Department of Pediatric Surgery, Research Institute at Nationwide Children's Hospital, Nationwide Children's Hospital, Center for Perinatal Research, Ohio State University College of Medicine, Columbus, Ohio, USA.
J Surg Res. 2012 Oct;177(2):359-64. doi: 10.1016/j.jss.2012.05.016. Epub 2012 May 23.
We have previously demonstrated that mesenchymal stem cell (MSC) administration protects the intestines from injury in a mouse model of intestinal ischemia/reperfusion injury. We have also shown that heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a potent intestinal cytoprotective agent in vivo that can protect the intestines by way of its effects on stem cells. The goal of the present study was to examine the effects of HB-EGF on both amniotic fluid (AF)- and bone marrow (BM)-derived MSCs in vitro.
MSCs were isolated from the AF and BM of pan-EGFP mice, grown in MSC-specific culture medium, and purified by sequential passages according to their adherence properties. Pluripotency was confirmed by induced differentiation. After incubation of MSCs with HB-EGF, proliferation was quantified using the CyQuant cell proliferation assay kit under normoxic and anoxic conditions. Chemotaxis was quantified using the CHEMICON QCM cell migration kit, and apoptosis was determined using caspase-3 immunohistochemistry after exposure of the MSCs to anoxic stress.
AF-MSCs and BM-MSCs showed significantly increased proliferation and migration in response to HB-EGF. HB-EGF significantly protected AF-MSCs and BM-MSCs from anoxia-induced apoptosis. The proliferative and anti-apoptotic effects of HB-EGF were even more pronounced in AF-MSCs than in BM-MSCs.
These results have demonstrated that HB-EGF acts as a mitogenic and chemotactic agent for MSCs that protects MSCs from injury. These findings could have important implications for future experiments designed to use MSCs to protect the intestines from injury.
我们之前已经证明，间充质干细胞（MSC）的给药可以保护肠道免受肠缺血/再灌注损伤的损伤。我们还表明，肝素结合表皮生长因子样生长因子（HB-EGF）是一种有效的体内肠保护剂，可以通过对干细胞的作用来保护肠道。本研究的目的是研究 HB-EGF 对羊水（AF）和骨髓（BM）来源的 MSC 的体外作用。
从 pan-EGFP 小鼠的 AF 和 BM 中分离 MSC，在 MSC 特异性培养基中培养，并根据其黏附特性通过连续传代进行纯化。多能性通过诱导分化来确认。将 MSC 与 HB-EGF 孵育后，在常氧和缺氧条件下使用 CyQuant 细胞增殖检测试剂盒定量增殖。使用 CHEMICON QCM 细胞迁移试剂盒定量趋化性，并用 caspase-3 免疫组织化学法检测 MSC 暴露于缺氧应激后的凋亡。
AF-MSCs 和 BM-MSCs 对 HB-EGF 的反应表现出明显的增殖和迁移增加。HB-EGF 显著保护 AF-MSCs 和 BM-MSCs 免受缺氧诱导的凋亡。HB-EGF 的增殖和抗凋亡作用在 AF-MSCs 中比在 BM-MSCs 中更为明显。
这些结果表明 HB-EGF 作为 MSC 的有丝分裂和趋化因子，可保护 MSC 免受损伤。这些发现可能对未来使用 MSC 来保护肠道免受损伤的实验具有重要意义。
