Zeng X, Chen Q, Li B, Tan N
College of Stomatology, West China University of Medical Sciences.
Hua Xi Kou Qiang Yi Xue Za Zhi. 2000 Apr;18(2):88-90.
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.
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.
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.
The expression of Bax is involved in oral carcinogenesis and the compensative increase of Bax protein expression may be an early response.
Bax基因是一种重要的促凋亡基因。为研究Bax在口腔癌前病变及口腔鳞状细胞癌中的表达情况，采用标记链霉亲和素-生物素（LSAB）免疫组化法对38份样本进行评估。
共研究38份标本，包括正常口腔黏膜、癌前病变及鳞状细胞癌。标本经获取、取材，用10%缓冲甲醛固定，采用常规组织病理学技术包埋于石蜡中。将石蜡包埋组织切成3微米厚的切片，置于涂有5% 3-氨丙基三乙氧基硅烷（APES）的载玻片上，56℃过夜干燥，二甲苯脱蜡，经梯度酒精复水至磷酸盐缓冲液（PBS，pH 7.4）。进行抗原修复时，将玻片浸入10 mmol/L柠檬酸钠缓冲液（pH 6.0）中，在微波炉（800 W）中煮沸两次，每次5分钟。用3% H2O2、18%甲醇-PBS处理10分钟后，玻片在37℃用10%正常猪血清覆盖10分钟。然后玻片在37℃用一抗（兔抗Bax多克隆抗体）孵育60分钟，随后用预稀释的抗兔免疫球蛋白生物素化抗体及链霉亲和素-辣根过氧化物酶复合物在37℃孵育30分钟。洗涤后，以3,3'-二氨基联苯胺为显色剂、H2O2为底物检测过氧化物酶活性。测试标本中显示颗粒状染色的细胞被视为阳性。使用带有6×6网格的血细胞计数器，仅计数交叉处的阳性细胞。在十个随机选择的400倍显微镜视野中进行细胞计数，每个样本的数学平均值作为最终计数结果。使用SPSS 6.0软件包进行统计学评估。阳性对照为膀胱癌组织切片。每个标本通过省略一抗并用PBS替代制备空白对照。
在口腔癌发生过程中，各阶段均有Bax表达。阳性染色出现在细胞质中。在正常口腔黏膜标本中，Bax表达在棘层明显，但在基底层未见。病变组织中可见不同程度的Bax表达。角化过度病变的染色模式与正常口腔黏膜相似，但在基底层也可见Bax表达。在轻度、中度、重度发育异常及鳞状细胞癌中，各层均可见Bax表达，然而，轻度和中度发育异常时染色强度更大。在角化过度、轻度和中度发育异常组织中，阳性细胞数量倾向于随着细胞恶性程度的发展而逐渐增加（P < 0.05）。在鳞状细胞癌组织中，阳性细胞数量与正常口腔黏膜相比无明显差异。
Bax的表达参与口腔癌发生，Bax蛋白表达的代偿性增加可能是一种早期反应。
