肝素对大鼠骨髓基质干细胞来源的血管内皮细胞贴附ePTFE影响的实验研究

Seeding of ePTFE with endothelium cells induced from rat marrow mesenchymal stem cells in vitro

目的应用大鼠骨髓间充质干细胞(Mesenchymal stem cells,MSCs)来源的血管内皮细胞贴附ePTFE血管壁,并加入肝素共同培养,观察其对细胞贴附效率的影响,探讨提高细胞贴附ePTFE的方法。方法密度梯度离心法分离SD大鼠MSCs,加入血管内皮生长因子(vascular endothelial growth factor,VEGF)和碱性成纤维细胞生长因子(basic fibroblast growth facotr,bFGF)体外诱导,通过免疫组织荧光染色和电镜鉴定,将诱导的血管内皮细胞种植于人工血管上并分别加入浓度为0、10、20、30 mg.L-1的肝素培养,每4 h检测内皮细胞贴附率,绘制细胞贴壁曲线。结果获取的MSCs漩涡状生长,诱导后细胞的vWF免疫组织荧光染色呈阳性表达,电镜下可见血管内皮细胞特征性的W-P小体。诱导的血管内皮细胞在ePTFE的贴附率逐渐升高,于20h时间点达到最高,4组贴壁率分别是88.34%、96.48%、91.98%、85.69%。结论肝素对MSCs来源的血管内皮细胞有重要的影响,具有能够有效地提高血管内皮化效率的作用,为人工血管的进一步发展提供了新的思路。

Aim Application of rat bone marrow-derived mesenchymal stem cells from the vascular endothelial cells attached ePTFE wall, and joined the common culture with heparin for observation on the efficiency of cell-attached, and discussion the methods of improving the cells attaching. Methods The marrow were collected and cultured into the rarefied MSCs. Then differentiation into endotheliallike cells by vascular endothelial growth factor （VEGF）and basic fibroblast growth facotr（bFGF）. The cells were characterized by immunohistochemistry stainand and electronic microscopy. Then the cells cultivated in ePTFE with the heparin sodium which concentrations were 0,10,20,30 mg· L^-1 and detected the cells attaching rates every 4 hours. Results Sufficient MSCs could ptoliferated in vitro, after immunohistoehemical staining, the expression of vWF was positive in the induced cells and W-P bodies （Weibel-Palade bodies） were boserved by transminssion electronic microscopy observation. The rate of the cell attaching were increase gradually, and the 4 groups highest rate were 88.34% ,96.48% ,91.98% ,85.69% at the piont of 20th hours. Conclusion The heparin sodium could remarkable increase the effect of the endothelium cells induced from rat marrow mesenchymal stem cells attching.