脂肪组织来源干细胞的细胞生物学研究

Cell biological study of adipose-derived stem cells

目的探索从脂肪抽吸物中脂质部分分离、培养脂肪组织来源干细胞(ASCs)的方法,并通过其生长动力学、形态学、分化能力、细胞衰老和表面标记物轮廓5个方面的特征进行鉴定。方法酶消化法处理脂类抽吸物,分离培养ASCs,观察细胞形态;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其表面分子表达;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;MTT比色法活性测定均证实ASCs具有很强的增殖活性,细胞周期研究发现ASCs具有干细胞的特性。丫啶橙染色3、4、6、8代无明显衰老。流式细胞仪检测显示干细胞标志的CD29、CD44、CD34的表达均成阳性;HLA-DR、CD133表达为阴性;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“0”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 approach to isolate and culture adipose-derived stem cells （ASCs） from the adipose tissue of liposuction aspirates, and conduct observations of the cell growth kinetics, morphology, differentiation capability, cell aging, and surface marker profiles. Methods From the liposucfion aspirates, ASCs were isolated by means of enzymatic digestion, and the appearance of the cultured cells was observed. The cell viability was evaluated with MTT chromatometry and cell growth curve was generated. Flow cytometry was performed for cell cycle analysis, and acridine orange staining was utilized for cell aging evaluation. The expressions of the cell surface marker profiles were 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 with a fibroblastic appearance and strong proliferation capacity as shown by MTT chromatometry. ASCs also showed characteristic stem cell cycle. Acridine orange staining of the ASCs at passages 3, 4, 6, and 8 did not reveal evidence of obvious cell aging. The expressions of CD29, CD44, and CD34 were observed in ASCs by flow cytometry while HLA-DR or CD133 expression was not detected. Expressions of VIII 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. Conclusion 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.