Expression Of Caspase-9, Cytochrome C And Bcl-2 In Gastric Carcinomas And Their Relationship

Apoptosis malfunctions have very important in the pathogenesis of many Human malignant tumors, caspase - 9, Cytochrome c and Bcl - 2 etc. turner gene product participate in the regulation of cell apoptosis. Gastric carcinoma is a common malignant tumor of the digestive system. The etiology is unclear. It is suggested Apoptosis malfunctions resulting that cell normal death reduces is associated with the development of tumor. There are only a few reports are about caspase - 9 and gastric carcinoma. The relationship between the expression of Cytochrome c and Bcl - 2 is unclear. We detect the expression of Cytochrome c in gastric carcinoma and its relationship between Cytochrome c and Bcl -2. And we talk about its meaning. Therefore, Cytochrome c may provide a new adjuvant therapeutic method for gastric carcinoma.Materials and Methods1. Patients and specimen:Pancreatic carcinoma specimens were selected from paraffin wax embedded specimens with full clinicopathological data. All the patients underwent operations in the 2nd affiliated hospital of China Medical University and the postoperative pathologic diagnosis was gastric carcinoma from year 2001 - 2003 All the patients havent accepted radio or chemical therapy.2. Main reagents;Anti - caspase - 9, Anti - Bcl - 2, Anti - Cytochrome c monoclonal antibody and immunohistochemistry S -P kits (Zhongshan Corp) , Goat anti -rabbit IgG marked by Biotin ( Maixin Corp. ).3. Immunohistochemical staining:Specimens from the paraffin wax blocks were cut into 5um sections. The first section was routinely stained with hematoxylin - eosin for histological diagnosis and differentiation. Immunohistochemical staining for caspase - 9, Bcl -2, and Cytochrome c was performed with a standard avidin - biotin - peroxidase complex detection kit. The sections were dewaxed and rehydrated; antigen repair 1.5 min through high temperature and pressure method with 0.01M PH6.0 citrate buffer solution. Endogenous peroxidase activity and nonspecific bindings were blocked by incubation with 3 % hydrogen peroxide and non - immune serum, respectively. Slides were then incubated sequentially with primary antibodies overnight at 4C with a biotinylated secondary antibodies for 30 min and with SP complex for 30 min. Then the chromogen DAB test was performed to be able to localize the positive staining by microscope sequentially. The sections were counterstained with hematoxylin, dehydrated in graded ethanol, cleared in xy-lene and mounted. Positive staining of breast cancer was used as a positive control. Negative control sections were prepared by substituting primary antibodies with PBS.4. Evaluation of the results:The positive staining was defined as light yellow to brown stain in cytoplasm or nuclei. Locate the area where the density of positive cells are the most ,count 5 HPF in this area ( * 400) ,the percent of positive cells of 1500 cells is desiged as<5%-- (), >5%-- (+)5. Statistics analysis:Statistical significance was calculated by the x2 test. The level of significance was set at P <0. 05.ResultsThe expression of caspase - 9, Cytochrome c and Bcl - 2 in gastric carcinoma:By immunohistochemistry, the expression of caspase - 9, Cytochrome c protein was detected in the cytoplasm of nomal cells, big or small yellow paticle canbe detected. The expression of Bcl - 2 gene was detected in the cytoplasm of turmor cells, big or small yellow paticle can be detected too. In gastric carcinoma and carcinoma - around tissue, the positive rate of caspase -9 was 27. 8% and 61.1% respectively, the positive rate of Cytochrome c was 37. 0% and 79. 6% respectively, the positive rate of Bcl -2 was64. 8% and 25. 9% respectively. In gastric carcinoma and carcinoma - around tissue , the expression of caspase - 9, Cytochrome c and Bcl - 2 is different. The difference was significant (P<0.01).The correlation between expression of caspase - 9, Cytochrome c and Bcl -2 in gastric carcinoma and clinicopathologic factors;There was no significant association between th...