TELOMERASE RESTORING DNA, CELL REPLICATION AFTER ROS

亚致死性ROS对体外培养的人成纤维细胞的作用 加快端粒缩短和明显衰老的速度 细胞的数量增加了10倍。这些研究将(A)澄清 ROS损伤减少后续细胞的机制(S) 复制能力，以及(B)检验 端粒缩短和细胞复制能力的丧失。 我们将分析ROS诱导的端粒缩短的动力学 使用一种更新的更灵敏的技术，定量荧光 多肽核酸探针与端粒的原位杂交 重复着。DNA修复在ROS诱导端粒缩短中的作用 将被确定；即，是否由于直接损坏而缩短 DNA，或者它与端粒损伤的修复有关，阳极化它 ROS后需要至少一轮染色体DNA复制 侮辱。最后，为了测试ROS诱导的端粒是否 缩短直接限制细胞复制，建议 确定人成纤维细胞是否过度表达催化亚基 端粒(HRT)在ROS损伤后恢复复制能力。如果 ROS诱导的端粒缩短会随意降低复制能力 潜力，那么这个HRT转染的细胞系应该会恢复 端粒缩短与增殖潜能协调。

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.