| PREFACEThe pathogenesis of gastric cancer is a process of multiple steps and many stages, which concerned with oncogene, tumor suppressor gene(TSG) , mismatch repair gene (MMR) , telomere and telomerase, cellular adhesive factors etc. PTEN is a newly found TSG which located in 10q23. 3, PTEN is the TSG which possessed the activity of phosphatase. Frequent LOH and mutations of this gene have been found in various types of cancer, Our purpose was to investigate the LOH and mutations of PTEN in gastric cancer and precancerous lesions. Another gene variation type in the molecular mechanism of tumor was the inactiva-tion of DNA mismatch repair gene, It mainly expressed as microsatellite instability, gastric cancer has high incidence of MSI , Our purpose was to investigate the changes of MSI during the process of gastric cancer, with the same microsatellite marker and the same judging standard. Due to the long poly A repeat units in exon 7 and 8 of PTEN, we supposed that PTEN can be the target gene of MSI, Our purpose was to investigate the relationship between PTEN and MSI.MATERIALS and METHODSMATERIALS1. cases . Precancerous lesion samples were collected from endoscopy center of China Medical University from 2001 - 2002, Early and advanced gastric cancer samples were collected from the oncology department of China Medical U-niversity after operation . Tissue samples were collected fresh, snap frozen in liquid nitrogen, and kept frozen at -70℃2. Primers of PTEN LOH: D10S 215 , D10S541, D10S2491 and primers of MSI: BAT26, D2S123, D5S346, D17S799, D18S34 were from WWW. gdb. org. Primers of exon 5 and 8 were got from documents .3. Taq polymerase, buffer, dNTP, marker, and marasmatic materials were bought from TAKARA corporation, DaLian.4. S - P immunohistochemistry Kits and mouse - human monoclonal antibody (1;200) were supplied by MaiXin technique corporation, FuZhou.5. Instruments: 2400 PCR amplifier, BIO - RAD vertical electrophoresis bath were supplied by PE corporation, America. Agarose electrophoresis apparatus; Beijing analytic instrumental factory. TG - 16 table centrifuge: SHang-Hai analytic instrumental factory. Thermostate water bath : Nanjing chemical instrumental factory.METHODS1 . DNA were extracted by the method of saturated sodium chloride.2. PTEN LOH:PCR - SSCP denaturing PAGE gel electrophoresis and sliver staining .20μ1 PCR mixture contained 200ng of genomic DNA, 0.4μM of each primers, 10μbuffer 2μl, 1.5mmol/L Mg2+, dNTP200mM, TaqDNA polymerase 1 unit. PCR was carried out over 35 amplification cycles for 45s at 94℃, 45s at 55℃, 60s at 12℃, predegeneration 5 min at 95℃ and prolongation 10 min at 72℃ after amplification. PCR products were resolved on a 16% denaturing polyacrylamide gel and silver staining .3 . Mutations detection via PCR - SSCP sequencing .20μ1 PCR mixture contained 200ng of genomic DNA, 2.5μM of each primers , 10μbuffer 2μ1, 2.0mmol/L Mg2+ , dNTP200mM, TaqDNA polymerase 1 unit. PCR was carried out over 35 amplification cycles for 45s at 94℃, 30s at 58℃, 60s at 72℃, predegeneration 5 min at 95℃ and prolongation 10 min at 72℃ after amplification. PCR products were resolved on a 12% nondenaturing polyacrylamide gel and silver staining. The shifted SSCP bands were sequenced.4. MSI detection: PCR - SSCP denaturing PAGE gel electrophoresis and sliver staining. The detail was the same as 2.5. Immunohistochemistry of PTEN protein .S - P immunohistochemistry . Judgment of the results : PTEN positive cells were defined as brown granule deposited in the plasma . We selected 5 high visual fields , calculated the percentage of the positive cells: (-) , <5%; ( + ) , 5%-25% ;(++), 25%-50% ;(+++), >50%.Statistical AnalysisAll the data were showed as X+SD, t test was used to compare the difference, Grade data was analyzed by Spearman.RESULTS1. PTEN LOHThe percentage of PTEN LOH in gastric cancer and precancerous lesions were seen table 1 .Table 1 PTEN LOH in gastric cancer and precancero...
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