Human Chondrocyte Cells in Continuous Culture

描述（由申请人提供）：本拟议计划的主要目的是延长正常人软骨细胞培养物的寿命，以开发连续细胞系，用于分发给研究团体和组织工程。现有约50种软骨细胞株可用于研究。将确定人端粒酶逆转录酶（hTERT）组成型表达的结果。已将hTERT的人cDNA克隆克隆到逆转录病毒表达系统pLXSN中，以便于转染到所选的人细胞株中。已显示引入该基因可使某些系统的细胞永生化。需要软骨细胞系的报告。将在转染前后评估软骨细胞系的功能和倍增潜力。组织酶学，免疫组织化学和分子测试提出。将使用TRAP（端粒重复扩增方案）测定来确定hTERT的表达。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。阳性对照将由正常或以类似方式永生化但具有HPV 16 E6/E7基因的相关细胞系组成（现有）。因此，我们的细胞系生成和工程策略应该提供一套潜在的治疗人群，为进一步的发展和移植研究。

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.