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目的探讨构建预血管化细胞膜片新方法的可行性。方法取3周龄日本大耳白兔分离培养BMSCs,并在VEGF和bFGF作用下诱导其向血管内皮样细胞(endothelial like cells,ECs)分化(实验组),以未诱导细胞作为对照组,倒置显微镜下观察细胞形貌,并行血管性假血友病因子(von Willebrand factor,vWF)及CD31免疫荧光染色观察。将第2代BMSCs以9×104个/cm2密度体外培养14 d形成未分化细胞膜片,将BMSCs诱导14 d获得的ECs以5×104个/cm2密度接种在细胞膜片上(A组)培养3、7、14 d,以单纯未分化的兔BMSCs膜片为B组,BMSCs诱导14 d获得的ECs单独培养为C组。倒置显微镜下观察血管网络形成,并行CD31免疫荧光染色及组织学观察。结果原代BMSCs呈长梭形;诱导分化后细胞形态发生改变,呈“铺路石”样。实验组vWF及CD31免疫荧光染色均呈阳性,而对照组均呈阴性。A组BMSCs诱导14 d后的ECs接种至未分化的BMSCs膜片上3、7、14 d后,细胞形态发生改变,镜下可见空泡样、网状结构形成;CD31免疫荧光检测显示了进行性管腔形成过程;HE染色显示了微血管管腔的形成。B、C组未观察到类似改变。结论 BMSCs在合适条件下可诱导形成ECs;将由BMSCs诱导分化形成的ECs接种至未分化BMSCs膜片上,可在体外形成具有血管网络结构的预血管化膜片,为工程化血管组织构建提供了新方法。
Objective To explore the feasibility of constructing a new method of pre-vascularization membrane. Methods BMSCs were isolated and cultured from 3-week-old Japanese white rabbits and induced to differentiate into vascular endothelial cells (ECs) under the action of VEGF and bFGF (experimental group). The untreated cells were used as the control group and inverted Cell morphology was observed under a microscope, and von Willebrand factor (vWF) and CD31 immunofluorescence staining were performed. The second generation BMSCs were cultured in vitro at a density of 9 × 104 cells / cm2 for 14 days to form undifferentiated membranes. The ECs obtained after 14 days of BMSCs were seeded onto the membrane of cells at a density of 5 × 104 cells / cm2 (group A) On the 7th and 14th day, the simple undifferentiated rabbit BMSCs membrane was in group B, and the ECs obtained after 14 days of BMSCs induction were cultured in group C alone. Inverted microscope was used to observe the formation of vascular network, parallel CD31 immunofluorescence staining and histological observation. Results The primary BMSCs presented long fusiform shape. After differentiation, the morphology of cells was changed and appeared as “paving stones”. The experimental group vWF and CD31 immunofluorescence staining were positive, while the control group were negative. A group of BMSCs induced 14 days after ECs were seeded on undifferentiated BMSCs membrane 3,7,14 d after the change in cell morphology, the vacuole-like, reticular formation can be seen microscopically; CD31 immunofluorescence test showed that The formation of sex lumen; HE staining showed the formation of microvascular lumen. B, C group did not observe similar changes. CONCLUSIONS: BMSCs can induce ECs formation under appropriate conditions. ECs induced by BMSCs were seeded onto undifferentiated BMSCs membrane and formed a pre-vascularized membrane with vascular network structure in vitro, which provided the basis for construction of engineered vascular tissue new method.