胶原补片上生长因子释放及对人骨髓干细胞增殖与分化的影响

背景：动物实验证明心肌细胞移植可改善心脏功能，但移植细胞效率较低，为减少移植细胞的流失，构建合适的移植载体与细胞共同移植于受体心脏已成为学界的共识。目的：观察经碳化二亚胺法处理，共价结合生长因子胶原补片上生长因子的释放情况，以及其对人骨髓干细胞增殖与分化的影响。方法：采用碳化二亚胺法将胶原补片激活后，对照组胶原补片直接保存于PBS内，实验组胶原补片继续浸于含有血管内皮生长因子和碱性成纤维细胞生长因子的PBS内，使其与生长因子共价结合，检测共价结合于胶原补片上生长因子的量；保存后1 d、3 d、7 d、2周、3周、4周，采用ELISA法检测上清液内血管内皮生长因子和碱性成纤维细胞生长因子的含量。将0.5＆#215;106的人骨髓间充质干细胞均匀接种于两组补片上，采用苏木精-伊红染色计数细胞，MTT法及Brdu增殖实验对比补片上的细胞增殖情况；以RT-PCR法检测两组细胞内Ⅰ、Ⅲ型胶原表达情况，SMA免疫荧光染色对比两组补片上的细胞分化情况。结果与结论：胶原补片上血管内皮生长因子和碱性成纤维细胞生长因子的共价结合率分别为42.4%，24.5%。保存4周内，胶原补片上的血管内皮生长因子和碱性成纤维细胞生长因子均呈现持续、缓慢的释放模式。与单纯胶原补片比较，共价结合生长因子的胶原补片可在体外有效促进人骨髓间充质干细胞的增殖并抑制其分化。

Release and effect of growth factors conjugated into collagen patches on human bone marrow mesenchymal stem cell proliferation and differentiation

BACKGROUND： Preclinical trials in animals have demonstrated myocardial cell transplantation can improve heart function, but it is hindered by the low retention of transplanted cells. Therefore, it becomes a common strategy to generate a proper cell platform to promote cell retention and vasculature formation.
OBJECTIVE： To evaluate the cytokine release of a novel cytokine-conjugated collagen patch prepared with 1-ethyl-3-3-dimethylaminopropyl] carbodiimide hydrochloride （EDC） and its physiological effects on proliferation and differentiation of seeded human bone marrow mesenchymal stem cells.
METHODS： After activating and crosslinking by EDC, the control patches （EDC-patch） were preserved in PBS and the experimental patches （GF-patch） were immersed into PBS containing vascular endothelial growth factor and basic fibroblast growth factor for cytokine conjugation. Collagen patches without EDC treatment underwent same process as internal control. ELISA was performed to detect the content of vascular endothelial growth factor and basic fibroblast growth factor in the supernatant of GF-patch at 1, 3, 7 days, 2, 3, 4 weeks after patch preparation. The 0.5×106 human bone marrow mesenchymal stem cells were evenly seeded on both kinds of patches and the cell proliferation was identified by hematoxylin-eosin staining followed by cell counting, MTT assay or BrdU staining test. Accordingly, cell differentiation was illustrated by RT-PCR for collagen I and collagen III expression, and immunofluorescent staining for SMA in seeding cells.
RESULTS AND CONCLUSION： 42.4% vascular endothelial growth factors and 24.5% basic fibroblast growth factors were successfully conjugated into collagen patches and they both exhibited the constant, controlled-release mode during the 4-week observation period. By contrast, the patch without EDC treatment just showed the physical bonding of both cytokines. In comparison with EDC-patch, GF-patch presented the capability to induce cell proliferation while retard cell differ