CORE--CELL CULTURE

核心--细胞培养

• 批准号: 6300865
• 负责人: JAMES RHEINWALD
• 金额: $ 18.13万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6300865
• 关键词: athymic mouse; biomedical facility; cell growth regulation; cell line; human tissue; keratinocyte; mouth neoplasms; neoplastic growth; oral pharyngeal neoplasm; squamous cell carcinoma; tissue /cell culture; transfection

Conditions have been optimized for growing in culture human oral squamous cell carcinoma (SCC) cells and normal oral mucosal keratinocytes, the cell type from which this cancer originate. Previous studies, including those by the Cell Culture Core director, have identified a number of growth and differentiation control systems in normal keratinocytes, assayable in culture, that are typically defective in advanced oral SCCs. Substantial variability with respect to p53 mutations, cyclin D1 and cdk4/6 expression, and integration of papillomavirus DNA sequences has been noted among SCCs from different patients. Progress to identify the specific regulators of cell cycle control and gene transcription that become inactivated or over expressed by mutation and other genetic events at each stage of neoplastic progression in this accessible and clinically important cancer has been hampered by lack of availability of representative, well-characterized normal and neoplastic human oral epithelial cell lines, and by the complexity of methods for culturing and generating stable experimental gene transfectants of them. Identification of early genetic and phenotypic changes during oral cancer development is especially important, but few cell lines from pre-malignant lesions and early stage cancers have been cultured and studied. The Core Director's experience in this field, and his extensive collection of normal and malignant oral cell lines are unique and valuable resource for the Program Project. Of special importance are six cell lines from dysplastic oral lesions and three from early invasive SCCs which we have recently cultured. Several of these exhibit properties in culture intermediate between normal keratinocytes and advanced SCC cells, consistent with the acquisition of early neoplastic genetic alterations. We will culture more lines from oral lesions and early cancers and characterize their in vitro phenotypes such as replicative life span, mitogen and growth inhibitor, sensitivities, and histogenic potential, and we will assess their tumorigenicity in athymic mice. We will culture oral keratinocytes from cyclin D1- over expressing transgenic mice and from dysplastic lesions and SCCs that arise in these animals. We will make stable transductants of normal and pre-neoplastic oral keratinocytes and SCC cells to over express cdk6, cyclin D1, doc-1, and oral HPV E6 and E7 proteins. These cells will be used by project investigators to test key hypotheses.

条件已被优化，以在人类口腔鳞状文化中生长 细胞癌（SCC）细胞和正常口服粘膜角质形成细胞，细胞 这种癌症起源的类型。以前的研究，包括 通过细胞培养核心主管确定了许多增长和 正常角质形成细胞中的分化控制系统，可在 培养物，通常在晚期口服SCC中有缺陷。重大的 关于p53突变，细胞周期蛋白D1和CDK4/6的变异性 已经注意到乳头瘤病毒DNA序列的表达和整合 在来自不同患者的SCC中。进步以确定特定的 细胞周期控制和基因转录的调节剂 在每个突变和其他遗传事件中灭活或过度表达 在该可访问和临床上的肿瘤进展阶段 由于缺乏可用性，重要的癌症受到了阻碍 代表性的，良好的正常和肿瘤性人口腔 上皮细胞系，以及通过培养和 产生稳定的实验基因转染剂。鉴别 口腔癌发展过程中的早期遗传和表型变化是 尤其重要，但几个细胞系带有临床前病变和 早期癌症已经进行了培养和研究。核心导演的 在这个领域的经验，以及他广泛的正常收藏和 恶性口腔细胞系是该程序独特而宝贵的资源 项目。特别重要的是来自发育不良口服的六个细胞系 病变和三个来自我们最近有的早期侵入性SCC 培养。其中几种在培养中间体中表现出特性 在正常角质形成细胞和晚期SCC细胞之间，与 早期肿瘤遗传改变的获取。我们将更多地文化 口腔病变和早期癌症的线条，并表征其体外 表型，例如复制寿命，有丝分裂原和生长抑制剂， 敏感性和组织生成潜力，我们将评估它们 无胸腺小鼠的肿瘤性。我们将从 细胞周期蛋白D1-超过表达转基因小鼠，发育不良病变和 这些动物出现的SCC。我们将制造 正常和肿瘤前的口服角质形成细胞和SCC细胞过度表达 CDK6，Cyclin D1，DOC-1和口服HPV E6和E7蛋白。这些细胞会 项目调查人员可用于检验关键假设。