Establishment of A Novel System for Malignancy Grading of Oral Cancer Using Molecular Biological Technique

为了研究口腔鳞状细胞癌（OSCC）原发部位的肿瘤细胞和转移的肿瘤细胞之间的差异，我们尝试使用独立于患者的原发肿瘤和转移肿瘤建立细胞系。此外，还对来自同一患者的口腔粘膜的正常人角质形成细胞（NHK）进行细胞系建立，其用作理想的对照细胞。在1993-1996年成功建立的5个细胞系中，在无蛋白培养基中同时建立了口腔鳞状细胞癌（OSCC）、口腔鳞状细胞癌（NHK）和正常角质形成细胞（NK）的原代细胞系、转移细胞系和正常角质形成细胞系，用PCR方法对OSCC和NHK细胞的转录本进行了差异显示，以寻找OSCC和NHK细胞特异表达的基因。我们报道了一个蛋白质，该蛋白质在口腔鳞状细胞癌（OSCC）和NHK细胞中的表达，并与正常角质形成细胞系的表达进行了比较。一种能识别未知血管内皮细胞的单克隆抗体， 关于我们 PKC eta是已知在上皮细胞中表达的PKC的亚型。为了了解细胞的生物学行为，使用腺病毒载体在上皮细胞中过表达PKC eta。结果发现，12-O-十四烷酰佛波醇13-乙酸酯（TPA）可抑制上皮细胞的生长，而成纤维细胞在相同条件下不受抑制，提示可能的基因治疗OSCC。我们以前报道过，p53抑癌基因在OSCC中经常发生突变，并被认为与该肿瘤的发生有关。为了了解突变的作用，在口腔鳞癌中发现的p53突变体进行了检查转染试验。第138位密码子的瓦尔突变体为温度敏感突变体。为建立p53基因突变的分子诊断体系，对p53基因突变的基因状态、临床过程和组织学恶性程度进行了详细的比较。虽然p53基因突变频率很高，但我们没有发现突变的存在与临床或组织学恶性程度之间有明显的相关性，提示p53基因突变在口腔鳞癌的发生中起重要作用。少

To investigate the difference between tumor cells of the primary site and metastasized tumor cells of oral squamous cell carcinoma (OSCC), we tried to establish cell lines using primary and metastasized tumors independently from a patient. In addition, normal human keratinocytes (NHK) from oral mucosa of the same patient was also subjected to cell line establishment, which served as ideal control cells. In 2 out of 5 cell lines, which were successfully established between 1993-1996, the primary and metastasized tumor cell lines and normal kratinocyte cell lines were simultaneously established in the protein free media.Transcripts from OSCC and NHK cells were differentially displayd by PCR method using sets of arbitrary primers to search for genes specifically expressed in OSCC or NHK cells.We reported a protein which was expressed by one of the OSCC cell line and was found to suppress the growth of lymphocytes.In addition, a monoclonal antibody, which recognize an unknown vascular endo … More epitherial protein, were prepared.PKC eta is a subtype of the PKC is known to be expressed in the epitherial cells. To understand the cell biological behavior, PKC eta was overexpressed in the epithelial cells using an adenovirul vector. It was found that the treatment by the 12-O-tetradecanoylphorbol 13-acetate (TPA) suppressed the growth of epithelial cells, while fibroblasts were not suppressed under the same condition, suggesting a possible gene therapy of OSCC.We previously reported that p53 tumor suppressor gene is frequently mutated in OSCC,and is considered to be responsible for the development of this tumor. To know the role of the mutation, the p53 mutants found in OSCC were examined by transfection assays. A mutant with Val of codon 138 were found to be a temperature sensitive mutant. To establish a system of molecular diagnosis using the p53 gene mutations, the status of the gene, clinical process and histological malignancy grade are compared in detail. Although the mutation frequency was so high, we could not found an obvious correlation between the existence of mutation and clinical or histological malignancy grade, suggesting that the p53 mutation is important in the initiation of OSCC. Less