Experiment Study Of CyclinE Expression In Osteosarcoma And Giant Cell Tumor Of Bone

PrefaceStudies based on molecular biology indicate that tumorigenesis is a complex procedure involved multi - gene mutation. Cell cycle regulators of all phases can be involved, especially Gl phase regulators. CyclinE is a pivotal regulator in Gl phase. In many human tumors, cyclinE overexpressed, but it is still unclear about the mechanism of tumorigenesis involved cyclinE. CyclinE forms a complex with cyclin dependent kinase 2 ( CDK2) before S phase. This complex has kinase activity and can activate retinoblastoma protein ( pRB) by phosphoryl-ating action. Hyperphosphorylated pRB then releases bound transcription factors of the E2F family to cause DNA synthesis, this is Gl phase CDKs/pRB/E2F path. CyclinE is both a regulator and a target of the E2F transcription family in an autoregulatory loop that is necessary for progression from the Gl to the S phase of the cell cycle. Geng Y demonstrated that CDKs/pRB/E2F path can make a connection between cellar external sign and the activity of cyclinE in the evolutional progression of multi - cell biome. This path can control the cell cycle through the expression of cyclinE, and it is suggested that this path is destroyed in almost every human tumor. So cyclinE play an important role in proliferation of malignant cells.Overseas authors have done a lot of job in the relationship be-tween cyclinE expression and tumor' s prognosis and histological type. The results suggested that cyclinE play a central role in tumorigenesis. There are hardly any articles reporting the expression of cyclinE in os-teosarcoma and giant cell tumor of bone either abroad or inland. Im-munohistochemical method was used in this study to investigate the expression of cyclinE in osteosarcoma and giant cell tumor of bone so as to analysis its role in genesis and development of both tumors and provide experimental data for molecular biological diagnosis and prognostic prediction.MaterialA. Tissue samples: The paraffin embedded specimens were obtained from the pathological department of The First Clinical College of China Medical University between 1992,1 and 2001,7. Specimens included 26 osteosarcomas, 34 giant cell tumors of bone and 22 osteo-chondromas. The diagnosis of each case was confirmed by three pathological experts according to hematoxylin and eosin (HE) stained sections. According to Jaffe classification criteria, there are 15 cases giant cell tumor of bone of grade I , 15 cases of grade II , 4 cases of grade Iff.B. Reagent: Mouse monoclonal antibody against cyclinE and streptavidin peroxidase ( SP) agent box were obtained from Maxim Biotech Inc.MethodA. All specimens were cut into 5um thick sections and mounted on glass slides. A serial section from each specimen was stained with HE for histological evaluation.B. Immunohistochemicai staining: The sections were deparaf-finized in xylene and rehydrated in ethanol, the sections were heated in citrate buffer at 92 - 981 for 150 seconds to retrieve antigen. Next, endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 minutes. After the sections were incubated with normal goat serum to reduce nonspecific binding, they were treated with anti- cyclinE antibody (first antibody) at 4C overnight. Then applied biotinylated second antibody goat anti - mouse IgG and HRP - strepta-vidin (third antibody) at 37C for 30 minutes respectively. The sec-dons were baptized three times by PBS during the interval between each step after retrieving antigen, 5 minutes each time. Staining was visualized with diaminobenzidine tetrachloride ( DAB). We use the known positive section as positive control, PBS instead of first antibod-y as negative control.C. Result judgement: OLYMPUS biomicroscope was used to observe the stained sections. A cell was regarded as positive if the nucleus was stained clear brown. For evaluation, ten fields were selected and a total of 1000 tumor cells were counted. The results of cyclinE expression were graded as: Less than 25% positive stained is ( + ) ,...