| Objective:To conduct the culture and passage of adipose-derived stem cells(ADSCs)and to observe and identify them,to study the biological characteristics of adipose-derived stem cells in the process of differentiation and culture and proliferation,the proliferation activity and differentiation ability of the adipose-derived stem cells were compared and analyzed.It lays a foundation for further therapeutic experimental research.Methods:adipose-derived stem cells were isolated and subcultured,and the isolated cells were identified by flow cytometry(FCM)detection by observing morphological characteristics and surface markers CD29,SCA-1,CD34 and CD45.P3,P6,P10 and P15 generations with good growth were selected from cultured adipose-derived stem cells,and the proliferation ability of adipose-derived stem cells was identified by using CCK8 kit detection,flow cytometry detection after PI staining,and western blot detection of CyclinD1 and CDK4 expression.Different generations of P3,P6,P10 and P15 adipose-derived stem cells were selected,and osteogenic differentiation was detected by alizarin red staining after osteogenic induction and differentiation,and expression of osteogenic genes Runx-2,ALP and Osterix were detected by QPCR.Results:1.adipose-derived stem cells were obtained from rat adipose tissue after separation and purification,after subculture,it was observed that the adipose-derived stem cells were transferred to the third generation with high purity and active growth,at this time,the adipose-derived stem cells were polyfusiform,spiral-shaped and S-shaped.The isolated adipose-derived stem cells were identified by flow cytometry after the reaction of CD29.SCA-1,CD34 and CD45 antibodies with adipose-derived stem cells.The results showed that the cell content of both CD29 and SCA-1 proteins was 97.567%,both of which were positive,and CD34 positive cells were 6.919%.CD45 positive cells were 0.839%and CD34 and CD45 were both negative.The isolated cells were identified as adipose-derived stem cells.2.CCK8 kit detected the cell growth of adipose-derived stem cells P3,P6,P10 and P15 in different generations,and showed that the proliferation activity of P3 and P6 cells was vigorous at 24h.48h,72h and 96h,while there was no significant difference between P3 and P6.The proliferation activity of P10 cells was weakened,and that of P15 cells was further weakened.Flow cytometry was used to detect the cycle distribution of adipose-derived stem cells in P3,P6,P10 and P15 generations,and the results showed that there were fewer cells in the G0/G1 phase in P3 and P6 generations than those in P10 and P15 generations.Western blot analysis detected CyclinDl and CDK4 in P3,P6,P10 and P15 generations,showing that CyclinDl and CDK in P3 and P6 generations were in low expression,while the expressons of CyclinD1 and CDK in P10 and P15 generations were relatively high.3.Alizarin red staining after the osteogenic differentiation of adipose-derived stem cells from P3,P6,P10 and P15 showed that the low generation of adipose-derived stem cells were more easily induced and differentiated than those from P10 and P15.The expression of osteogenic genes Runx-2,ALP and Osterix in adipose-derived stem cells of different generations of P3,P6,P10 and P15 was detected by QPCR,and the results showed that the expression of Runx-2,ALP and Osterix genes in the lower generation of P3 and P6 cells was significantly higher than that of P10 and P15 cells.Conclusion:1.Rat adipose tissue was isolated,dedifferentiated and subcultured,and the cultured cells were identified as adipose-derived stem cells by observation and detection.2.In the proliferation ability test of adipose-derived stem cells of different generations of P3,P6,P10 and P15,the proliferation activity of the lower generation of P3 and P6 cells was significantly higher than that of P10 and P15 cells.3.In the osteogenic induction and differentiation test of adipose-derived stem cells in different generations of P3,P6,P10 and P15,the differentiation ability and differentiation degree of adipose-derived stem cells in the lower generation of P3 and P6 were significantly higher than those in the P10 and P15 generations.4.From the perspective of proliferative activity and differentiation ability,the low generation adipose-derived stem cells have stronger proliferative activity and differentiation ability,and are more suitable for culture,further experiment and treatment. |
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