Human Chondrocyte Cells in Continuous Culture

描述（由申请人提供）：该计划的主要目标是延长正常人类软骨细胞培养的寿命，以发展连续的细胞系，用于研究社区和组织工程。现有的50种软骨细胞菌株可供研究。人类端粒酶逆转录酶（hTERT）组成表达的结果将被确定。在逆转录病毒表达系统pLXSN中克隆了hTERT的人cDNA克隆，准备转染到选定的人细胞株中。该基因的引入已被证明可以使某些系统的细胞永生。需要有关软骨细胞系的报告。将在转染前后评估软骨细胞系的功能和加倍潜力。建议进行组织酶学、免疫组织化学和分子检测。hTERT的表达将使用TRAP（端粒重复扩增协议）测定。为了评估hTERT的组成性或周期性表达的功能后果，将进行一系列结构和表型测试。阳性对照将由相关系组成，正常或以类似方式永生化，但带有HPV16 E6/E7基因（手头）。因此，我们的细胞系生成和工程策略应该为进一步的发育和移植研究提供一组潜在的治疗群体。

DESCRIPTION (provided by applicant): The primary objective of this proposed program is to extend the life-span of normal human chondrocyte cell cultures to develop continuous lines for distribution to the research community and for tissue engineering. Some existing 50 chondrocytic strains are available for study. The consequence(s) of constitutive expression of human telomerase reverse transcriptase (hTERT) will be determined. The human cDNA clone for hTERT has been cloned in the retroviral expression system pLXSN for ready transfection to selected human cell strains. Introduction of this gene has been shown to immortalize cells of some systems. Reports with chondrocyte cell lines are needed. Functionality and doubling potential of chondrocytic lines will be assessed both before and after transfection. Histoenzymological, immunohistochemical, and molecular tests are proposed. The expression of hTERT will be determined using the TRAP (Telomeric Repeat Amplification Protocol) assay. To assess the functional consequences of the constitutive or periodic expression of hTERT, a series of structural and phenotypic tests will be performed. Positive controls will consist of related lines normal or immortalized in similar fashion but with the HPV16 E6/E7 genes (on hand). Thus, our cell line generation and engineering strategy should provide sets of potentially therapeutic populations for further developmental and transplantation research.