Study On The Induced Differentiation Of Adipose Tissue Derived Mesenchymal Stem Cells Into Chondrocytes In Three Dimensional Culture Systems

Objective:1.In order to establish a stable extraction method for adipose tissue derived mesenchymal stem cells(ADMSCs),the research was carried out on three respects:donor age,location of fat obtained and extraction way.2.To Study the suitable conditions of the induced differentiation of adipose tissue derived mesenchymal stem cells into chondrocytes in three dimensional culture systems.Methods:1.Study on the ADMSCs' extraction:①ADMSCs were extracted with the enzyme-digestion-separate method,cultured in monolayer and passenged,identified the phenotype by detected marker molecule of stem cells:CD44,CD105 with immunofluorescence.②Cells morphous was observed by phase-contrast microscope; Cytoactive was observed by stained with trypan blue;Cells growth was detected by cytometry;Cell life cycle was detected by flow cytometry.③The yield of extracted ADMSCs and the biological activity of cultured cells were compared among the groups with different extraction methods,from different locations and ages.2.Study on the induced differentiation of adipose tissue derived mesenchymal stem cells into chondrocytes in three dimensional culture systems:①The third passenged cells were incubated in pellet culture and in suspension culture variously.Induced or common medium was added into the experiment group or the control group,collect samples on the 7,14,21 days.②The histological structure of neocartilage was observed by species stained with H&E;③Immunofluorescence analysis was performed to detect the expression of collagenⅡ;④The production of matrix was estimated from the determination of hydroxyproline content and Alcian Blue method.;⑤The collagenⅡand GAGs DNA expression level of neocartilage was measured by real time PCR.Results:1.The eugonic ADMSCs could be stably extracted from adipose tissue with the multi-digestion-separate method,and the yield is high,the cells activity was good.The proliferation activity of extracted ADMSCs was similar among the rabbits from different ages and locations,but to select adult individuality,to draw the materials among muscles, and in groin where the adipose tissue is abundant and unitary could raise the yield significantly.2.Immunofluorescence results show that most cells display masculine of CD44,CD105,it confirmed a good purity;growth curve exhibited cells proliferation pace doesn't step down with generation increased.3.ADMSCs formed cartilage-like tissue with distinct differentiated zones and regions in three dimensional culture systems in vitro.4.In three dimentional culture system and induction conditions,immunofluorescence results show that the 14,21 days samples of experiment group display masculine of collagenⅡ,the 7 days samples of experiment group and all the samples of control group display negative;The determination of hydroxyproline content and Alcian Blue method displayed in the experiment group the collagenⅡand GAGs content increased along the time,while the control group didn't show a tendency;Real time PCR.results display the expression of collagenⅡand GAGs rose along with culture in experiment group,and the pellet culture was super than the suspension culture.Conclusion:1.To select adult individuality,draw the materials from where the adipose tissue is abundant and unitary,extract with the multi-digestion-separate method could raise the yield significantly,and keep cells in good activity.2.ADMSCs can differentiated into the chondrocytes under the action of derivative in these two three dimensional cuture,and secret the chondrocyte charatered protein.3.Pellet culture or high-density culture could be helpful to enhanced cartilage character matrix production compare with the Suspension,culture or low-density culture,it can stimulate the induction effect.