Ball Andrew J, Levine Fred
UCSD Cancer Center, 9500 Gilman Drive, La Jolla, CA 92093-0816, USA.
Aging Cell. 2005 Feb;4(1):21-30. doi: 10.1111/j.1474-9728.2004.00137.x.
Fetal cardiomyocytes have been proposed as a potential source of cell-based therapy for heart failure. This study examined cellular senescence in cultured human fetal ventricular cardiomyocytes (HFCs). HFCs were isolated and identified by immunocytochemistry and RT-PCR. Cells were found to senesce after 20-25 population doublings, as determined by growth arrest, morphological changes and senescence-associated beta-galactosidase activity. Using the telomeric repeat amplification protocol assay, telomerase activity was undetectable in primary HFCs. Cells were transduced to express the human reverse transcriptase subunit (hTERT) of telomerase. This resulted in greatly increased telomerase activity, but no significant lifespan extension. Analysis of telomere length in primary HFCs revealed that the senescent phenotype was not accompanied by telomere shortening. Telomeres in hTERT-positive cells were elongated in comparison with primary cells, and elongation was retained in senescent cells. Levels of the tumor suppressor protein p16INK4A increased in all senescent cells whether telomerase-positive or -negative. Senescence was accompanied by a decline in transcript levels of the polycomb gene Bmi-1, Ets1 and Ets2 transcription factors, and Id1, Id2 and Id3 helix-loop-helix proteins, suggesting roles for these genes in maintenance of cardiomyocyte proliferative capacity. In addition to offering novel insights into the behavior of human fetal cardiomyocytes in culture, these findings have implications for the development of a cell-based therapy for cardiac injury using primary fetal heart tissue.
胎儿心肌细胞已被提议作为心力衰竭基于细胞治疗的潜在来源。本研究检测了培养的人胎儿心室心肌细胞(HFCs)中的细胞衰老情况。通过免疫细胞化学和逆转录聚合酶链反应(RT-PCR)分离并鉴定了HFCs。通过生长停滞、形态变化和衰老相关β-半乳糖苷酶活性测定发现,细胞在经历20 - 25次群体倍增后会衰老。使用端粒重复序列扩增协议检测法,在原代HFCs中未检测到端粒酶活性。对细胞进行转导以表达端粒酶的人类逆转录酶亚基(hTERT)。这导致端粒酶活性大幅增加,但细胞寿命没有显著延长。对原代HFCs中端粒长度的分析表明,衰老表型并未伴随着端粒缩短。与原代细胞相比,hTERT阳性细胞中的端粒延长,并且在衰老细胞中仍保持延长状态。无论端粒酶阳性还是阴性,所有衰老细胞中肿瘤抑制蛋白p16INK4A的水平都会升高。衰老伴随着多梳基因Bmi-1、Ets1和Ets2转录因子以及Id1、Id2和Id3螺旋-环-螺旋蛋白转录水平的下降,表明这些基因在维持心肌细胞增殖能力中发挥作用。除了为培养的人胎儿心肌细胞行为提供新见解外,这些发现对使用原代胎儿心脏组织进行心脏损伤的基于细胞治疗的发展具有重要意义。