Attar Armin, Khosravi Maharlooi Mohsen, Khoshkhou Sara, Hosseini Ahmad, Jaberipour Mansoureh, Dehghan Arman, Monabati Ahmad
Student Research Committee, Shiraz University of Medical Sciences (SUMS), Shiraz, Iran.
Cellular and Molecular Research Club, Shiraz, Iran.
Iran Biomed J. 2013;17(3):146-51. doi: 10.6091/ibj.1100.2013.
Endothelial progenitor colony forming unit-endothelial cells (CFU-EC) were first believed to be the progenitors of endothelial cells, named endothelial progenitor cells. Further studies revealed that they are monocytes regulating vasculogenesis. The main hindrance of these cells for therapeutic purposes is their low frequency and limited replicative potentials. This study was undertaken to determine telomerase activity and alternative splicing variants in CFU-EC as a potential cause of limited replicative capacity in these cells.
CFU-EC were isolated from peripheral blood using a standard cell culture assay. Colonies were detached mechanically and alternative splicing variant mRNA were evaluated using real-time PCR. Telomerase enzyme activity was assessed using telomerase repeat amplification protocol. The same procedures were done on the cancer cell line Calu6 as the positive control.
The cultured peripheral blood mononuclear cells formed colonies with spindle-shaped monocytic cells sprouted from the clusters. These morphological characteristics fulfill the definition of CFU-EC. Telomere length amplification protocol assay revealed no telomerase activity and real-time PCR showed no expression of telomerase enzyme mRNA in CFU-EC. Both parameters were significantly higher in the cancer cell line Calu6 taken as the positive control.
The absence of telomerase activity in the CFU-EC is a result of pre-transcriptional regulation of gene expression rather than other mechanisms for controlling telomerase activity such as post-transcriptional modifications. This finding can explain the limited proliferative activity of CFU-EC cells. We propose that absence of telomerase activity in CFU-EC can be attributable to their more mature monocytic nature that needs further investigations.
内皮祖细胞集落形成单位 - 内皮细胞（CFU - EC）最初被认为是内皮细胞的祖细胞，称为内皮祖细胞。进一步研究表明，它们是调节血管生成的单核细胞。这些细胞用于治疗目的的主要障碍是其低频率和有限的复制潜能。本研究旨在确定CFU - EC中的端粒酶活性和可变剪接变体，作为这些细胞复制能力受限的潜在原因。
使用标准细胞培养测定法从外周血中分离CFU - EC。机械分离集落，并使用实时PCR评估可变剪接变体mRNA。使用端粒酶重复扩增协议评估端粒酶活性。对癌细胞系Calu6进行相同程序作为阳性对照。
培养的外周血单核细胞形成集落，从簇中长出纺锤形单核细胞。这些形态特征符合CFU - EC的定义。端粒长度扩增协议测定显示CFU - EC中无端粒酶活性，实时PCR显示CFU - EC中无端粒酶mRNA表达。作为阳性对照的癌细胞系Calu6中的这两个参数均显著更高。
CFU - EC中缺乏端粒酶活性是基因表达转录前调控的结果，而非控制端粒酶活性的其他机制，如转录后修饰。这一发现可以解释CFU - EC细胞有限的增殖活性。我们提出CFU - EC中缺乏端粒酶活性可归因于其更成熟的单核细胞性质，这需要进一步研究。
