even if 4 *106 Anti-7901 cells were injected subcutaneously.Conclusion:We succeedly constructed recombinant plasmid using subclone technique...
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