| Backgrounds and ObjectivesThe gastric cancer is one of the most common malignant tumor in the world, being characteristic of high incidence,high mortality,low early diagnosis rate and low survival rate.Nowadays, the main therapie of the developmental stomache cancer was surgery, accompany with chemotherapy.But the questions were low efficiency, high recurrence rate and durg resistance.So that to find a new way of less side effects and better therapiey effect is imperative. With the deepgoing study of the mechanism of the gastric cancer, it is improved that the progress of the gastric cancer have various procedures and phages, and involve in many factors. And the variation of oncogenes and the signal transduction are on intimate terms with the development of the gastric cancer. The way of protein-tyrosine kinase receptors is the most important conduction ways of the signal transductions.This is exactly the first link of the signal transductions of the hepatocyte grouth factors and their receptors.The protein encoded by protooncogene c-Met which belongs to the family of protein-tyrosine kinases(PTKs), is the Specificity receptor of Hepatocyte Growth Factor(HGF). The study shows that c-Met express continue abnormality greatly in stomach carcinoma. Abnormal expression of c-Met can activate downstream molecules to induce the multiplicative, division, adhesion and imbibition of the tumor cells and the formation of the tumor blood vessels. It plays an important role in the growth development and transferance of the stomach carcinoma.The RNA interference can introduce the dsRNA,which is homology complementary with the target gene,into the cells to specific degrade this mRNA,and furthering lose the corresponding functionality phenotype.It pertain topost-transcriptional gene silence(PTGS). The RNA interference can lead to specific and effective gene silence,and restrain definite gene with high efficiency,which have rapid and extensively application in identification of gene function, expression and regulation after transcription.It provid new thread to the gene therapy of many diseases, especially in gene therapy of tumor.In this study,we use the RNA interference to inhibit the expression of c-Met gene. Compare the expression of mRNA and protein of c-Met gene and the generation ability of stomach carcinoma cell SGC-7901 before and after transfection.Investigate the influence of c-Met gene silence using RNA interference on the cytobiology of stomach carcinoma, providing new methods and threads for the therapy of stomach carcinoma.Materials and Methods1 Human stomach carcinoma cell SGC-7901 was bought from Academia Sinica Shanghai life sciences cell bank. Cultivate the cells with the RPMI1640 complete culture media containing 10%fetal bovine serum in the 37℃,80%moisture,5%CO2 saturated humidity incubator. According to the c-Met cDNA sequence to design and synthesis the sequence of 3 target gene by Shanghai GenePharma company.2 Transfect the c-Met-siRNA into the Human stomach carcinoma cell SGC-7901 mediated by Lipofectamine TM 2000.Meanwhile, install the blank and empty vector group. Detect the transient transfect efficiency under the inverted Fluorescence microscope.3 48h after the transfection, observe the morphology of each group under the inverted microscope.Detect the c-Met mRNA and protein by RT-PCR and Western blot respectively.Detect the generation activity of each cell group using MTT method and draw the growth curve. Results1. The RT-PCR and Western blot showed that the expression level of c-Met mRNA and portein was obviously lower than the control group.2.The cell generation and the cell number was decreased while the cell aggregation was inhibited in the transfected cells 48h after the transfection observed under the inverted microscope.3.The results of MTT showed that there are difference (p<0.05) between the transfected cells and the control cells in the OD of the time 24h,48h,72h,96h after transfection. The aggregation of experimental group was apparently attenuated, significantly in the 48h when the inhibition ratio of the reproductive activity was >50%.Conclusions1. Successfully transfect the c-Met-siRNA into the Human stomach carcinoma cell SGC-7901 by Lipofectamine TM 2000.2.After transfection,the mRNA and protein expression and the generation ability of experimental group were inhibited greatly.3. The RNA interference can sucessfully inhibit the exprssion of c-Met gene in Human stomach carcinoma cell SGC-7901, providing foundation of the stomach carcinoma therapy.And this technique will be a hoping new strategy in the stomach carcinoma gene therapy. |
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