Comparing The Expression Of Markers In Different Stages Of ATDC5 Cell Proliferation And Differentiation

Cartilage injury is a common clinical disease,including degenerative lesions that occur after wear and aging,it is difficult to repair the cartilage once it is damaged,due to the special organizational structure,such as Lack of blood vessels and nerves.ATDC5 is an important chondrocyte model,which has been widely applied to the study of chondrocyte proliferation and differentiation.However,there are few studies on the changes in the markers of ATDC5 cells in the stages of proliferation and differentiation.This experiment will clarify that whether the ATDC5 cell line has the same staging characteristics as chondrocytes,such as resting,proliferating,hypertrophic and calcification.Through the detection of ATDC5 cell markers in different days,identify the differential expression of relevant marker genes and proteins at each stage of chondrocyte differentiation,and defined the stage of proliferation and differentiation of the cell line.To confirm whether ATDC5 cells can mimic the proliferation and differentiation process of chondrocytes in vitro,and provide a reference for the further experimental study of ATDC5 cells.In this experiment,ATDC5 cells were used as an in vitro model for chondrocytes,and the expression of the marker factors at various stages of proliferation and differentiation of chondrocytes was examined at four time points,4 days,14 days,and 21 days.The ATDC5 cells were cultured in different days,and the cell proliferation,activity and morphological changes of ATDC5 cells were determined from the cell level.The cells were stained with aliline blue and alizarin red S,and the proteoglycan secretion and cell calcification were detected respectively;the changes in the activity of alkaline phosphatase were detected at each time point,too.The relative expression levels of Fn1,Sox9,type II collagen(Col2),Aggrecan,Runx2,type X collagen(Col10)and type I collagen(Col1)gene were detected by reverse transcription polymerase chain reaction and Western blotting at the gene and the protein level.The results showed that compared with 4 days,alcian blue staining was stained deeper on days 14 and 21,the alizarin red showed that the number of calcified nodules increased with the number of culture days.Through further detection of gene and protein levels,the mRNA expression of Fn1 and Sox9 were increased significantly at 7 days and decreased significantly after the cell entered the mature stage.The expression of Sox9 protein showed the same trend with the trend of mRNA.The mRNA and protein expression of Col2 peaked at 14 day,the expression of Col2 began decreased at 21 days of culture(P<0.001).Compared with cultured for 4 day and 21 day,the mRNA expression of Aggrecan was significantly increased in 7 day and 14 day,and the protein expression of Aggrecan was increased significantly at 14 days and 21 days compared with 4 days and 7 days of culture(P<0.01).The mRNA expression level of Runx2 peaked at 14 day,and was significantly different from that at 7 day(P<0.01)and 21 day(P<0.001).The protein expression level of Runx2 was significantly increased at 14 day and 21 day.The mRNA expression of Col10 was significantly increased in 14 days and 21 days compared with 4 days and 7 days(P<0.001),the expression of protein was in accordance with the trend of gene expression.The mRNA expression level of Col1 was increased at 21 days,which was extremely significant compared with 4 days,7 days,and 14 days(P<0.001).The activity of ALP was the highest in 14 days,and remained the same high activity until 21 days,there was a downward trend compared with 14 days,the difference was not significant.At the end of differentiation,ATDC5 cells undergo hypertrophy and secrete Col10,the activity of ALP was increased.Col2,Aggrecan,Col10,Runx2 and ALP are the hallmarks of different stages of chondrocyte proliferation and differentiation.These cell markers are present in the extracellular matrix.The trend of these factors will reflect the stage of ATDC5 cell proliferation and differentiation.The experimental results show that the ATDC5 cells were cultured to 7 days,ATDC5 cells were in the resting period;after 14 days of culture,ATDC5 cells were in the proliferative phase,and the expression of prehypertrophic markers increased;After 21 days,ATDC5 cells mainly showed the characteristics of the hypertrophic chondrocytes,and have a tendency to transform into osteoblasts.Therefore,this study suggests that ATDC5 cells can complete the entire process of endochondral osteogenesis by cell fusion,aggregation,formation of calcifications and mineralization of cells,and the process include chondrocyte differentiation,cell proliferation,and chondrocyte mineralization.