Kang Mo K, Park No-Hee
School of Dentistry, David Geffen School of Medicine, and Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA, USA.
Methods Mol Biol. 2007;371:151-65. doi: 10.1007/978-1-59745-361-5_12.
Normal human somatic cells undergo limited cell division cycles and enter irreversible replication arrest called senescence. Cellular senescence of many human cell types is regulated by the length and status of telomeric sequences, which is shortened after each round of DNA replication. Telomeres can be rejuvenated by telomerase, an enzyme which carries out de novo synthesis of telomeric DNA. Telomerase is a ribonucleoprotein complex composed minimally of telomere reverse transcriptase gene (hTERT) and RNA template (hTR), and its enzyme activity in cells is primarily limited by the level of hTERT expression. Therefore, telomerase activity in cells can be reconstituted by overexpression of hTERT, frequently resulting in extension of replicative life span or immortalization. It is well established that the effect of telomerase reconstitution on cellular life span is clearly cell type-dependent because telomere shortening is not the only limiting factor of cellular life span. However, telomerase activity appears to be a requirement for cellular immortalization, irrespective of the cell types. In this article, we discuss the detailed methods to extend the in vitro replicative life span of primary human cells by ectopic expression of hTERT.
正常人类体细胞经历有限的细胞分裂周期，进入称为衰老的不可逆复制停滞状态。许多人类细胞类型的细胞衰老受端粒序列长度和状态的调控，端粒序列在每一轮DNA复制后都会缩短。端粒酶可使端粒恢复活力，端粒酶是一种进行端粒DNA从头合成的酶。端粒酶是一种核糖核蛋白复合体，至少由端粒逆转录酶基因（hTERT）和RNA模板（hTR）组成，其在细胞中的酶活性主要受hTERT表达水平的限制。因此，通过hTERT的过表达可在细胞中重建端粒酶活性，这通常会导致复制寿命延长或永生化。众所周知，端粒酶重建对细胞寿命的影响明显依赖于细胞类型，因为端粒缩短并非细胞寿命的唯一限制因素。然而，无论细胞类型如何，端粒酶活性似乎是细胞永生化的必要条件。在本文中，我们讨论了通过hTERT的异位表达来延长原代人类细胞体外复制寿命的详细方法。
