目的进一步研究血管内皮细胞(vascular endothelial cells,VECs)和脂肪干细胞(adipose-derivedstromal cells,ADSCs)联合培养对ADSCs成骨分化的影响,为联合培养VECs和ADSCs作为骨组织工程种子细胞的可能性提供实验依据。方法分别自足月妊娠SD大鼠和18周龄SD大鼠制备脐血来源的VECs和ADSCs,行形态学观察及免疫荧光染色鉴定。取第3代ADSCs与诱导培养6周的VECs按细胞比例分别为3∶1、1∶1和1∶3设为实验组A、B、C组,ADSCs、VECs单独培养设为对照组D、E组。培养第7、14天取各组细胞行ALP和饱和茜素红钙染色,进行ALP和骨钙定性检测;第4、7、14天定量测定各组细胞ALP和骨钙素(osteocalcin,OC)含量。结果诱导培养6周的脐血来源VECs可见短梭形和多角形细胞混合生长,免疫荧光染色可见血管性血友病因子为阳性。ADSCs培养可见贴壁单个核细胞形态单一,呈梭形生长,无重叠现象;免疫荧光染色可见CD90+。各组细胞培养第7天,ALP染色示A、D、E组呈阴性反应,B、C组可见部分阳性细胞;第14天时D、E组仍呈阴性反应,A、B、C组可见片状阳性细胞。培养第7天,茜素红染色示各组均未发现红色阳性细胞;第14天时A、B、C组可见极少数阳性细胞,D、E组未发现阳性细胞。各组ALP含量随时间延长均逐渐增高,其中B组各时间点含量均最高,与其余各组比较差异均有统计学意义(P<0.01),各时间点A、C组与D、E组比较差异均有统计学意义(P<0.01),A、C组间及D、E组间比较差异无统计学意义(P>0.05)。各组OC含量随时间延长逐渐增高,培养第7、14天时B组含量最高,各时间点B组与其余各组比较差异均有统计学意义(P<0.01),第4、14天时C、D组间比较差异均有统计学意义(P<0.01),第14天时A、C组和E组比较差异有统计学意义(P<0.05),其余各时间点各组间比较差异均无统计学意义(P>0.05)。结论大鼠脐血来源的VECs能在体外诱导ADSCs向成骨细胞方向分化,1∶1比例分化能力最强。 Objective To further investigate the effect of co-culture of vascular endothelial cells (VECs) and adipose-derived stromal cells (ADSCs) on the osteogenic differentiation of ADSCs and to provide a possibility for the joint culture of VECs and ADSCs as a seed tissue for bone tissue engineering Experimental basis. Methods Umbilical cord blood-derived VECs and ADSCs were prepared from full-term pregnant SD rats and 18-week-old SD rats, respectively. Morphological observation and immunofluorescence staining were performed. The third generation of ADSCs and induced VECs cultured for 6 weeks were divided into groups A, B and C according to the cell ratio of 3: 1, 1: 1 and 1: 3 respectively. ADSCs and VECs were cultured as control group D, Group E On the 7th and 14th day after culture, ALP and saturated alizarin red calcium staining were performed in all the groups. ALP and osteocalcin were detected qualitatively and ALP and osteocalcin (OC) content. Results VECs cultured for 6 weeks showed mixed growth of short fusiform cells and polygonal cells. Immunofluorescent staining showed that vWF was positive. Adherent adherent mononuclear cells showed a single morphology with spindle-shaped growth and no overlap in ADSCs. Immunofluorescence staining showed CD90 +. On day 7 of each group, ALP staining showed negative reaction in groups A, D and E, and some positive cells were seen in group B and C. On day 14, D and E still showed negative reaction, while group A, B and C showed visible film Positive cells. On day 7 of culture, alizarin red staining showed no red positive cells in each group. On the 14th day, a few positive cells were found in group A, B and C, but no positive cells in groups D and E. The content of ALP in each group gradually increased with time, of which the content of B in each time point was the highest, the difference was statistically significant compared with the other groups (P <0.01), at each time point A, C group and D, E group The difference was statistically significant (P <0.01). There was no significant difference between A, C and D, E groups (P> 0.05). The content of OC in each group gradually increased with time, the content of B was the highest on the 7th and the 14th day of culture, the difference between the B group and other groups at each time point was statistically significant (P <0.01) There was significant difference between the two groups (P <0.01). On the 14th day, the difference between the groups A, C and E was statistically significant (P <0.05). There was no statistical difference among the groups at other time points Significance (P> 0.05). Conclusion VECs derived from umbilical cord blood can induce ADSCs to differentiate into osteoblasts in vitro, with the highest ratio of 1: 1 differentiation.