| Background:The appearance of tissue engineering technology have provided a revolutionary routine for the problem of settling tissue defect thoroughly in clinic.However,the key of cellural component of tissue engineering is how to obtain the cells which have the characters of widely in origin,sufficient in quantity and well in quality.In recent years,the adipose derived stem cells(ASCs) from liposuction have become a hot spot.But ASCs is a mixed populatione residing within adult adipose tissue.Only about 40%of them can differentiate into adipocytes.So it can not satisfy the development of adipose tissue engineering,too.It is still a hot spot of finding a better kind of cells which is isolated easy,highly expandable in culture and highly differentiatable to adipocytes.Adipose tissue is heterogeneous.It contains fully mature adipocytes,vascular endothelial cells,fibroblasts,histiocytes and stem cells et al.Large lipid accumulations make mature adipocytes naturally buoyant and therefore difficult to culture.Little research has been conducted on the nature of them.They have also been considered to be in the terminal stage of differentiation and lacking proliferative activity.Studies,concluded by foreigners,suggested that mature adipocytes could be induced to undergo lipolysis,reinitiate proliferative mechanisms and begin to proliferate in vitro.We have termed this physiological transition,adipocyte dedifferentiation.We think that this may prove to be an emerging area of cell physiology and may amend present thinking about tissue renewal capabilities.However,study regarding the cell biology of the dedifferentiated adipocytes are still scarce.In our experiments we attempt to isolate pure mature adipocytes from the fatty portion of liposuction and induce mature adipocytes to dedifferentiate in order to get dedifferentiated adipocytes (DA).At the same time,the growth kinetics,morphology,surface marker profiles, differentiation capability and rate of adipose differentiation of the DA were compared to ASCs derived from the fatty potion.It provides a new approach to study the DA and learn more data about the DA,which will be used in adipose tissue engineering as cells for the first time.OBJECTIVE:1,To find a method which can be used for isolation,abstraction and cultivation of mature adipocytes from liposuction,to induce the dedifferentiation phenomenon of human mature adipocytes cultured in vitro,so we can get dedifferentiated adipocytes(DA).2,To provide the laboratory evidence for DA to be used extensively in tissue engineering by charactering and comparing DA and ASCs in five aspects:the growth kinetics,morphology,surface marker profiles,differentiation capability and rate of adipose differentiation.METHODS:Mature adipocytes and ASCs were harvested from human fat aspirates via liposuction.Mature adipocytes were cultured and induced to DA by ceiling adherent culture method,cell morphology were observed during the whole process.Viabilities of DA and ASCs were compared by MTT chromatometry and cell growth curves were drew based on it.Cell surface markers of DA and ASCs were detected by flow cytometry.The adipogenic,osteogenic and chondrogenic ability of DA and ASCs were assessed by oil red O staining,alizarin bordeaux staining and alcian blue s taining,respectively.The adipogenic ability of DA and ASCs were compared by microscope observation,absorption spectrometry assay of oil red O staining,and cell counting ofoil red O stained cells.RESULTS:Human mature adipocytes can dedifferentiate into fibroblast-shaped DA.MTT chromatometry assay demonstrated that DA and ASCs both have strong reproductive activity,there's no significant difference between them.Flow cytometry assay demonstrated that both DA and ASCs express HLA-ABC,CD29 and CD44,while don't express CD45,CD34 and CD106.After two weeks of osteogenic differentiation,calcium salts mineralization in DA and ASCs can be detected by alizarin bordeaux staining.After two weeks of chondrogenic differentiation,matrix of cartilage cells in DA and ASCs can be detected by alcian blue staining.After two weeks of adipogenic differentiation,lipid droplets can be displayed by oil red O staining in both DA and ASCs.We can see under microscope that there are obviously much more lipid droplet in DA than in ASCs.Absorption spectrometry assay of oil red O staining showed significant lipid droplet aggregation after DA was induced for 4 days.However,the same phenomenon cannot be seen in ASCs until 10 days' differentiation.After 12 days' differentiation,the optical density of DA is higher than that of ASCs,with significant difference.Oil red O stained cell counting demonstrated that the average adipogenic rate of DA and ASCs were 65% and 35%respectively,between which have a statistical significant difference. CONCLUSIONS:We can isolate and extract DA from the fatty portions of autologous liposuction aspirates of human.Mature adipocytes can dedifferentiate into DA in vitro.DA and ASCs have similar characters in growth dynamics,morphology,surface marker profiles and differentiation ability,etc.DA has strong reproductive activity,expresses some of the stem cell-related cell surface proteins,has osteogenic ability, chondrogenic ability and strong adipogenic ability.It is a promising seed cell of adipose tissue engineering. |
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