| Background Gastric cancer is most commen malignant tumor of digestivetract.because of low defect-discovery rates, most of clinical gastric cancer is atadvanced stages.It cannot be cured once and for all with the surgery,and soChemotherapy has become one of the main treatment for recurrence and metastasis ofadvanced gastric cancer.But Multidrug resistance generation lead to the effect ofchemotherapy is far from ideal, and even it has became the major cause of failure oftreatment of gastric cancer. Studying for the MDR mechanism of the phenomenon andto overcome MDR is one of the main ways to improve the effect of chemotherapy,itMay cause a breakthroug for the tumor effect of chemotherapy. Since the proteomicsbirth and Proteome Project implementation, Proteomics research has brought new waysof thinking and research areas for the tumor MDR research. It is from this new point ofview of the organization or the overall level of cellular proteins to study tumor MDRmechanisms, this will may lead to new breakthroughs in cancer MDR mechanisms.The group had applied proteomics technology on proteomics of human gastriccancer cell line SGC7901, doxorubicin resistance cell strains SGC7901/ADR, andSalvia reversed after the drug-resistant cell lines differences, HPLC-ESI-MS/MS hadalso been applied to identify the differentially expressed proteins. The results showedthat high expression of S100A6in human gastric cancer cell line SGC7901, expressedin multidrug resistant cell line SGC7901/ADR down, no or low expression, Upregulatedafter the Salvia reversal of multidrug resistance.After this, through the use ofimmunocytochemistry, Western-blot analysis, RT-PCR,it was further verified that thisdifference in expression, and found that S100A6in these two cells there was nodifference in mRNA expression. To study the mechanism of this phenomenon, theexperiment will apply Gene transfection techniques to introduce the plasmids whichcontain the S100A6gene into SGC7901/ADR cells. By Immunocytochemical methods and RT-PCR, S100A6expression will be detected in SGC7901/ADR of non-transfectedand transfected.It may lay the foundation for the further study of the mechanism ofgastric cancer MDR.Objective Respectively detect the differential expression of S100A6inSGC7901/ADR of non-transfected and transfected,further investigate the mechanism ofS100A6downregulation in SGC7901/ADR cells.Methods Transforme the plasmids Containing the S100A6gene into theprepared DH5α competent E. coli, then extracte the plasmids whith E.Z.N.A.TMPlasmid Mini Kit I. Subcultured human gastric cancer doxorubicin resistant cell lineSGC7901/ADR cells, apply Gene transfection techniques to introduce the plasmidswhich contain the S100A6gene into SGC7901/ADR cells. By the application of cellcrawl-chip technology, make the subcultured SGC7901/ADR and transfectedSGC7901/ADR cells grow in6-well plates pretreated coverslip. Culture for24hours ormore, rapid immunohistochemistry method was adopted to observe the differentialexpression level of S100A6in the two kinds of cells at the protein level. Extracte thetotal RNA in the non-transfected and transfected SGC7901/ADR cells respectively,semi-quantitative RT-PCR was used to detect the expression of S100A6at mRNAlevels in the two cells. And then analyse the possible causes that S100A6expressionwas decreased in SGC7901/ADR cells.Results Immunocytochemistry method detected that S100A6is showed positiveexpression of brown color in non-transfected SGC7901/ADR cytoplasm and alsoshowed positive expression of brown color in transfected SGC7901/ADR cytoplasm.Semi-quantitative RT-PCR result: Extracte the total RNA in the non-transfected andtransfected SGC7901/ADR cells respectively,then Semi-quantitative RT-PCR, theRT-PCR product was subjected to1.5%agarose gel, detecte the gene straps IOD,thenon-transfected SGC7901/ADR mRNA IOD x:1.92e5,the transfected SGC7901/ADRmRNA IOD x:3.07e5, the average expression amount of S100A6mRNA innon-transfected SGC7901/ADR is0.426±0.027,in transfected SGC7901/ADR is0.530±0.007, T-test was adopted to analyze the differential expression,P<0.05.Conclusions1. The plasmids had been transfected in SGC7901/ADR canamplify the Gene of S100A6.The expression of transfected SGC7901/ADR S100A6mRNA is significantly higher than non-transfected SGC7901/ADR.2. There is no differential expression at S100A6protein expression levels in thenon-transfected and transfected SGC7901/ADR cells. 3. The phenomenon that S100A6elevated at mRNA levels and it do not matchwhith the protein expression levels May be related to protein degradation mechanisms. |
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