| Background: Gastric cancer is one of common malignant tumors in China, and the incidence rate is the top among different types of cancer in China. Clinical treatments are surgery, radiationtherapy, chemotherapy. Chemotherapy has its own unique in eradication of cancer, or preventing recurrence of distant metastasis, and cancer treatment is an indispensable component. Chemotherapy is more and more widely used in cancer therapy, but due to the production of multidrug resistance (Multi-drug Resistance, MDR), the effect is often poor, severely affected the quality of life of patients. MDR mechanism of malignant is complex, but proteomics for studying the phenomenon of MDR tumors provides a new way of thinking and research. In the early 90s the implementation of the Human Genome Project began, and with the gradual human genome project completed, the scientists made after further Genome Project. Proteomics is a very important content to provide a new platform for life science research, which study new mechanism of tumor MDR from the view of overall level of the protein.Those days ,our group applied two-dimensional gel electrophoresis (Two-dimensional Gel Electrophoresis, 2-DE) technology separate effectively and show the whole protein of SGC7901, SGC7901/ADR and SM SGC7901/ADR cells . Find 14 kinds of proteins which were closely related to MDR in the 2-DE map, and 7 downregulated proteins were identified by high performance liquid chromatography electrospray ionization tandem mass spectrometry flight in SGC7901/ADR cells. The results show that Myotrophin is of 118 amino acids, and its theory is Mw12.9KDa, and is transcriptional regulation cell factor. In recent years, the gene structure, biological function, the mechanism in some diseases of Myotrophin are in-depth research, but the relationship between Myotrophin and MDR mechanism has not been reported.To further investigate the relationship of MDR between Myotrophin with gastric cancer ,and clarify the mechanism of gastric cancer.This study uses technologies of immunocytochemistry, Western-blot and RT-PCR to detect the expression level of Myotrophin in SGC7901 and SGC7901/ADR cells.Objective: Verify and detect the differential expression of Myotrophin in human gastric cancer cell line SGC7901 and SGC7901/ADR, and deeply explore the role of Myotrophin in the development of MDR in human gastric cancer.Methods: Human gastric cancer SGC7901 cells and SGC7901/ADR cells were inoculated on cover slips placed in 6-well plates in normal culture over 24 hours, immunocytochemistry MaxWisinTM method staining Myotrophin in the expression of two different cell lines. Dilution of rabbit polyclonal anti-human antibody was 1:50, and compared the results. Chemistry lyses methods extracted the total cellular protein, and measured by Coomassie brilliant blue method of protein concentration to analyze the results from the protein level in the cell line SGC7901 and SGC7901/ADR. The total RNA was extracted by RNAiso Reagent lysates from two kinds of cells, using RT-PCR to analyze the expression of Myotrophin in cells from the mRNA level in the cell line SGC7901 and SGC7901/ADR.Results: 1. Immunocyrochemistry results: Myotrophin in SGC7901 cells was high, dyeing as strong positive (+ + +). Hyper chromatic in cell cytoplasm and nucleus was tan-yellow-brown. Expression of Myotrophin in SGC7901 / ADR cells dropped, dyeing as weak positive (+).Cell cytoplasm and nucleus were hyper chromatic dyed pale yellow.2. Western blot results: Extract the whole protein of SGC7901 and SGC7901 / ADR cells, and make use of Coomassie brilliant blue to determinate protein concentration. The full protein of concentration in SGC7901 was 3.23ug/ul, while the concentration in SGC7901 / ADR was 3.16ug/ul. Use SDS-PAGE electrophoresis, western-blot immune protein-mark experimental method and make use of the gel quantitative software Quantity One and SPSS Statistics software to find that the average amount of Myotrophin protein in SGC7901 was 0.707±0.035, and0.24±0.07 in SGC7901 /ADR. Applying t-test of two independent sample mean to do statistical analysis, which showed that P = 0.02, P < 0.05, so there was statistical significance and had significant difference. Expression of Myotrophin in SGC7901 was high, while in SGC7901 / ADR cells declined. 3. RT - PCR results: Extract total RNA of SGC7901 and SGC7901 / ADR, and detect total RNA concentration of two cells with ultraviolet absorption spectrometry. Apply experimental methods of semi-quantitative RT-PCR agarose gel electrophoresis, and the gel quantitative software Quantity One and SPSS Statistics software. The results showed that the average total amount of Myotrophin genes mRNA in SGC7901 was 0.988±0.022 ,and 0.865±0.011 in SGC7901 / ADR cells. Do two independent sample t-test statistical analysis, P =0.04, P<0.05. So there was statistical significance and had significant difference. Expression of Myotrophin mRNA in SGC7901 was high, while in SGC7901 / ADR cells declined.Conclusions: 1. Expression of Myotrophin is different in SGC7901 and SGC7901/ADR cells. Express high in SGC7901 and the expression in SGC7901/ADR cells declines.2. The mRNA of Myotrophin in the human gastric cancer cell lines SGC7901 and SGC7901 / ADR has different expression, which is high in SGC7901 and low in the SGC7901/ADR cells. The difference between SGC7901 and SGC7901/ADR cells of expression may be caused by the reasons that the process of transcription is repressed, or a partly degradation of mRNA happens. The reasons result in a reduction of mRNA.3 .The different expression of Myotrophin in SGC7901 and SGC7901/ADR cells may be related with gastric cancer MDR. |
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