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医学信息[ISSN:1006-1959/CN:61-1278/R]

卷:

33卷

期数:

2020年01期

页码:

89-93

栏目:

论著

出版日期:

2020-01-01

文章信息/Info

Title:

Study on Biological Characteristics and Differentiation of Human Fat Stem Cells

文章编号:

1006-1959（2020）01-0089-05

作者:

李 莹; 李小兵

（天津市第一中心医院整形与烧伤外科，天津 300000）

Author(s):

LI Ying; LI Xiao-bing

(Plastic and Burn Surgery,Tianjin First Central Hospital,Tianjin 300000,China)

关键词:

脂肪干细胞; 定向诱导分化; 细胞增殖

Keywords:

Adipose stem cells; Directional induced differentiation; Cell proliferation

分类号:

R329

DOI:

10.3969/j.issn.1006-1959.2020.01.027

文献标志码:

A

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。

Abstract:

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.Results ① 48 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. ② Surface 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. ③ hADSCs 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 cells entered an exponential growth period from the 24 h on average. Comparison of cell proliferation functions between the three generations: P4> P5, P6> P5, the difference was statistically significant (P<0.05). Conclusion The target cells were successfully isolated and cultured from human adipose tissue this time. After preliminary identification, they were confirmed to be hADSCs. At the same time, hADSCs were successfully induced into adipogenic, osteogenic and chondrogenic differentiation. The proliferative capacity of hADSCs may decrease with the increase of the number of passages; serum-free stimulation may affect the proliferative function of hADSCs and reduce their proliferative capacity.

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更新日期/Last Update: 2020-01-01