兔骨髓诱导的内皮细胞和纤维蛋白凝胶体外共培养

Coculturation of endothelial cells induced from rabbit marrow cells and fibrin sealant in vitro

目的将兔骨髓诱导的内皮细胞接种于纤维蛋白凝胶内并观察其生长增值情况。方法梯度离心分离兔骨髓单个核细胞(MNCs),直接向内皮细胞诱导培养,传代培养至第2代细胞,倒置显微镜观察细胞形态及生长特点,免疫组化鉴定;以105密度接种于纤维蛋白凝胶中培养并定期观察细胞生长情况,单纯细胞及胶原内的细胞分别用MTT法检测其生长并绘制生长曲线,胶原切片并作苏木精-伊红染色观察细胞在胶原内的生长。结果诱导的内皮细胞为铺路石样,呈单层生长,第2代细胞CD31、八因子相关抗原免疫组化均为阳性,摄取低密度脂蛋白和结合凝集素实验均阳性;细胞在纤维蛋白凝胶内成立体生长,细胞在纤维蛋白凝胶内3d后成梭形铺开,6d后自发形成管腔样结构;细胞在纤维蛋白凝胶内生长曲线成“S”形。结论骨髓诱导的内皮细胞在纤维蛋白凝胶内生长增值良好,纤维蛋白凝胶可以作为接种骨髓诱导的内皮细胞的基质材料。

Objective To research the growth and proliferation of induced endothelial cells（ECs）in fibrin sealant in vitro.Methods The marrow mononuclear cells（MNCs）in rabbit marrow were isolated by the gradient centrifugation and cultivated to isolate and induce ECs. The changes of ECs morphology,growth and proliferation, were observed during the passage cultivation. The immunohistochemical staining of CD31 and vWF and function of ECs were checked. 105 cells were seeded into lml fibrin sealant,the growth and proliferation of induced ECs were observed under microscope and HE, and the growth curve were drawn by the way of MTY. Results Induced ECs were monolayer arrayed to form cobblelike shape.The cells of 2nd passage showed positive staining of CD31 and vWF,and positive labeling of DiI-LDL and UEA.The cells grow in three-dimensional in fibrin sealant,become fusiform in 3th day and emerge tubeform after 6 days. The growth curve show ＂S＂ form. Conclusion Induced ECs grow and froliferate well in fibrin sealant in vitro. It is feasible that fibrin sealant can act as matrix in the process of seeding the induced ECs in bone tissue engineering in future.