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 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.