人脂肪间充质干细胞的分离及培养和鉴定

张文瑾; 孙建军

doi:10.13201/j.issn.1001-1781.2016.09.014

人脂肪间充质干细胞的分离及培养和鉴定

张文瑾^1,2,
孙建军^1,2, ,

- 1. 安徽医科大学研究生学院(合肥, 230032);
- 2. 海军总医院全军耳鼻咽喉-头颈外科中心
基金项目:
国家自然科学基金资助项目（No：81170904）

详细信息

通讯作者: 孙建军,E-mail:jjsun85@sina.com

中图分类号: R394.2

收稿日期: 2016-02-14

Isolation, culture and identification of human adipose-derived mesenchymal stem cells

ZHANG Wenjin^1,2,
SUN Jianjun^1,2, ,

- 1. Graduate College of Anhui Medical University, Hefei, 230032, China;
- 2. Department of Otolaryngology-Head and Neck Surgery, Navy General Hospital, PLA

More Information

Corresponding author: SUN Jianjun, E-mail: jjsun85@sina.com

Received Date: 14 February 2016

摘要

摘要: 目的：建立体外分离培养人脂肪间充质干细胞（hADSCs）的方法，观察其形态及生物学特性，探讨其作为颞骨上皮组织工程种子细胞的可行性。方法：整形外科吸脂术获得女性腹部皮下脂肪，0.3%Ⅰ型胶原酶消化，贴壁培养hADSCs，进行成脂、成骨诱导验证其多向分化潜能；流式细胞术检测细胞表面分子；MTT法绘制细胞增殖曲线。结果：该实验获得形态均一的长梭形、成纤维细胞样生长的hADSCs，具有成脂、成骨分化潜能；流式细胞术检测细胞高表达CD105、CD29、CD90，低表达CD34、CD45；细胞有明显的指数生长期。结论：该实验分离获得的细胞具有hADSCs特性，可作为颞骨上皮组织工程种子细胞。
- 脂肪间充质干细胞 /
- 分离 /
- 培养 /
- 鉴定
Abstract: Objective: To establish isolation and culture methods of human adipose derived mesenchymal stem cells(hADSCs) in vitro and to investigate the basic biological characteristics of hADSCs as seed cells involved in repair of epithelium during temporal bone surgery.Method: Human adipose tissue was collected after liposuction surgery from the cosmetic and plastic surgery center,and then was digested with 0.3% collagenase Ⅰ. hADSCs were isolated and adherent cultured. Multilineage differentiation potential of hADSCs were determined by differentiating cells into adipocytes and osteoblast. Phenotypes of cells were detected by flow cytometer, and cell growth curve was observed by MTT.Result: Obtained hADSCs were similar size, spindle-shaped fibroblasts-liked cells and had differentiation potential to adipocytes and osteoblast. Flow cytometry showed that cells were highly positive for CD105, CD90, CD29 and weakly positive for CD34 and CD45. MTT exhibited that hADSCs had a exponential growth phase.Conclusion: Isolated stem cells from human adipose tissue show the typical characterization of hADSCs, and can be used as seed cells involved in repair of epithelium during temporal bone surgery.
- adipose-derived mesenchymal stem cells /
- isolation /
- cultivation /
- identification

HTML全文

参考文献(18)

[1]	DAHER S R,JOHNSTONE B H,PHINNEY D G,et al.Adipose stromal/stem cells:basic and translational advances:the IFATS collection[J].Stem Cells,2008,26:2664-2665.
[2]	PREMACHANDRA D J,WOODWARD B M,MILTON C M,et al.Treatment of postoperative otorrhoea by grafting of mastoid cavities with cultured autologous epidermal cells[J].Lancet,1990,335:365-367.
[3]	PREMACHANDRA D J,WOODWARD B,MILTON C M,et al.Treatment of chronic mastoiditis by grafting of mastoid cavities with autologous epithelial layers generated by in vitro culture of buccal epithelium[J].J Laryngol Otol,1991,105:413-416.
[4]	SOMERS T,VERBEKEN G,VANHALLE S,et al.Treatment of chronic postoperative otorrhea with cultured keratinocyte sheets[J].Ann Otol Rhinol Laryngol,1997,106:15-21.
[5]	SOMERS T,DUINSLAEGER L,DELAEY B,et al.Stimulation of epithelial healing in chronic postoperative otorrhea using lyophilized cultured keratinocyte lysates[J].Am J Otol,1997,18:702-706.
[6]	BAER P C,GEIGER H.Adipose-derived mesenchymal stromal/stem cells:tissue localization,characterization,and heterogeneity[J].Stem Cells Int,2012,2012:812693-812693.
[7]	LINDROOS B,SUURONEN R,MIETTINEN S.The potential of adipose stem cells in regenerative medicine[J].Stem Cell Rev,2011,7:269-291.
[8]	GU J,LIU N,YANG X,et al.Adiposed-derived stem cells seeded on PLCL/P123 eletrospun nanofibrous scaffold enhance wound healing[J].Biomed Mater,2014,9:035012-035012.
[9]	FRASER J K,WULUR I,ALFONSO Z,et al.Fat tissue:an underappreciated source of stem cells for biotechnology[J].Trends Biotechnol,2006,24:150-154.
[10]	MCINTOSH K R,LOPEZ M J,BORNEMAN J N,et al.Immunogenicity of allogeneic adipose-derived stem cells in a rat spinal fusion model[J].Tissue Eng Part A,2009,15:2677-2686.
[11]	KILROY G E,FOSTER S J,WU X,et al.Cytokine profile of human adipose-derived stem cells:expression of angiogenic,hematopoietic and pro-inflammatory factors[J].J Cell Physiol,2007,212:702-709.
[12]	SALGADO A J,REIS R L,SOUSA N J,et al.Adipose tissue derived stem cells secretome:soluble factors and their roles in regenerative medicine[J].Curr Stem Cell Res Ther,2010,5:103-110.
[13]	ZOQRAFOU A,PAPADOPOULOS O,TSIQRIS C,et al.Autologous transplantation of adipose-derived stem cells enhances skin graft survival and wound healing in diabetic rat[J].Ann Plast Surg,2013,71:225-232.
[14]	LIN Y C,GRAHOVAC T,OH S J,et al.Evaluation of a multi-layer adipose-derived stem cell sheet in a full-thickness wound healing model[J].Acta Biomater,2013,9:5243-5250.
[15]	DOMINICI M,LE BLANC K,MUELLER I,et al.Minimal criteria for defining multipotent mesenchymal stromal cells.The international society for cellular therapy position statement[J].Cytotherapy,2006,8:315-317.
[16]	LINDROOS B,AHO K L,KUOKKANEN H,et al.Differential gene expression in adipose stem cells cultured in allogeneic human serum versus fetal bovine serum[J].Tissue Eng Part A,2010,16:2281-2294.
[17]	LINDROOS B,BOUCHER S,CHASE L,et al.Serum-free,xeno-free culture media maintain the proliferation rate and multipotentiality of adipose stem cells in vitro[J].Cytotherapy,2009,11:958-972.
[18]	闵敏,张雪静,马红,等.人脂肪间充质干细胞的原代培养及体外成骨成脂诱导分化[J].江苏大学学报(医学版),2013,23(3):185-190.