| Aim:COX is the rate-limiting enzyme in the synthesis of PCs from arachidonic acid. COX-2, one of the isoforms, has been described. Glioma is the main tumor of brain tumor. The up-regulation of cyclooxygenase-2 (COX-2) expression is a frequent occurrence in a variety of different tumors. To evaluate the expression of COX-2, VEGF and factor VIII in human gliomas and normal brain tissues and demonstrate whether or not there is a relationship with tumor phenotype in this study. Material and Methods:1. MaterialSixty two tissue specimens were obtained from patients with astrocytomas in the first affiliated hospital, Zhejiang University school of Medicine. Three normal brain tissues were obtained from the other patients as the control group. There were 37 male and 25 female patients for astrocytoma groups with an age ranged from 4 to 71 years(mean 38.12 years). The tumors were diagnosed as low-grade astrocytoma in 19 cases, anaplastic astrocytoma in 20 cases, and glioblastoma in 23 cases according to the World Health Organization criteria(1990).2. Western Blot Analysis for COX-2Western blot analysis was performed as described previously to detecte COX-2 protein in three normal brain tissues and nine human gliomas. Briefly, cell lysates (50 ng/lane) wereseparated on 12% SDS polyacrylamide gel and then were electrophoretically transferred to a polyvinylidene difluoride membrane. COX-2 protein was detected by a mouse monoclonal IgG and visualized by the enhanced chemiluminescence system. 3. Immunohistochemical staining3. 1 Paraffin tissue sections (4-6 urn) of tumor and normal brain specimens were used for COX-2 immunohistochemical studies. Slides were deparaffinized, and endogenous peroxidase activity was blocked by incubation in 1% H2O2 in PBS for 30 min. Antigen retrieval was performed by incubating slides in 10 mM sodium citrate at 100癈 in a microwave for 1 5 min. A mouse monoclonal antibody against human COX-2 was then applied at a dilution of 1 :300 overnight at 4癈, rabbit polyclonal antibody against human VEGF (1:50 dilution ), and rabbit polyclonal antibody against human FVIIIRAg (1:200 dilution). After rinsing with PBS, the biotinylated secondary IgG antibody was applied for 30 min at room temperature. Immunoperoxidase staining was performed using streptavidin-biotin-peroxidase method(S-P method), and sections were counterstained with hematoxylin.3.2 As an additional negative control, primary antibody was omitted. Tissue from a human colon carcinoma known to overexpress COX-2 was used a positive control.3.3 The percentage of positive tumor cells was determined semiquantitatively by assessing the whole tumor section, and each sample was assigned to one of the following categories: 0 (0-4%), 1 (5-24%), 2 (25-49%), 3 (50-74%), or 4 (75-100%). The intensity of immunostaining was determined as 0 (negative), 1+ (weak), and 2+ (strong). Additionally, an immunoreactive score was calculated by multiplication of the percentage of positive cells and the staining intensity, as proposed by Krajewska et al. For heterogenous staining patterns, each component was scored independently and the results were summed. For example, a specimen containing 25% tumor cells with strong intensity (1 x 2+ = 2), 25% tumor cellswith weak intensity (1 x 1+ = 1), and 50% tumor cells without immnoreactivity received ascore of 2 + 1 + 0 = 3.3.4 SPSS 10.0 statistical software was applied and the correlations between the levels ofCOX-2 & VEGF expression and the malignancy or MVD were considered to be significantat a probability value of less than 0.05.Results:1. western blot analysisExpression of COX-2 are expressed in three normal brain and nine glioma specimens, regardless of histological grade.2. Immunohistochemical staining2. 1 The relationship between COX-2 expression and tumor malignancy hi normal brain tissues COX-2 expresed in the neuron. In the low grade astrocytoma, COX-2 is low and the tumor cells are mainly light-stained. COX-2 are strongly expressed in glioblastoma. C... |
PDF Full Text Request