The Effects On Proliferation And Differentiation To Osteoblast Of Human Adipose Derived Stem Cells In Vitro Of Asperosaponin ?

Objective: 1 In this study,to hold the potential of differentiation while maintaining a large number of cells and provide a stable h ADSCs culture system for the next experiment,an effective method was developed for the extraction of human adipose derived stem cells(h ADSCs)in vitro.2 Through studying the effect of traditional Chinese medicine monomer asperosaponin ? for h ADSCs proliferation and osteoblast differentiation.Evaluting the impact of asperosaponin ? induces human adipose derived stem cells to the osteogenetic differentiation process.Exploring the possibility of asperosaponin ? as a cell growth factor was applied to bone tissue engineering,at the same time,it provided the experimental basis for the optimal selection of the seed cells of bone tissue engineering.Methods: 1 Isolation and cultivation of human adipose derived stem cells(h ADSCs)by enzyme digestion and centrifugation.Cell morphology was observed by inverted phase contrast microscope.The absorbance value of each period was detected by Thermo Scientific Microplate Reader and the characteristics of cell growth curve were analyzed.Differentiation into osteoblasts,adipocytes,and then alizarin red staining,oil red O staining.To ensure the cells were h ADSCs,and as the seed cells of follow-up experiments.2 The ASD dissolved into L-DMEM medium containing 10% FBS,the cell culture medium was prepared into four groups of c,at the same time,h ADSCs was induced by osteogenic inducer(0.1mol/L?-GP,10-8mol/L Dex,50mg/L VC).MTT method was used to detect the proliferation and differentiation of h ADSCs in different concentration ASD groups and positive control group.3 ALP method was used to detect the effects of different concentrations of ASD on osteogenic differentiation of h ADSCs,determining the optimal concentration;And then set up the blank group,the optimal ASD group,the positive control group,the optimal ASD and the positive control group,the difference of h ADSCs differentiation into osteogenesis was detected by ALP and alizarin red staining,to observe the ability of ASD to induce differentiation of h ADSCs into osteogenesis.Result: 1 With 0.1% collagenase type I to digest human adipose derived stem cells which were received from liposuction.2 days later,firstly changed cell-culture medium,cells were passaged when reached 80% degrees of fusion.The cell growth curve showed that the cell growth was "S" model,that is the stagnation period,logarithmic growth phase and plateau phase.After osteogenic and adipogenic induction were performed,alizarin red staining and oil red O staining were all positive.2 Different concentrations of ASD group and positive control group were co cultured with h ADSCs,MTT showed that ASD can promote the proliferation of h ADSCs in a certain concentration range(10-5mol/L?10-6mol/L),compared with the control group,the difference was statistically significant(P<0.05).Compared with the control group,there was no significant difference between the concentration of 10-5mol/L?10-6mol/L ASD groups and positive control group(P>0.05).Positive control group could not promote h ADSCs proliferation.3 ALD activity was measured after co-culture with h ADSCs for 1st,3rd,5th and 7th day in ASD and positive control.The concentration of 10-6 mol/L and 10-5 mol/L ASD were positive,compared with the blank group,there was statistical significance(P<0.05).The concentration of 10-5 mol/L was significantly different from 10-6 mol/L group(P<0.05),10-5 mol/L was the best induction group.Compared with the blank group,the optimal ASD group,the positive control group and the best ASD group and the positive control group were able to improve the activity of ALP on the 3rd?6th?9th day(P<0.05).At the same time point,the difference was statistically significant between the three groups: the best ASD group,the positive control group,the best ASD group and the positive control group.On the 12 th day,the ALP activity of each group decreased,the best ASD group + positive control group and the other three groups were statistically significant(P <0.05).After 2 weeks of induction,alizarin red staining was carried out.The result of staining was negative in the blank group.The best ASD group showed a small amount of calcified nodules with small number and small area.Positive control group and the best ASD group + positive control group showed obvious red staining calcification nodules,was orange,the best ASD group + positive control group compared with the positive control group,the larger area.Conclusion: 1 This experiment successfully separated and cultured h ADSCs from liposuction of the human adipose tissue.The cells can be stably passaged and proliferate in vitro,also differentiate into osteoblasts and adipocytes.2 ASD can promote the proliferation of third generation h ADSCs in vitro and differentiate into osteoblasts,increase ALP activity and up-regulate the expression of BMP2 gene.Therefore,ASD can be used as an inducing factor for osteogenesis differentiation of h ADSCs.As a cell growth factor used in bone tissue engineering has a certain feasibility,in order to further study the ASD to provide experimental basis.