中国组织工程研究

中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (19): 2953-2957.doi: 10.12307/2022.370

• 脂肪干细胞 adipose-derived stem cells •    下一篇

原代大鼠脂肪间充质干细胞的体外培养扩增及鉴定

刘海琴1，马华根2，唐元瑜3    

  1. 福建中医药大学，1中西医结合学院，3中医学院，福建省福州市   350122；2北京中医药大学中医学院，北京市   102401
  • 收稿日期:2021-07-21 修回日期:2021-07-22 接受日期:2021-09-18 出版日期:2022-07-08 发布日期:2021-12-28
  • 通讯作者: 唐元瑜，博士，副教授，福建中医药大学中医学院，福建省福州市 350122
  • 作者简介:刘海琴，女，2000年生，福建省龙岩市人，汉族，本科。
  • 基金资助:
    国家自然科学基金(81072714)，项目负责人：唐元瑜；福建省科技厅自然科学基金(2017J01545)，项目负责人：唐元瑜

Expansion and identification of primary rat adipose-derived mesenchymal stem cells in vitro

Liu Haiqin1, Ma Huagen2, Tang Yuanyu3   

  1. 1College of Integrated Traditional Chinese and Western Medicine, 3College of Traditional Chinese Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, Fujian Province, China; 2College of Traditional Chinese Medicine, Beijing University of Traditional Chinese Medicine, Beijing 102401, China
  • Received:2021-07-21 Revised:2021-07-22 Accepted:2021-09-18 Online:2022-07-08 Published:2021-12-28
  • Contact: Tang Yuanyu, MD, Associate professor, College of Traditional Chinese Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, Fujian Province, China
  • About author:Liu Haiqin, College of Integrated Traditional Chinese and Western Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, Fujian Province, China
  • Supported by:
    the National Natural Science Foundation of China, No. 81072714 (to TYY); the Natural Science Foundation of Fujian Science and Technology Department, No. 2017J01545 (to TYY)

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摘要：

文题释义：
脂肪间充质干细胞：是一类存在于脂肪组织中、具有自我更新和多潜能分化特征的特异性细胞群，该细胞能调控内分泌代谢，维持脂肪细胞周围微环境稳态，参与脂肪细胞的增殖与修复，在人体脂质代谢和肥胖症的发生发展中起着重要作用。
三系分化潜能：指当间充质干细胞生长的微环境发生改变时，能分化为成骨细胞、软骨细胞和脂肪细胞的生物学特性，这也是鉴定间充质干细胞的重要指标之一。

背景：脂肪间充质干细胞具有调控内分泌代谢、维持脂肪细胞周围微环境稳态、参与脂肪细胞发育等功能，在脂质代谢和肥胖症发生发展中起着重要作用。目前脂肪间充质干细胞的体外培养主要采用Ⅰ型胶原酶消化法，但其存在消化时间较长、效率低的缺点，故如何选择合适消化酶，以缩短消化时间、提高实验效率，值得关注和探索。
目的：建立简单、高效的体外大鼠脂肪间充质干细胞原代培养方法，为临床开展干细胞治疗和基因治疗提供重要的细胞载体。
方法：无菌条件下取出大鼠双侧腹股沟和附睾处的脂肪垫组织，用虹膜剪剪碎成糊状，加入2 mL 0.1% Ⅱ型胶原酶消化45 min，洗涤离心后接种于含体积分数为10%胎牛血清的低糖型DMEM完全培养基中，置于CO2培养箱内进行原代培养。待细胞达80%-90%融合时，以1∶3的比例进行传代培养。倒置显微镜下连续观察细胞形态变化，并对第4代目的细胞进行细胞表面标志物特异CD分子检测及成脂、成骨诱导分化实验。
结果与结论：①原代培养3 d后，少量短梭形细胞爬出；5 d后，细胞呈集落或团簇样生长，形态为长梭形；6-8 d时，细胞集落相互融合，密度达80%-90%，呈漩涡状排列；传至第3代时，细胞增殖迅速，接种48 h后即可再次传代，且细胞形态均一，呈现明显的鱼群样生长；②流式细胞仪检测结果显示CD90、CD73和CD29表达阳性，CD34、CD45和CD11b/c呈阴性表达；③第4代脂肪间充质干细胞成脂诱导7-10 d后，细胞形态逐渐由长梭形变为多边形或圆形，胞浆内积聚了大小不等的脂肪滴，经油红O染色后脂肪滴呈鲜艳深红色；而成骨诱导21 d后，细胞逐渐由长梭形变为多边形，呈聚集样生长，表面有大小不等的黑色小颗粒，局部有矿化样的结晶物；经茜素红染色后可见散在分布、大小不等的“蘑菇样”深红色球形结节；④由结果可见，该实验成功建立了一种简单、高效的大鼠脂肪间充质干细胞原代培养方法。

