Study Of Effects Of Fat-1 On Human Oral Squmous Cell Carcinoma Cells

n-3 polyunsaturated fatty acids(PUFAs)are essential components required for normal cellular function and play a particularly important role in pharmaceutical and nutritional fields, which are concerned by scientists for a long time. It is necessary to maintain the appropriate proportion of n-3 PUFAs physiological and it can serve to prevent cardiovascular disease, neurodegenerative diseases and even cancers. The shift in n-6/n-3 fatty acid ratio, especially the deficiency of n-3 fatty acids, might have imposed a risk of modern diseases. These fatty acids are termed essential fatty acids because they could not be produced by the body (whether animal or human) and must be supplied by the diet. The two classes of PUFAs are metabolically and functionally distinct and often have important opposing physiological functions and their balance is important for homeostasis and normal development.The Fat-1 gene encodes an n-3 fatty acid desaturase, which is an important fatty acid desaturase in the biosynthesis of PUFAs.This enzyme can catalyze the conversion of n-6 PUFAs to n-3 PUFAs by introducing an n-3 double bond into their hydrocarbonchains. It increases the n-3 PUFA content of products endogenously. Many findings as presented here suggest that gene transfer of the n-3 fatty acid desaturase not only elevate tissue concentrations of n-3 PUFAs quickly and effectively, but also decreases the level of excessive endogenous n-6 PUFAs.Oral squamous cell carcinoma (OSCC) is most common oral cancer, which is a serious threat to human health with a very high incidence and a low survival rate. To test the biological function of Fat-1 gene in human oral squamous carcinoma, we firstly synthetic Fat-1 gene using SOE-PCR method, then reconstruct the gene vector pcDNA3.1-Fat-1 based on the construction of eukaryotic expression vector pcDNA3.1(+). Restriction Analysis of pcDNA3.1-Fat-1 by Double Cut with Nheâ… and Kpnâ… indicated the recombinants were correct. The human oral squamous carcinoma cells line Tca8113 was transferred using cationic lipid-transfer method, and the positive transgenic cells are obtained after G418 selection. After testing, the PCR test proved that foreign genes had been integrated within the genome. The fatty acids content of the transfected Tca8113 cells was detected by using gas chromatograpHy technology. The data of Gas Chromatography showed that the cells that expressed the Fat-1 gene had a lower n-6/n-3 PUFA ratio compared with the cells that expressed the control vector.The proliferation of Tca8113 cells was determined by MTT assay and cell growth curve. The absorbance at 490 nm was measured by using an ELISA plate reader. The data showed that the proliferation of Tca8113 cells transfected with the pcDNA3.1-Fat-1 reconbinants was inhibited significantly, compared with normal cells and the cells that were transfected with the pcDNA3.1(+)vector and the inhibitory effeet at 72h was most significant. Then Fat-1 positive cells and control cells were stained by DAPI and detected by flow cytometry to measure the cell apoptosis. Data was collected from the two groups which showed that the expression of Fat-1 gene can induce cell apoptosis efficiently, but the control cells did not show the obvious apoptosis character.In total, through the three experiments presented above, we successfully constructed the pcDNA3.1-Fat1 reconbinants, and confirmed that it has significant effect on the PUFAs content of Tca8113 cells. Also, the data from MTT assay and flow cytometry obviously indicated the expression of Fat-1gene had a significant effect on cell proliferation and apoptosis of Tca8113 cells, which make a foundation in the future study on the internal mechanism of biological function of Fat-1 gene in human oral squamous carcinoma.