从脂肪抽出物的脂类和液体部分提取脂肪来源干细胞的细胞生物学研究

目的 探索从抽吸物中脂质和液体部分分离、体外培养脂肪组织来源干细胞的新方法,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比较.方法 抽脂术后抽吸物分解为脂质和液体部分.脂质和液体部分分别用酶消化法和直接离心过滤法分离、培养ASCs,观察其在体外培养的形态学和生物学特点;MTT比色法测细胞活性,统计学分析;流式细胞仪测定细胞周期;随机选取3、4、6、8代做丫啶橙染色检测细胞的衰老;用流式细胞仪、免疫组织化学染色法鉴定其表面分子表达;成脂、成骨定向诱导分化,油红O染色、茜素红染色定性.结果 从吸脂抽吸物的脂质和液体两部分中都能培养出大量的ASCs,分别为PLA和LAF,呈成纤维细胞样贴壁生长,MTY测定细胞活性及细胞周期研究发现PLA、LAF这两种细胞的活力与增殖能力是非常相似的;丫啶橙染色3、4、6、8代细胞无明显衰老;流式细胞仪检测显示干细胞标志的CD29、CIM4、CD34的表达均呈阳性;免疫化学染色发现Ⅷ因子、CD31、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.成骨诱导2周后,细胞可见白色矿化钙盐沉积,茜素红染色可见成骨细胞红染.结论 本实验建立了一种自人体脂肪抽吸物中脂质和液体部分分离和培养ASCs的新方法,经济简便实用,从成人脂肪抽吸物液体部分中也可以分离得到大量的可为脂肪组织工程所利用的ASCs,其细胞量与脂质来源的ASCs的量基本相同.贴壁的LAF与PLA细胞在细胞的生长动力学、形态学、细胞衰老、表面标志物和分化能力等方面具有非常相似的特性,都具有很强的增殖活性且衰老率较低,能稳定表达干细胞表面标志并能实现定向成脂、成骨多向诱导分化.这种经过最小限度人工干预的ASCs可能是将来脂肪组织工程比较理想的种子细胞之一.

Cell biological study of cultured cells derived from the fatty and fluid portions of liposuction aspirates

Objective To explore an approach to isolate and culture the Adipose derived stem cens (ASCs)from the fatty and the fluid portions of liposuction aspirates,and to investigate the growth kinetics,morphology,differentiation capability,cell senescence,surface marker profiles of the ASCs.Methods The liposuction aspirates were divided into fatty portion and liquid portion.ASCs were isolated from each portion by collagenase digestion and directly centrifugate and cuhured to observe the morphology and biology characters in vitro.Cell activity was studied by MTT chmmatometry and analyzed statistically.Cell cycle was detected by flow cytometry.Cells were randomly selected from the 3rd,4th,6th,8th generation cells to dectect senescence of ASCs by acridine orange staining.The cell surface markers were detected by flow cytometry and immunohistochemistry.Adipogenic and osteogenic lineage differentiation of ASCs was assessed by Oil Red O and alizarin bordeaux staining respectively.Results A large amount of ASCs could be islated and cultured both from the fatty portion and the liquid portion.including PLA cells and LAF ceHs which had fibroblastic characters with strong viability and proliferative activity.The statistical result indicated that the cell activity of PLA cells and LAF cells was very similar.ASCs from passage 3、4、6、8 didn't show insenecence.CD29、CD44、CD34,which were the markers of mesenchymal stem ceils,vWF、CD31、CD105、SMA were all expressed in ASCs.Adipogenic differentiation of ASCs was assessed by Oil Red O staining after 2 weeks.The cells contained many lipid-filled droplets.After 2 weeks'osteogenic induction,cells were positively stained by alizarin Bordeaux.Conclusions The method can isolate ASCs by directly centrifugate from the fatty and the fluid portions of human liposuetion aspirates.The way of culture is convenient and economical.ASCs isolated from the liquid and fatty portions of liposuction aspirateS show identical in cells numbers and quality.LAF cells and PLA cells have similar characters in growth dynamics,morphology,cell senescence,surface marker profiles and differentiation ability,etc.Expression of the ceil surfaee marker of stem cells is also observed in ASCs.ASCs can differentiate into adipose and osteogenesis directionally.The results suggest that the ASCs,which are isolated with minimum intervention,may be the ideal seed cells for adipose tissue engineering in future.