A Preliminary Study On Transdifferentiation Between Osteoblast And Adipocyte Under The Condition Of Transwell Indirect Co-culture System

Objective: Constructing a co-culture system of osteoblast and adipocyte, under this condition, we will study the trend of osteoblast transdifferentation into adipocytes. Using this experimentation investigate the possibility and mechanism of osteoporosis,and this pre-experiment is prepare for futher study about reduction in micro environment.Methods: Chose Logarithmic phase preadipocyte 3T3-L1, induce the preadipocyte to adipocyte.After identifition of adopocyte in the way of oil red Ostainig, we constrct the system of Transwell inoculating 3T3-L1(adipocyte group) in the bottom chamber, and inoculation MC3T3-E1(treatment group) in top chamber, according to a ratio of 1:4.Seperate culture control control group.Cells were observed on 7d, 14 d, 21 d, we study on the change of cell morphology, experiment different of red oil O staining, alizarin red staining, MTT inhibition rate,expression of the relative content of triglyceride(TG) on 0h, 24 h, 36 h,expression of PPARgama2.Result:1.After culturing 2weeks,pre-adipocyte chanced from spindle to round, lipid droplet became larger in the cytoplasm of cells,after oil red Ostaining, we reserved the adipocyte. 2.Observating in microscope,cell of Bgroup rarely changed in 7d and 14 d,on 21 d,MC3T3-E1 shape has changed from long spindle to round,more and more lipid droplet has comeout. The control group cells cultured for up to 21 days no significant cell morphological change; a group of cells were always carry bright fat droplets of round cells had no obvious change. 3.Oil red O staining showed in group B cell staining area presenting increasing trend, and is proportional to the time, cells in control group no significant changes. Triglyceride relative amount detection results showed that treatment group cell triglyceride accumulation amount of presenting increasing trend, its growth is proportional to time compare with intergroups and the control group difference was statistically significant(P< 0.05), the changes in control group were no statistical significance(P > 0.05). 4. Alizarin red staining results showed that treatment group cells from 7 days, 14 days, 21 days showstaining region decrease trend; control group cells had no significant changes. 5.MTT inhibitory rate test results show that treatment group cell proliferation inhibition rate is proportional to the time, the difference between the C group and the comparison group, there is statistical significance(P<0.05), compared with the control group,the difference was statistically significant(P<0.05). 6.Relative content of triglyceride showed that the amount of TG increased over time,treatment group compare with control group,the different is stastically sig nificant(P<0.05). 7.Western blot showed in treatment group cell PPAR gamma 2 protein expression quantity increased progressively, and is proportional to the time, between groups and adipocyte groups and control group were compared, differences were statistically significant(P<0.05), adipocyte group PPAR gamma 2 protein expression did not change significantly, the difference was not statistically significant(P > 0.05).Conclusion: 1.In Transwell indirect co culture system, osteoblast under the mature adipocytes induced, will shows the ability of dedifferentiation, PPAR gamma2 express ion amount is proportional to the time, inversely proportional to the amount of calcium nodules; 2.mature fat cells compared to cross to differentiation into bone cells in the early stage of target protein PPAR gamma 2 expression andadipocyte, there is a clear difference occurred, is horizontal differentiation into adipocyte to be the next step research;3.adipocyte metabolism and cytokines can significantly inhibited the proliferation of osteoblast.