【作者】 刘流； 王福科； 赵德萍； 代晓明； 李逸松； 李宗芳；

【Author】 LIU Liu1 WANG Fu-Ke1 ZHAO De-ping2 DAI Xiao-ming1 LI Yi-song1 LI Zong-fang3 (1Dept.of head and neck surgery,2The experimental center,3Dept. of MRI The 1st Affiliated Hospital of Kunming Medical College,Kunming 650032,China.)

【机构】 昆明医学院第一附属医院头颈外科； 昆明医学院第一附属医院实验中心； 昆明医学院第一附属磁共振室；

【摘要】 目的:为进一步研究血管内皮细胞(Vascular Endothelial Cells,VECs)和脂肪干细胞(Adipose-Derived Stromal Cells,ADSCs)联合培养对细胞增殖和成骨分化的影响。为联合培养VECs和ADSCs作为组织工程骨种子细胞的可能性提供实验依据。方法:取ADSCs与VECs按照VECs组、3:1、1:3、1:1、ADSCs组共同培养;分别于7、14天观察联合细胞的形态变化;各组细胞分别于1～15天,改良MTT法描出生长曲线;7、14天碱性磷酸酶和饱和茜素红钙染色;4、7、14天定量测定各组细胞碱性磷酸酶和骨钙素含量。结果:①1:1和3:1组14天可见细胞之间出现许多突触连接,部分细胞融合成团块状,其余组未见细胞团出现。②各组吸光度逐渐升高,ADSCs、3:1、1:3、1:1组12天时处于高峰,1:1组吸光度最高;VECs组第10天吸光度达到高峰;随后各组吸光度逐渐下降,差异有统计学意义。③第7天时ALP染色ADSCs、3:1、VECs组呈阴性反应,1:3和1:1组可见部分细胞阳性;茜素红骨钙检测各组均未发现阳性细胞。14天时ALP染色ADSCs、VECs组仍呈阴性反应,3:1、1:3和1:1组可见片状阳性细胞;茜素红骨钙检测3:1、1:3和1:1组可见极少数阳性细胞,其余各组未发现阳性细胞④各组ALP检测量随时间延长逐渐增高,各时间1:1组ALP最高;ADSCs组和VECs组ALP基本没有变化;1:1组和其它各组之间两两比较均有显著统计学意义(P<0.001)⑤各组OC检测量随时间延长逐渐增高7天和14天时1:1组OC最高;1:1组和其它各组之间两两比较均有显著统计学意义(P≤0.001)。结论:①VECs和ADSCs联合培养细胞相互促进增殖;1:1比例增殖能力最强。②VECs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。更多还原

【Abstract】 Objective:The purpose of this study was to further study the influence of the co-cultivation of vascular endothelial cells and adipose-derived stromal sells on cell proliferation and differentiation of osteoblasts 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:To co-culture VECs and ADSCs by the proportion of VECs、3:1、1:3、1:1 and ADSCs,morphological changes were observed by inverted microscope.On the 7th day and 14th day.The adhesive and proliferation conditions of bone marrow stromal stem cells were evaluated by MTT.Alkaline phosphatase(ALP) and Von Kossa staining were done respectively on the 7th day and 14th day,ALP and osteocalcin(OC) were detectedrespectively on the 4th day,7th day and 14th day.Result:①There were many synaptic connections in the co-culture cell and part of the cell form cell clusters;no cell clusters observed in other groups.②The absorbance of every group increased gradually,it reached the peak in ADSCs、3:1、1:3 and 1:1 ratio group on 12th day and the highest was the 1:1 group,but the time was 10th day in the VECs group,the differences had statistical significance.③ALP staining of the ADSCs,3:1 and VECs group were negative on 7th day, but part of the cell were positive in the 1:3 and 1:1 group;,(?)on Kossa staining of every group was negative on 7th day.On the 14th day ,ALP staining was also negative in the ADSCs and VECs group and it was positive in the 3:1,1:3 and 1:1 group,Von Kossa staining was positive in the little cell of the 3:1,1:1 and 3:1 group and other groups were negative.④The ALP value of every group increased gradually,it was highest in the 1;1 group on the 4th,7th and 14th day,there were no changes on the ADSCs and VECs group;multiple comparison of the value showed remarkable increase between the 1:1 group and other groups(P<0.001)⑤The OC value of every group increased gradually,the OC value of the 1:1 group was highest on the 7th and 14th day,multiple comparison of the value showed remarkable increase between the 1:1 group and other groups(P=0.001).Conclusion:①The co-culture cell can stimulate proliferation each other in the system of co-culture VECs and ADSCs in vitro,it can reach the best result when the proportion is 1:1.②ADSCs are ossified by VECs in the system of co-culture VECs and ADSCs in vitro,the influence on ossification is the strongest in the 1:1 group.更多还原