组织工程与重建外科杂志

组织工程与重建外科杂志 ›› 2012, Vol. 8 ›› Issue (1): 1-5.doi: 10.3969/j.issn.1673-0364.2012.01.001

• 论著 •    下一篇

CD204阴性人脂肪来源细胞体外成软骨潜能的初步研究

丁金萍，孙恒赟，陈谨君，张文杰，刘伟，曹谊林，周广东   

  1. 上海交通大学医学院附属第九人民医院整复外科上海市组织工程研究重点实验室
  • 发布日期:2020-07-23

A Preliminary Study:Chondrogenesis of CD204(-) Human Adipose-Derived Stem Cells In vitro

DING jinping,SUN hengyun,CHEN jinjun,ZHANG Wenjie,LIU Wei,CAO Yilin,ZHOU Guangdong   

  1. Department of Plastic and Reconstructive Surgery,Shanghai Ninth People′s Hospital,Shanghai Key Laboratory of Tissue Engineering Research,National Tissue Engineering Center of China,Shanghai 200021,China.
  • Published:2020-07-23
  • Contact: 国家自然科学基金　　（30772264,30973131）；上海市曙光计划　　（08SG）；教育部新世纪优秀人才　　（NCET-09-0561）；上海市优秀学科带头人计划　　（XBR2011050）

摘要： 目的比较CD204+和CD204-两种人脂肪来源细胞的干细胞特性和体外诱导成软骨的能力。方法分离培养人脂肪来源细胞并进行流式细胞分选,分别获得CD204+和CD204-两种细胞。将这两种细胞培养扩增至第2代,观察其形态学特点;成脂、成骨定向诱导分化,油红O染色、茜素红染色定性;同时将两种细胞进行pellet成软骨诱导分化,比较体外诱导成软骨的能力,并以HE、Safranin O和Ⅱ型胶原免疫组化染色等进行鉴定。结果经流式细胞分选,脂肪来源细胞中CD204表达阳性率约为10%。分选后的CD204+和CD204-细胞均呈成纤维细胞样贴壁生长,但CD204-细胞具有更强的增殖能力。成脂诱导10 d后,两组细胞油红O染色均可见胞浆内有大量红染脂滴,但CD204+细胞具有更强的成脂潜能。成骨诱导2周后,两组细胞茜素红染色均可见钙结节红染,而CD204-细胞具有更强的成骨潜能。同时,细胞pellet成软骨诱导4周后,大体观可见CD204-细胞组pellet形成白色透明样软骨成分,而CD204+细胞组pellet外观微黄。HE和Ⅱ型胶原免疫组化染色均显示CD204-组pellet可见成熟软骨陷窝形成,Safranin O染色显示CD204-组软骨特异性细胞外基质沉积;而CD204+细胞组pellet则呈纤维化改变。结论脂肪组织来源的CD204-细胞具有干细胞的生物学特点,且有良好的成软骨潜能,可以成为软骨组织工程良好的种子细胞来源。

关键词: CD204(巨噬细胞清道夫受体)脂肪组织来源干细胞, 软骨潜能, 细胞分选

Abstract: Objective To compare the characteristics and chondrogenesis between CD204(-) ADSCs and CD204(+) ADSCs in vitro.Methods ADSCs were isolated and cultured,then the CD204(+) ADSCs and CD204(-) ADSCs were sorted by flow cell sorting.After expansion to passage 2,the morphology of CD204(+) and CD204(-) cells were observed in vitro.Adipogenic and osteogenic lineage differentiation in two cell groups were assessed by Oil Red O and alizarin red staining.Meanwhile,the two cell groups were cultured in chondrogenic media forming cell pellet and assessed by HE,Safranin O and collagen II staining.Results The expression of CD204 in adherent cells reached about 10% by FACS.Both of them all demonstrated fibroblast-like morphology and CD204(-) cells showed better proliferation ability.After adipogenic induction for 10 days,large amount of lipid-filled droplets were observed in two cell groups by Oil Red O staining,but CD204(+) cells demonstrated a better adipogenic ability.After osteogenic induction for 2 weeks,stronger positive staining was observed in CD204(-) cell group by alizarin red staining.After chondrogenic induction,the pellets formed by CD204(-) cells had a transparent white cartilage-like morphology while the CD204(+) cell pellets had a yellowish appreance.Histologically,the CD204(-) group showed a homogeneous cartilage-like structure with typical lacuna,strong safranin-O staining and strong collagen II staining,but pellets of CD204(+) group were almost fibrosis.Conclusion CD204(-) cells have the same characteristics with stem cells and high chondrogenic potential.CD204(-) cells could be an ideal seed cells in cartilage tissue engineering.

Key words: CD204, Adipose-derived stem cells, Chondrogenic potential, Cell sorting

中图分类号: