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。
Objective To compare tile characteristics and chondrogenesis between CD204（-） ADSCs and CD204（＋） ADSCs in vitro. Methods ADSCs were isolated and cuhured, 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 tile C1）204（＋） cell pellets had a yellowish appearance. 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.
出处
《组织工程与重建外科杂志》
2012年第1期1-5,共5页
Journal of Tissue Engineering and Reconstructive Surgery
基金
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)
