TELOMERASE RESTORING DNA, CELL REPLICATION AFTER ROS

亚致死活性氧对年轻人成纤维细胞的作用 加速端粒缩短和表观衰老的速度 细胞数量增加了10倍。 这些研究将（a）澄清 ROS损伤降低随后的细胞凋亡的机制 复制能力，和（B）测试之间的因果关系 端粒缩短和细胞复制能力的丧失。 ROS诱导端粒缩短的动力学将被分析 使用更新的更灵敏的技术，定量荧光In 端粒肽核酸探针原位杂交 重复。 DNA修复在活性氧诱导端粒缩短中的作用 将被确定;即，由于直接断裂， DNA，或者它与端粒损伤的修复有关， 在ROS之后需要至少一轮染色体DNA复制 侮辱。 最后，为了测试ROS诱导的端粒 缩短直接限制了细胞复制，建议 确定人成纤维细胞是否过表达催化亚基 端粒（hrT）在ROS损伤后恢复复制能力。 如果 ROS诱导的端粒缩短是偶然减少复制性的， 潜在的，那么这个hrT转染的细胞系应该恢复 端粒缩短和增殖潜能协调。

Sublethal ROS treatment of young human fibroblast cells in vitro accelerates the rate of telomere shortening and apparent senescence of the cells by as much as 10 fold. These studies will (a) clarify the mechanism(s) by which ROS damage reduces subsequent cellular replicative capacity, and (b) test for a causal relationship between telomeric shortening and the loss of cellular replicative capacity. The kinetics of ROS-induced telomeric shortening will be analyzed using a newer more sensitive technique, quantitative Fluorescence In Situ Hybridization with a peptide Nucleic Acid probe to telomeric repeats. The role of DNA repair in ROS induced telomere shortening will be determined; i.e., is shortening due to direct breakage of DNA, or is it associated with repair of telomeric lesions, anodes it require at least one round of chromosomal DNA replication after ROS insult. Finally, in order to test if the ROS-induced telomeric shortening directly limits cell replication, it is proposed to determine if human fibroblasts over expressing the catalytic subunit telomere (hrT) recover replicative ability following ROS damage. If ROS-induced telomere shortening is casually reducing replicative potential, then this hrT-transfected cell line should restore shortened telomeres and proliferative potential coordinately.