体外血管内皮细胞和脂肪干细胞联合培养对脂肪干细胞成骨分化的影响  

INFLUENCE OF CO-CULTURING VASCULAR ENDOTHELIAL CELLS AND ADIPOSE-DERIVED STROMAL CELLS ON OSTEOGENIC DIFFERENTIATION IN VITRO

作　　者：王福科[1] 刘流[1] 赵德萍[2] 何晓光[1] 代晓明[1] 李逸松[1] 李玉晓[1] 

机构地区：[1]昆明医学院第一附属医院头颈外科,昆明650032 [2]昆明医学院第一附属医院实验中心,昆明650032

出　　处：《中国修复重建外科杂志》2010年第4期399-405,共7页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(30960388);云南科技厅联合专项基金资助项目(2007C0007R);昆明医学院博士创新基金项目(KM2007D03)~~

摘　　要：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,Objective To further study the influence of the co-cultivation of vascular endothelial cells（VECs）and adipose-derived stromal cells（ADSCs）on cell osteogenic differentiation in vitro and provide experimental evidences of the probability of the co-cultivation of VECs and ADSCs as the seed cells of tissue engineering.Methods The VECs derived from cord blood and ADSCs were prepared by full-term pregnancy SD rats and 18-week-old SD rats,to carry on the morphological observation and immunohistochemical staining identification.The third generation of ADSCs and the VECs induced by conditioned medium for 6 weeks were cultured and were divided into groups A,B,and C as the experimental group according to cell ratios of 3：1,1：1,and 1：3,respectively.ADSCs or VECs was cultured alone in groups D and E as control groups.ALP and alizarin red staining were done respectively on the 7th day and 14th day;ALP and osteocalcin（OC）were detected respectively on the 4th day,7th day,and 14th day.Results The VECs derived from cord blood showed mixed growth of short spindle and polygonal cells after 6 weeks of induction,the immunofluorescent staining result of von Willebrand factor was positive.ADSCs showed adherent mononuclear cells and spindle-shaped growth without duplication;the immunofluorescent staining result of CD90 was positive and no positive cells were seen in the control group.On the 7th day of cell culture,ALP staining showed that the results were negative in groups A,D,and E,and some positive cells were seen in groups B and C;on the 14th day,the results were still negative in groups D and E,and positive cells fused to sheet form in groups A,B,and C.von Kossa staining showed that the results were negative in all groups on the 7th day;few positve cells were seen in groups A,B,and C,and no positive cells were seen in groups D and E on the 14th day.The ALP contents increased gradually in all groups,which was highest in group B at every time point,showing significant difference（P0.01）between group B and other gr

关 键 词：血管内皮细胞 脂肪干细胞 ALP 骨钙素 联合培养 大鼠 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]