丝素蛋白-左旋聚乳酸微载体扩增脂肪来源干细胞的实验研究  

Experimental study on adipose-derived stem cells amplified by silk fibroin/poly-L-lactic acid microcarriers in vitro

作　　者：黄元亮 穆琳 林燕娴 蒋海越 滕利 HUANG Yuanliang;MU Lin;LIN Yanxian;JIANG Haiyue;TENG Li(Department of Craniomaxillofacial Surgery,Plastic Surgery Hospital,Chinese Academy of Medical Sciences&Peking Union Medical College,Beijing,100144,P.R.China)

机构地区：[1]中国医学科学院整形外科医院颅颌面中心,北京100144

出　　处：《中国修复重建外科杂志》2021年第5期611-619,共9页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：中国医学科学院医学与健康科技创新工程(2017-I2M-1-007)。

摘　　要：目的探讨丝素蛋白-左旋聚乳酸(silk fibroin-poly-L-lactic acid,SF-PLLA)微载体对脂肪来源干细胞(adipose-derived stem cells,ADSCs)的扩增作用及对其分化能力的影响。方法使用脂肪抽吸术患者自愿捐赠脂肪组织,经酶消化法提取ADSCs。取第3代ADSCs分别接种于CultiSpher G和SF-PLLA微载体(分别设为A、B组),细胞-微载体复合物应用旋转生物反应器培养,并采用正常二维平面传代培养的ADSCs作为对照组(C组)。应用扫描电镜观察两种微载体结构及细胞生长状况;Live/Dead染色观察细胞在两种微载体上分布及成活情况;DNA定量法检测两种微载体上细胞增殖情况;培养18 d实时荧光定量PCR(real-time fluorescence quantitative PCR,qRT-PCR)检测3组ADSCs成软骨、成骨、成脂相关基因表达,行流式细胞术鉴定MSCs表面标志物,并行成软骨、成骨、成脂三系分化鉴定。结果 ADSCs在两种微载体上均可黏附并实现高效扩增,扩增18 d时两种微载体上ADSCs总扩增量差异无统计学意义(P>0.05)。对扩增所得ADSCs行qRT-PCR检测示,与C组比较,B组成软骨相关基因(蛋白聚糖、软骨寡聚基质蛋白、软骨特异性基因SOX9)呈显著上调,A组成脂相关基因(过氧化物酶体增殖物激活受体γ、脂蛋白酯酶、脂联素基因)呈显著上调,差异均有统计学意义(P<0.05)。流式细胞术检测与三系分化鉴定显示两种微载体扩增后细胞仍为ADSCs,仍具有三系分化能力。结论 SF-PLLA微载体可用于扩增ADSCs,且扩增所得细胞成软骨分化趋势显著提高,成脂分化趋势降低。Objective To investigate the effect of silk fibroin-poly-L-lactic acid(SF-PLLA) microcarriers on the expansion and differentiation of adipose-derived stem cells(ADSCs). Methods ADSCs were extracted from adipose tissue donated voluntarily by patients undergoing liposuction by enzymatic digestion. The 3 rd generation ADSCs were inoculated on CultiSpher G and SF-PLLA microcarriers(set up as groups A and B, respectively), and cultured in the rotary cell culture system. ADSCs cultured in normal two-dimensional plane were used as the control group(group C). Scanning electron microscope was used to observe the microcarriers structure and cell growth. Live/Dead staining and confocal fluorescence microscope was used to observe the distribution and survival condition of cells on two microcarriers. DNA quantification was used to assess cell proliferation on two microcarriers. Real-time fluorescence quantitative PCR(qRTPCR) was used to detect chondrogenesis, osteogenesis, and adipogenesis related gene expression of ADSCs in 3 groups cultured for 18 days. Flow cytometry was used to identify the MSCs surface markers of ADSCs in 3 groups cultured for 18 days, and differential experiments were made to identify differentiation ability of the harvested cells. Results ADSCs could be adhered to and efficiently amplified on the two microcarriers. After 18 days of cultivation, the total increment of ADSCs of the two microcarriers were similar(P>0.05). qRT-PCR results showed that chondrogenesis related genes(aggrecan, cartilage oligomeric matrix protein, SOX9) were significantly up-regulated for ADSCs on SF-PLLA microcarriers and adipogenesis related genes(peroxisome proliferator-activated receptor γ, lipoprotein lipase, ADIPOQ) were significantly up-regulated for ADSCs on CultiSpher G microcarriers, all showing significant differences(P<0.05). Flow cytometry and differentiation identification proved that the harvested cells of the two groups were still ADSCs.Conclusion The ADSCs can be amplified by SF-PLLA microcarriers, and the chon

关 键 词：丝素蛋白-左旋聚乳酸 微载体 动态培养 脂肪来源干细胞 

分 类 号：R318.08[医药卫生—生物医学工程]