| In order to study the effect of leptin on the proliferation and differentiation of ATDC5 cell,ATDC5 cell was used as chondrocyte model,and 0 ng/m L(control group),10 ng/m L and 100ng/m L were used in three different concentrations of leptin stimulate ATDC5 cells.ATDC5 cells were cultured 14 d,carry out Alcian blue staining,The relative expression of type Ⅱ collagen,Sox9 and Aggrecan mRNA was detected by RT-PCR,The relative expression of Sox9 and Aggrecan protein was detected by Western blot;After 21 d,carry out alizarin staning,the expression level of typeⅩ collagen mRNA was detected by RT-PCR,the relative expression of collagen type X was detected by Western blot.After that,measured the effect of leptin on alkaline phosphatase activity.The results showed that Alcian blue staining was not enough to prove the the impact of leptin on proteoglycan secretion;After alizarin red S calcified nodules staining,by checking the number of nodules found that with the increasing concentration of leptin,the number of calcified nodules was reduced.The expression of Sox9 mRNA decreased with the increase of leptin concentration,and there was significant difference between the experimental group and the control group(P<0.01),the difference between the two groups was significant(P<0.01).The relative expression of the Sox9 protein in the leptin concentration was 10 ng/m L and 100 ng/m L,which was significantly higher than that in the control group(P<0.01);With the increase of leptin concentration,the expression of type Ⅱcollagen m RNA increased at the concentration of 10 ng/m L,which was significantly different from the control group(P<0.05)and the expression level was decreased when 100 ng/m L,there was on difference with the control group(P>0.05),but it was significantly higher than the 10 ng/m L group(P<0.05).Compared with the control group,the expression of Aggrecan m RNA decreased with the increase of leptin concentration,and when the leptin concentration was 10 ng/m L,the gene expression level was significantly different from that of the control group(P<0.05),the leptin concentration was 100 ng/m L,the expression of m RNA was higher than the control group(P<0.01),the two leptin-treated groups was significant differently(P<0.01),the expression of protein was consistent with the expression of the gene,the expression of 100 ng/m L group was significantly higher than that of the control group(P<0.05),there was no significant difference between the two groups(P>0.05).The above results indicated that the increase of leptin concentration could inhibit the expression of Sox9 gene,but the expression of protein was increased.In a certain range,the increase in leptin concentration willpromote the expression of type Ⅱ collagen,but when the leptin concentration exceeds a certain range,type Ⅱ collagen secretion was declined;higher concentrations of leptin will inhibit the Aggrecan gene and protein expression;Sox9 can enhanced the activity of type Ⅱ collagen and Aggrecan promoter,while leptin decreased the expression of Sox9 gene,so the expression of typeⅡ collagen and Aggrecan decreased.Compared with the control group,the expression of type X collagen mRNA increased significantly with the increase of leptin concentration,and when the leptin concentration was 10 ng/m L,there was no difference between the control group(P>0.05),and the difference was significant with control group and other experimental group(P<0.01)at the leptin concentration was 100 ng/m L,the expression of collagen type X was significantly higher at the leptin concentrationthan was 10 ng/m L and 100 ng/m L,and there was significant difference between the two experimental groups and the control group(P<0.01).The results of alkaline phosphatase activity showed that ALP activity increased with the increase of leptin concentration.Chondrocyte differentiation in the hypertrophy,secretd type X collagen,leptin concentration will promote the secretion of collagen type X,inhibition of calcification,alkaline phosphatase activity increased.Type II collagen,Sox9,Aggrecan,type Ⅹ collagen and alkaline phosphatase was different marker factors secreted at different stages of cell proliferation and differentiation,which were present in the extracellular matrix.These changes reflected the state of the bone and joint,The combination of the indicators of the test results show that a certain concentration of leptin could inhibit ATDC5 cell proliferation and differentiation.Normal cartilage tissue tissue leptin content is very low,the experimental supply of exogenous leptin,equivalent a higher concentration of leptin levels.Therefore,the higher concentration of leptin in this study will inhibit the proliferation and differentiation of ATDC5 cells,and the higher the role of the stronger the higher the level of leptin,extracellular matrix structure damage,long-term cell hypertrophy stage,calcification blocked,Leading to bone formation disorders.The results of this experiment show that a certain concentration of leptin will inhibit the proliferation and differentiation of ATDC5 cells.Normal cartilage tissue tissue leptin content is very low,the experimental supply of exogenous leptin,the equivalent of a higher concentration of leptin levels.Therefore,this experiment is considered within a certain range,the higher concentration of leptin will inhibit the proliferation and differentiation of ATDC5 cells,and the higher the role of the stronger the higher the level of leptin,extracellular matrix structure damage,long-term cell hypertrophy stage,Calcification blocked,leading to bone formation disorders. |
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