| Ovarian cancer is the most common malignancy in the female genital tract, and the epithelial ovarian cancer is the most common type of ovarian cancers. Because most of the patients had reached an advanced stage when they found they were sick, it' s difficult to cut all the cancer nests. At present, the most common treatments are operation, chemotherapy and radiotherapy, but all of these therapies have their limits, so the treatment of ovarian cancer is an important topic for us. Immunotherapy is under studying, its effect on tumor hasn' t be known exactly. This study indicates the alteration of PKC (protein kinase C) activity and alpha isoform, thus supply a pathway to know the mechanism of tumor genesis from the aspect of molecular biology and supply the theoretical basis for the use of PKC and its inhibitors on anti-tumor.Protein kinase C (PKC) is a family of calcium- dependent, activated by diacylglycerol (DAG), phorbol esters, and phospholipids serine/threonine protein kinases. PKC consists of at least 12 isoenzymes according to their structural and biochemical characteristics. PKC isoenzymes can be subdivided into three major groups. These isozymens also differ in tissue expression, subcellular localization,and substrate specificity. When protein kinase C (PKC) is activated by diacylglycerol(DAG), phorbol esters, and phospholipids, it will catalysis many proteins that have physiologic activity and involved in many physiologic process , such as intracellular signal transduction, secretion, cell proliferation, differentiation and cancer genesis and so on. Now, it has become the focus of many scientists in biologic and medical fields. At present, there hasn' t any report on the relationship of PKC and ovarian epithelial neoplasm in our country.Materials and MethodsMaterials1. Anti-PKC alpha antibody and the AKP-conjugated secondary antibody were purchased from Sigma.2. Harvesting of specimens.Normal ovarian tissues and the benign and malignant human epithelial ovarian neoplasm tissues were obtained from patients surgically treated by the Gynecologic Department of the First and the Third hospital in the China Medical University, each kind of tissues is 15. The patients in this study didn' t accept prior hormonal therapy, chemotherapy and radiotherapy. Tissues were collected at the time of surgery, placed in a Dewar flask containing liquid nitrogen, and stored at -70℃ until further analysis. Methods1. Protein fractionation of the cytosol and membrane in the tissues.All the operation of the experiment must be performed in 4℃. Tissues of the same weight were lysed using an ice-cold buffer which weighs five times to the specimens. Lysates were cut and then be sonicated on ice for cycles, centrifuged(13000 rpm.) for 1 hour and supernatants were collected as soluble fraction. The remaining pellets were resuspended in lysis buffer, and sonicated for cycles too. After centrifugation (13000 rpm. 4℃), supernatants were collected as particulate fractions.2. We detectived the concentration of the protein in the fractions of the cytosol and membrane in the tissues.3. PKC activityWe detectived the cpm of the specimens and then know the PKC activity by a certain formula.4. Analysis of PKC alpha expressionWestern blot analysis was performed in five independent series to know the PKC alpha expression in the epithelial ovarian cells. Equal amounts of protein were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane. The signal intensity of the bands was analyzed using Chemi Imager 5500. And the IDV which we got is in inverse to the protein quantitation.5. Statistical analysis.We used one-way ANOVA and SNK-q test to analysis the results by SAS6. 12 software. Statistical significance was indicated when P<0.01.Results1. Protein kinase C activityProtein kinase C activity in the cytomembrane and cytosol in the malignant ovarian epithelial cancer tissue is significantly higher t...
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