脂肪组织来源干细胞的分离培养及鉴定

Isolating culture and identification of adipose-derived stem cells

目的探讨脂肪组织来源干细胞(adipose-derived stem cells,ASCs)的分离、培养、分化和鉴定的方法,为进一步的实验研究提供理论依据。方法酶消化法处理脂类抽吸物,分离培养ASCs,观察细胞形态;四甲基偶氮唑盐(MTT)比色法测细胞活性并绘制细胞生长曲线,流式细胞仪测定细胞周期,丫啶橙染色检测细胞的衰老;流式细胞仪、免疫组织化学染色法鉴定表面分子表达;成脂定向诱导分化后油红"O"染色定性。结果原代培养的ASCs呈成纤维细胞样贴壁生长,具有很强的增殖能力;细胞周期研究发现ASCs具有干细胞的特性。流式细胞仪检测显示干细胞标志的CD29、CD44、CD34的表达均呈阳性;免疫化学染色发现VШ因子、CD31、CD34,CD105、SMA表达阳性;成脂诱导分化2周后,细胞内可见有大量脂滴,油红"O"染色可见胞浆内有大量红染颗粒。结论自人体脂类抽吸物中通过酶消化法能分离和培养出具有干细胞特性的成纤维细胞样细胞群,该细胞具有很强的增殖活性且不易衰老,能稳定表达干细胞表面标志并能实现定向成脂分化。

Objective To explore a way to isolate and culture adipose-derived stem cells（ASCs） from the liposuction aspirates,and observe the cell growth kinetics,morphology,differentiation capability,cell aging,and surface marker profiles.Methods From the liposuction aspirates,ASCs were isolated by enzymatic digestion,and the cultured cells appearance observed.The cell viability was evaluated with MTT chromatometry.Flow cytometry was performed for cell cycle analysis,and acridine orange staining was utilized for cell aging evaluation.The surface molecule expression was detected by flow cytometry and immunohistochemistry.Adipogenic differentiation of ASCs was assessed by Oil Red O staining.Results Primarily cultured ASCs adhered to the culture plates had a fibroblastic appearance and strong proliferation capacity as shown by MTT chromatometry.ASCs also showed characteristics of stem by cell cycle analysis.The expressions of CD29,CD44,and CD34 were observed in ASCs by flow cytometry.Expressions of VШ factor,CD31,CD34,CD105 and SMA were observed in ASCs by immunohistochemistry.Oil Red O staining of the ASCs after 2 weeks of culture demonstrated numerous intracellular lipid droplets.Conclusions ASCs can be isolated from autologous liposuction aspirates of human via enzymatic digestion and cultured ex vivo.These cells are fibroblast-like cells expressing cell surface markers of stem cells with strong proliferative ability,and can be induced to differentiate into adipose tissue.