Researcher, Laboratory for Cell Technologies; Research Institute for Complex Issues of Cardiovascular Diseases, 6 Sosnovy Blvd, Kemerovo, 650002, Russia.
Senior Researcher, Laboratory of Genome Medicine; Research Institute for Complex Issues of Cardiovascular Diseases, 6 Sosnovy Blvd, Kemerovo, 650002, Russia.
Sovrem Tekhnologii Med. 2022;14(3):15-19. doi: 10.17691/stm2022.14.3.02. Epub 2022 May 28.
was to assess the suitability of endothelial colony-forming cells in the development of tissue engineering constructs based on the study of the gene expression profile compared to mature endothelial cells.
In the experiment, we used the endothelial colony-forming cells (ECFC) obtained from the peripheral blood of patients who underwent percutaneous coronary intervention. The cells were isolated on a Histopaque 1077 density gradient (Sigma-Aldrich, USA), and then cultured in EGM-2MV culture medium (Lonza, Switzerland). A commercial culture of primary human coronary artery endothelial cells (HCAEC) was used as a control. The cells were unfrozen and cultured according to the manufacturer's recommendations in MesoEndo Cell Growth Medium (Cell Applications, USA).The experiment was carried out in specialized μ-Luer plates in the perfusion system (IBIDI, Germany), which provided a continuous unidirectional flow of the culture medium with a shear stress of 5 dyn/cm. Control plates were cultured under standard conditions for a similar period of time. Total RNA was isolated from cell samples. The expression of the genes , , , , , , , , , , , , , was assessed using a quantitative real-time polymerase chain reaction. The expression of the genes was calculated by the ΔCt method and expressed on a logarithmic (log10) scale as a fold change relating to the control samples.
In mature endothelial cells HCAEC when exposed to a laminar flow, only the transcription factor and venous differentiation marker values increased significantly. ECFC showed statistically significant growth in , , , and , as well as expression decrease. In addition, we observed the overexpression of , , , and in ECFC in relation to HCAEC and hypoexpression. overexpression characteristic of progenitor cells was also found. An increase in expression associated with type IV collagen synthesis was a characteristic feature of ECFC.
The gene expression profile of endothelial colony-forming cells is quite close to that of primary endothelial cells of the human coronary artery, and thus, the cells obtained from patients' peripheral blood can be used to develop personalized tissue-engineered constructs.
通过比较基因表达谱，评估内皮祖细胞（endothelial colony-forming cells，ECFC）在基于组织工程构建的成熟内皮细胞中的适用性。
本实验中，我们从接受经皮冠状动脉介入治疗的患者外周血中分离出 ECFC。使用 Histopaque 1077 密度梯度（Sigma-Aldrich，美国）分离细胞，然后在 EGM-2MV 培养基（Lonza，瑞士）中培养。使用商业培养的原代人冠状动脉内皮细胞（human coronary artery endothelial cells，HCAEC）作为对照。按照制造商的建议，将细胞在 MesoEndo Cell Growth Medium（Cell Applications，美国）中进行无冻存培养。实验在灌注系统中的特殊 μ-Luer 板（IBIDI，德国）中进行，该系统提供具有 5 dyn/cm 切应力的培养基单向持续流动。对照板在相似的时间段内按标准条件培养。从细胞样本中提取总 RNA。使用定量实时聚合酶链反应评估基因的表达。通过 ΔCt 法计算基因的表达，并以对数（log10）尺度表示，与对照样本相比为倍数变化。
在成熟的内皮细胞 HCAEC 中，当暴露于层流时，仅转录因子和静脉分化标志物的值显著增加。ECFC 表现出统计学上显著的生长，以及和的表达下降。此外，我们观察到 ECFC 相对于 HCAEC 和的表达下调，以及、和的过度表达。还发现了祖细胞特征的过表达。与 IV 型胶原合成相关的表达增加是 ECFC 的特征。
内皮祖细胞的基因表达谱与人类冠状动脉的原代内皮细胞非常接近，因此，从患者外周血中获得的细胞可用于开发个性化的组织工程构建物。
