PubMed2000
OBJECTIVE: Bax gene is an important apoptosis-promoting gene. In order to investigate the expression of Bax in oral premalignant lesions and oral squamous cell carcinomas, a total of 38 samples are evaluated using a labelled streptavidin-biotin (LSAB) immunohistochemical assay. METHODS: A total of 38 specimens were studied, including normal oral mucosa, premalignant lesions and squamous cell carcinomas. The specimens were obtained and blocked, fixed with 10% buffered formalin and embedded in paraffin using conventional histopathological techniques. 3 microns-thick sections of paraffin-embedded tissues were cut, mounted onto slides coated with 5% APES (3-aminopropyltriethoxysilane), dried overnight at 56 degrees C, dewaxed in xylene and rehydrated through descending graded alchols to phosphate-buffered saline (PBS, pH 7.4). For antigen retrieval, slides were immersed in 10 mmol/L sodium citrate buffer (pH 6.0) and boiled twice for 5 min in a microwave oven (800 W). After treated for 10 min with 3% H2O2, 18% methanol in PBS, the slides were covered with 10% normal porcine serum for 10 min at 37 degrees C. Then slides were incubated with primary antibody (bax rabbit polyclonal antibody) for 60 min at 37 degrees C and were subsequently incubated with prediluted biotinylated antibody against rabbit immunogobulins, and streptavidinhorseardish peroxidase conjugate for 30 min at 37 degrees C. After washing, peroxidase activity was detected using 3,3'-diaminobenzidine as chromogen with H2O2 as substrate. The cells in the test specimens which demonstrated granular staining were considered as positive. A haemocytometer counter with 6. 6 framework were applied and only the positive cells on the cross were counted. The cell counting were processed in ten randomly chosen 400* microscopic fields and the mathematic mean was presented as the final counting of each sample. Statistical valuations were performed using the version 6.0 SPSS package. Positive controls were sections of bladder cancer tissues. Blank controls were fabricated for each specimen by the omission of the primary antibody, which was replaced with PBS. RESULTS: In the process of oral carcinogenesis, each stage had Bax expression. The positive staining appeared in cytoplasm. In the normal oral mucosal specimens Bax expression was evident in the prickle layer, but not in the basal cell layers. Various degrees of Bax expression were seen in the diseased tissues. The staining pattern of hyperkeratotic lesions was similar to the normal oral mucosa, but Bax expression were also seen in the basal cell layer. In the mild, moderate, severe dysplasia and squamous cell carcinomas, Bax expression were seen in all layers, however, the intensity of staining were greater in mild and moderate dysplasia. The number of positive cells tended to increase gradually with the development of cell malignancy in the tissues of hyperkeratosis, mild and moderate dysplasia (P < 0.05). In the tissue of squamous cell carcinomas the number of positive cells had no marked difference comparing with the normal oral mucosa. CONCLUSION: The expression of Bax is involved in oral carcinogenesis and the compensative increase of Bax protein expression may be an early response.