https://orcid.org/0000-0002-6112-5443(刘海琴) 

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

关键词: 干细胞, 脂肪间充质干细胞, 酶消化法, 原代培养, 鉴定, 大鼠

Abstract: BACKGROUND: Adipose-derived mesenchymal stem cells can regulate endocrine metabolism, maintain the homeostasis of microenvironment around adipocytes, participate in the development of adipocytes, and play an important regulatory role in lipid metabolism and the occurrence and development of obesity. At present, type I collagenase digestion is mainly used for the culture of adipose-derived mesenchymal stem cells in vitro, but it has the disadvantages of long digestion time and low efficiency. How to select appropriate digestive enzymes to shorten digestion time and improve experimental efficiency is worthy of attention and exploration.  
OBJECTIVE: To establish a method for primary culture of rat adipose-derived mesenchymal stem cells in vitro, and to provide important cell carriers for clinical development of stem cell therapy and gene therapy. 
METHODS: Under aseptic condition, the adipose tissue from the bilateral groin and epididymis of rats was taken out, cut into paste with iris scissors. Totally 2 mL of 0.1% type II collagenase was added for digestion for 45 minutes. After washing and centrifugation, the adipocytes were inoculated in low-sugar DMEM complete medium containing 10% fetal bovine serum and placed in the CO2 incubator for primary culture. When the fusion rate reached 80%-90%, the cells were subcultured at the ratio of 1:3. The morphological changes of cells were observed under the inverted microscope. The fourth generation of adipose-derived mesenchymal stem cells was detected for specific cell surface marker CD molecules, and the adipogenic and osteogenic differentiation experiments were carried out. 
RESULTS AND CONCLUSION: (1) After 3 days of culture, a small number of short spindle cells crawled out. 5 days later, the cells grew like colonies or clusters, and the shape was long spindle. After 6-8 days, the colonies fused 80%-90% with each other, arranged in a vortex and then subcultured. In the third generation, the cells grew rapidly and could be subcultured again 48 hours after inoculation, and the cells had uniform morphology and showed obvious fish like growth. (2) Flow cytometry showed that CD90, CD73, and CD29 were positive, while CD34, CD45, and CD11b/c were negative. (3) At 7-10 days after adipogenic differentiation of the fourth generation of adipose-derived mesenchymal stem cells, cell morphology gradually changed from a long spindle to a polygonal or round shape, and fat droplets of various sizes accumulated in the cytoplasm. Oil red O staining exhibited that fat droplets were bright and deep red. At 21 days after osteogenic differentiation, the cells gradually changed from a long spindle shape to a polygonal shape, growing in an aggregated shape, with small black particles of varying sizes on the surface, and mineralized crystals were visible in some parts. Alizarin red staining displayed “mushroom-like” dark red spherical nodules scattered in different sizes. (4) It is concluded that this experiment successfully established a simple and efficient method for primary culture of rat adipose-derived mesenchymal stem cells.

Key words: stem cells, adipose-derived mesenchymal stem cells, chemical enzyme digestion method, primary culture, identification, rat

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