Xie Shang-zhe, Fang Ning-tao, Liu Shui, Zhou Ping, Zhang Yi, Wang Song-mei, Gao Hong-yang, Pan Luan-feng
Laboratory of Molecular Biology, Shanghai Medical College, Fudan University, Shanghai 200032, China.
J Zhejiang Univ Sci B. 2008 Dec;9(12):923-30. doi: 10.1631/jzus.B0820257.
A major shortcoming in tissue engineered blood vessels (TEBVs) is the lack of healthy and easily attainable smooth muscle cells (SMCs). Smooth muscle progenitor cells (SPCs), especially from peripheral blood, may offer an alternative cell source for tissue engineering involving a less invasive harvesting technique.
SPCs were isolated from 5-ml fresh rat peripheral blood by density-gradient centrifugation and cultured for 3 weeks in endothelial growth medium-2-MV (EGM-2-MV) medium containing platelet-derived growth factor-BB (PDGF BB). Before seeded on the synthesized scaffold, SPC-derived smooth muscle outgrowth cell (SOC) phenotypes were assessed by immuno-fluorescent staining, Western blot analysis, and reverse transcription polymerase chain reaction (RT-PCR). The cells were seeded onto the silk fibroin-modified poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (SF-PHBHHx) scaffolds by 6x10(4) cells/cm2 and cultured under the static condition for 3 weeks. The growth and proliferation of the seeded cells on the scaffold were analyzed by 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT) assay, scanning electron microscope (SEM), and 4,6-diamidino-2-phenylindole (DAPI) staining.
SOCs displayed specific "hill and valley" morphology, expressed the specific markers of the SMC lineage: smooth muscle (SM) alpha-actin, calponin and smooth muscle myosin heavy chain (SM MHC) at protein and messenger ribonucleic acid (mRNA) levels. RT-PCR results demonstrate that SOCs also expressed smooth muscle protein 22alpha (SM22alpha), a contractile protein, and extracellular matrix components elastin and matrix Gla protein (MGP), as well as vascular endothelial growth factor (VEGF). After seeded on the SF-PHBHHx scaffold, the cells showed excellent metabolic activity and proliferation.
SPCs isolated from peripheral blood can be differentiated into the SMCs in vitro and have an impressive growth potential in the biodegradable synthesized scaffold. Thus, SPCs may be a promising cell source for constructing TEBVs.
组织工程血管（TEBVs）的一个主要缺点是缺乏健康且易于获取的平滑肌细胞（SMCs）。平滑肌祖细胞（SPCs），尤其是来自外周血的，可能为组织工程提供一种替代细胞来源，且获取技术侵入性较小。
通过密度梯度离心从5毫升新鲜大鼠外周血中分离出SPCs，并在含有血小板衍生生长因子-BB（PDGF BB）的内皮生长培养基-2-MV（EGM-2-MV）中培养3周。在接种到合成支架上之前，通过免疫荧光染色、蛋白质印迹分析和逆转录聚合酶链反应（RT-PCR）评估SPC衍生的平滑肌生长细胞（SOC）表型。将细胞以6×10⁴个细胞/平方厘米的密度接种到丝素蛋白修饰的聚（3-羟基丁酸酯-co-3-羟基己酸酯）（SF-PHBHHx）支架上，并在静态条件下培养3周。通过3-（4,5-二甲基噻唑-2-基）-二苯基四氮唑溴盐（MTT）法、扫描电子显微镜（SEM）和4,6-二脒基-2-苯基吲哚（DAPI）染色分析接种在支架上的细胞的生长和增殖情况。
SOCs呈现出特定的“峰谷”形态，在蛋白质和信使核糖核酸（mRNA）水平上表达SMC谱系的特定标志物：平滑肌（SM）α-肌动蛋白、钙调蛋白和平滑肌肌球蛋白重链（SM MHC）。RT-PCR结果表明，SOCs还表达收缩蛋白平滑肌蛋白22α（SM22α）、细胞外基质成分弹性蛋白和基质Gla蛋白（MGP）以及血管内皮生长因子（VEGF）。接种到SF-PHBHHx支架上后，细胞表现出优异的代谢活性和增殖能力。
从外周血中分离出的SPCs可在体外分化为SMCs，并在可生物降解的合成支架中具有显著的生长潜力。因此，SPCs可能是构建TEBVs的一种有前景的细胞来源。
