目的:观察3-羟基丁酸-3-羟基己酸共聚酯(PHBHHx)能否促进多能干细胞(m ESC)的存活、增殖以及分化,为心肌梗死的治疗提供新的治疗策略.方法:培养m ESC,传代培养至3代后与PHBHHx材料制成的生物薄膜共同培养72h,CCK-8试剂盒测定m ESC的活性及增殖,DAPI染色观察细胞,电镜观察m ESC生长.将小鼠m ESC向心肌细胞定向分化,15d后免疫荧光和流式细胞术定量测定心肌肌钙蛋白(c Tn T).结果:CCK-8活性检测显示PHBHHx膜培养m ESC可以促进细胞更好的增殖;电镜扫描显示,m ESC在PHBHHx膜上能够粘附生长,细胞形态正常.加入干细胞分化培养基使干细胞向心肌细胞定向分化15d后,通过免疫荧光显示心肌细胞特异性标志蛋白c Tn T表达阳性;流式细胞术测得PHBHHx膜培养比传统的细胞培养方式能够促进细胞更好的分化,测得的c Tn T表达量明显增加.结论:PHBHHx膜与m ESC有较好的组织相容性,并且能够促进m ESC的增殖和分化.%Objective: To study the effect of polyhydroxyalkanoates cultivation on adhesion, proliferation and differentiation of embryonic stem cells via co-culture in vitro, to find a suitable polymeric biomaterials for m ESC attachment, proliferation and differentiation, forming myocardial patches as a new repair method for myocardial infarction. Methods: m ESC were recovered, passaged and cultured with PHBHHx films together.The morphology of m ESCs attached on the film was visualized under scanning electron microscope (SEM) The cell vitality was detected by CCK-8. DAPI fluorescence staining to observe cell proliferation. The embryonic stem Cells were induced by differentiation medium that containing vitamin C. Control group did not add any inducer. The survival and differentiation of m ESC were observed through immunofluorescence, RT-PCR and flow cytometry. Results: Cell activity assy showed that PHBHHx film culture can promote m ESC proliferation better than traditional cultivating way. Scanning electron microscope (SEM) observation showed that lots of cells grew on the patch formed by m ESC and PHBHHx film. Cell shape was normal. After adding differentiation medium, immunofluorescence showed that myocardial cell marker protein c Tn T expressed positively. Flow cytometry technique measured that PHBHHx cell culture can promote m ESC differentiation than traditional culture way, the expression of c Tn T increased significantly. Conclusion: m ESCs had good histocompatibility with PHBHHx films. PHBHHx film cell culture can promote the proliferation and differentiation of miPSC.
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