摘要
目的研究从人脂肪组织中获取人脂肪干细胞(hADSCs)的方法,并观察其形态特点、生物学特性、多谱系细胞分化的特点及间充质来源细胞表面相关标志的表达,探讨脂肪干细胞作为组织工程种子细胞的临床应用前景。方法应用Ⅰ型胶原酶消化分离人脂肪组织,分时间段收集细胞并进行体外培养;采用流式细胞仪检测CD29、CD34、CD44、CD45、CD105等间充质来源细胞表面相关标志抗原的表达;利用成脂、成骨及成软骨诱导培养基对hADSCs进行定向分化诱导,观察细胞随诱导时间延长形态的变化,分别于诱导第11、14、21天进行油红O染色、茜素红染色及阿尔新蓝染色;采用Cell Counting Kit-8(CCK-8)细胞增殖检测试剂盒检测hADSCs的增殖功能。结果①原代培养的hADSCs接种48 h后大部分细胞贴壁,为多角形的单层细胞;第3代细胞分裂增殖旺盛,细胞细长且排列规律;第8~9代后细胞形态不规则,胞内颗粒明显增多。②第3代hADSCs的表面抗原标记结果:CD29、CD44、CD105阳性表达,阳性率分别为98.89%、93.73%、86.99%;CD34、CD45阴性表达,阳性率分别为0.16%、0.11%。③hADSCs成脂诱导第11天、成骨诱导第14天、成软骨诱导第21天时细胞分化达到高峰,油红O染色、茜素红染色、阿尔新蓝染色均为阳性;④第4、5、6代(P4、P5、P6)hADSCs随培养时间的延长,均呈增长趋势,细胞平均从第24 h开始进入指数增长期,三代之间细胞增殖功能比较:P4>P5,P6>P5,差异有统计学意义(P<0.05)。结论本次成功从人脂肪组织中分离、培养出目的细胞,经初步鉴定,证实其为hADSCs,同时成功对hADSCs进行了成脂、成骨及成软骨定向诱导分化。hADSCs的增殖能力可能会随传代次数的增加而有所下降;无血清刺激也可能会影响hADSCs的增殖功能,使其增殖能力有所下降。
Objective To study the method of obtaining human adipose stem cells(hADSCs)from human adipose tissue,and to observe its morphological characteristics,biological characteristics,characteristics of multi-lineage cell differentiation,and expression of relevant markers on the surface of mesenchymal-derived cells,and to explore adipose stem cells.Prospects for clinical application of tissue engineering seed cells.Methods Human adipose tissue was isolated and digested with type I collagenase,and cells were collected in time and cultured in vitro.Flow cytometry was used to detect the expression of surface-associated marker antigens on cells such as CD29,CD34,CD44,CD45,and CD105;Adipogenic,osteogenic,and chondrogenic induction media induce directional differentiation of hADSCs.Observe the morphological changes of cells with prolonged induction time.Oil red 0 staining,alizarin red staining,and Al New blue staining;Cell Counting Kit-8(CCK-8)cell proliferation detection kit was used to detect the proliferation function of hADSCs.Results①48 h after inoculation of primary cultured hADSCs,most of the cells were adherent,and they were polygonal monolayer cells;the third-generation cells had vigorous division and proliferation,and the cells were slender and arranged regularly.After the 8th to 9th generations,the cell morphology was irregular and the intracellular particles increased significantly.②Surface antigen labeling results of the third generation hADSCs:CD29,CD44,and CD105 were positively expressed,with positive rates of 98.89%,93.73%,and 86.99%,respectively;CD34 and CD45 were negatively expressed,with positive rates of 0.16%and 0.11%,respectively.③hADSCs reached the peak of cell differentiation on the 11th day of adipogenic induction,the 14th day of osteogenesis induction,and the 21st day of chondrogenic induction,and oil red O staining,alizarin red staining,and alcin blue staining were all positive;④The 4th,5th,and 6 th generation(P4,P5,P6)hADSCs showed an increasing trend with the increase of culture time.The c
作者
李莹
李小兵
LI Ying;LI Xiao-bing(Plastic and Burn Surgery,Tianjin First Central Hospital,Tianjin 300000,China)
出处
《医学信息》
2020年第1期89-93,共5页
Journal of Medical Information
关键词
脂肪干细胞
定向诱导分化
细胞增殖
Adipose stem cells
Directional induced differentiation
Cell proliferation
