Kishimoto Satoko, Inoue Ken-Ichi, Sohma Ryoichi, Toyoda Shigeru, Sakuma Masashi, Inoue Teruo, Yoshida Ken-Ichiro
Comprehensive Research Facilities for Advanced Medical Science, Research Center for Advanced Medical Science, Dokkyo Medical University, Mibu, Tochigi 321-0293, Japan.
Center of Regenerative Medicine, Dokkyo Medical University Hospital, Mibu, Tochigi 321-0293, Japan.
Stem Cells Int. 2020 May 18;2020:7219149. doi: 10.1155/2020/7219149. eCollection 2020.
The adipose-derived stromal vascular fraction (SVF) is an effective source for autologous cell transplantation. However, the quality and quantity of SVFs vary depending on the patient's age, complications, and other factors. In this study, we developed a method to reproducibly increase the cell number and improve the quality of adipose-derived SVFs by surgical procedures, which we term "wound repair priming." Subcutaneous fat from the inguinal region of BALB/c mice was surgically processed (primed) by mincing adipose parenchyma (injury) and ligating the subcutaneous fat-feeding artery (ischemia). SVFs were isolated on day 0, 1, 3, 5, or 7 after the priming procedures. Gene expression levels of the primed SVFs were measured via microarray and pathway analyses which were performed for differentially expressed genes. Changes in cellular compositions of primed SVFs were analyzed by flow cytometry. SVFs were transplanted into syngeneic ischemic hindlimbs to measure their angiogenic and regeneration potential. Hindlimb blood flow was measured using a laser Doppler blood perfusion imager, and capillary density was quantified by CD31 staining of ischemic tissues. Stabilization of HIF-1 alpha and VEGF-A synthesis in the SVFs were measured by fluorescent immunostaining and Western blotting, respectively. As a result, the number of SVFs per fat weight was increased significantly on day 7 after priming. Among the differentially expressed genes were innate immunity-related signals on both days 1 and 3 after priming. In primed SVFs, the CD45-positive blood mononuclear cell fraction decreased, and the CD31-CD45-double negative mesenchymal cell fraction increased on day 7. The F4/80-positive macrophage fraction was increased on days 1 and 7 after priming. There was a serial decrease in the mesenchymal-gated CD34-positive adipose progenitor fraction and mesenchymal-gated CD140A-positive/CD9-positive preadipocyte fraction on days 1 and 3. Transplantation of primed SVFs resulted in increased capillary density and augmented blood flow, improving regeneration of the ischemic limbs. HIF-1 alpha was stabilized in the primed cutaneous fat , and VEGF-A synthesis of the primed SVFs was on a peak on 5 days after priming. Wound repair priming thus resulted in SVFs with increased number and augmented angiogenic potential.
脂肪来源的基质血管成分（SVF）是自体细胞移植的有效来源。然而，SVF的质量和数量会因患者年龄、并发症及其他因素而有所不同。在本研究中，我们开发了一种通过手术程序可重复性增加细胞数量并提高脂肪来源SVF质量的方法，我们将其称为“伤口修复预处理”。通过切碎脂肪实质（损伤）并结扎皮下脂肪供血动脉（缺血），对BALB/c小鼠腹股沟区域的皮下脂肪进行手术处理（预处理）。在预处理程序后的第0、1、3、5或7天分离SVF。通过微阵列测量预处理后SVF的基因表达水平，并对差异表达基因进行通路分析。通过流式细胞术分析预处理后SVF的细胞组成变化。将SVF移植到同基因缺血后肢中，以测量其血管生成和再生潜力。使用激光多普勒血流灌注成像仪测量后肢血流量，并通过对缺血组织进行CD31染色来量化毛细血管密度。分别通过荧光免疫染色和蛋白质免疫印迹法测量SVF中HIF-1α的稳定性和VEGF-A的合成。结果，预处理后第7天，每克脂肪中SVF的数量显著增加。在预处理后第1天和第3天，差异表达基因中均有先天免疫相关信号。在预处理后的SVF中，第7天CD45阳性血液单核细胞分数降低，而CD31-CD45双阴性间充质细胞分数增加。预处理后第1天和第7天，F4/80阳性巨噬细胞分数增加。在第1天和第3天，间充质门控的CD34阳性脂肪祖细胞分数和间充质门控的CD140A阳性/CD9阳性前脂肪细胞分数呈系列下降。移植预处理后的SVF可增加毛细血管密度并增加血流量，改善缺血肢体的再生。预处理后的皮肤脂肪中HIF-1α稳定，预处理后SVF的VEGF-A合成在第5天达到峰值。因此，伤口修复预处理可使SVF数量增加且血管生成潜力增强。
