Liu Liang, Jin Xian, Zhou Zhong'e, Shen Chengxing
Department of Cardiology, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, 1665 Kongjiang Road, Shanghai 200092, China.
Department of Cardiology, Central Hospital of Minhang District, 170 Xinsong Road, Shanghai 201199, China.
Int J Mol Sci. 2016 Jul 20;17(7):1175. doi: 10.3390/ijms17071175.
Most mesenchymal stem cells (MSCs) die shortly after transplantation into a myocardial infarcted area. Dead MSCs (dMSCs) are phagocytized by macrophages (pMΦ) in vivo and in vitro; however, the effects of pMΦ on cardiac stem cells (CSCs) remain unknown.
MSCs, CSCs, and macrophages were obtained from bone marrow, hearts, and peritoneal cavity of mice, respectively. dMSCs were harvested after hypoxia for 24 h, and incubated with macrophages (2:1) for another 2 days with or without lipopolysaccharide (LPS, 50 ng/mL) and sorted by flow cytometry to obtain pMΦ. Viability and apoptosis of CSCs were respectively evaluated with the cell counting kit-8 (CCk-8) assay and Annexin V-PE/7-AAD staining at 0, 6, 12, and 24 h of culture with supernatant fluids from macrophages (MΦ), LPS-stimulated macrophages (LPS-pMΦ), pMΦ, and MSCs. GATA-4 and c-TnI expression was measured by flow cytometry on the seventh day. Expression of inflammation and growth factors was assessed by real-time polymerase chain reaction (RT-PCR) in MΦ, LPS-pMΦ, and pMΦ cells.
pMΦ expressed higher levels of interleukin-10 (IL-10) and transforming growth factor-β (TGF-β)and lower levels of tumor necrosis factor-α(TNF-α)and IL-6 than LPS-pMΦ, higher levels of growth factors and of GATA-4 and c-TnI at the 7th day, which were similar to those in MSCs. CSCs cultured with supernatant fluids of pMΦ exhibited higher proliferative, anti-hypoxic, and differentiation activities.
The supernatant fluids of macrophages that had phagocytized dead MSCs encouraged changes in phenotype and growth factor expression, enhanced proliferation, differentiation, and anti-hypoxic activity of CSCs, which is relevant to understanding the persistent therapeutic effect of MSCs after their massive demise upon transplantation in myocardial infarction. Furthermore, some miRNAs or proteins which were extracted from the supernatant fluids may give us a new insight into the treatment of myocardial infarction in the future.
大多数间充质干细胞（MSCs）在移植到心肌梗死区域后不久就会死亡。死亡的间充质干细胞（dMSCs）在体内和体外都会被巨噬细胞（pMΦ）吞噬；然而，pMΦ对心脏干细胞（CSCs）的影响仍然未知。
分别从小鼠的骨髓、心脏和腹腔中获取间充质干细胞、心脏干细胞和巨噬细胞。缺氧24小时后收获死亡的间充质干细胞，并与巨噬细胞（2:1）一起培养2天，添加或不添加脂多糖（LPS，50 ng/mL），然后通过流式细胞术分选以获得pMΦ。在与巨噬细胞（MΦ）、LPS刺激的巨噬细胞（LPS-pMΦ）、pMΦ和间充质干细胞的上清液培养0、6、12和24小时时，分别用细胞计数试剂盒-8（CCk-8）检测法和膜联蛋白V-PE/7-AAD染色评估心脏干细胞的活力和凋亡。在第7天通过流式细胞术检测GATA-4和c-TnI的表达。通过实时聚合酶链反应（RT-PCR）评估MΦ、LPS-pMΦ和pMΦ细胞中炎症和生长因子 的表达。
与LPS-pMΦ相比，pMΦ表达更高水平的白细胞介素-10（IL-10）和转化生长因子-β（TGF-β），以及更低水平的肿瘤坏死因子-α（TNF-α）和IL-6，在第7天表达更高水平的生长因子以及GATA-4和c-TnI，这与间充质干细胞中的情况相似。用pMΦ的上清液培养的心脏干细胞表现出更高的增殖、抗缺氧和分化活性。
吞噬死亡间充质干细胞的巨噬细胞的上清液促使表型和生长因子表达发生变化，增强了心脏干细胞的增殖、分化和抗缺氧活性，这与理解间充质干细胞在心肌梗死移植后大量死亡后的持续治疗效果相关。此外，从上清液中提取的一些微小RNA或蛋白质可能会在未来为我们治疗心肌梗死提供新的思路。
