| Background: With the improvement of living standards,the number of obese population in the world has been increasing by year,and excessive energy intake is the main cause of obesity.Obesity leads to an increase in adipose tissue.In addition to common white and brown fats in fat tissue,bone marrow adipose tissue(BMAT)appears as a “new” fat reservoir,which can account for 5% of the total fat,as a unique fat reservoir the importance of BMAT has been confirmed in bone-fat interactions.And as an endocrine organ,adipose tissue can secret adipokines whchi has a great effect on body.Recently studies have found that Adipokines whchi secreted by adipose tissue plays an important role in bone formation and metabolism.Adipokines can influence the formation of bone by regulating the differentiation process of bone marrow mesenchymal stem cells,osteogenic differentiation and adipogenic differentiation.The fat family includes leptin,adiponectin,s FRP5(secretory frizzled-related protein-5)and so on.As the function of structural characteristics,s FRP5 can be a member of the secreted Frizzled-related protein(s FRP)family,it is also an inhibitor ofWNT signaling pathway.Some studies have found that high-fat diet-induced obese mice have abnormal bone development and the serum s FRP5 expression is significantly increased.We selected C57 and ob/ob mice(leptin-deficient mice)for high-fat diet induction.The purpose of this study was to construct high-fat-induced obese mice by testing the association of s FRP5 with bone changes in high-fat mice.To investigate the effect of s FRP5 on the skeleton of obese mice and its mechanism.At the same time,we also examined changes in bone autophagic levels in obese mice and changes in autophagic levels during differentiation of bone marrow mesenchymal stem cells.PART I The changes of s FRP5 in bone and the changes of bone in obese mice induced by high fat dietObjective: to construct obese mice and detect the correlation between bone changes and s FRP5.Methods: 20 male C57 mouse and 20 ob/ob male mouse were divided into a high-fat diet group and a normal diet group and measured the weight weekly.After feeding 10 weeks,we collect the serum blood glucose,high-density lipoprotein,low density lipoprotein,triglyceride and other biochemical indicators,and measures the mouse of length,fat and liver weight.The side of femur were detected through Micro-CT.After thefemur was made into slices,the slices were treated by Masson staining HE staining,Alcian Blue and immunohistochemical.The other side of the femur was frozen in liquid nitrogen,and the RNA was extracted and PCR was performed to measure the lever of s FRP5 and related factors.Results: The weight and body length of the mouse induced by high-fat diet were increased than that of the control group after 10 weeks of feeding(P<0.05).The biochemical indexes were significantly higher than the control group(P<0.05).The obese mouse model was successfully constructed.Micro CT examination results showed a significant decrease in bone mass and bone trabecula in the control group(P<0.05).HE,Alcian Blue,Masson staining also found the same result.The results of PCR and immunohistochemistry showed that the high fat group of s FRP5 increased,and the expression of lipid-related factors increased.Conclusion: After high fat feeding,the bone mass of obese mice decreased..The expression of s FRP5 in the high-fat group was increased,and the Ob/ Ob changes more.PART II The effect and mechanism of s FRP5 on differentiationof bone marrow mesenchymal stem cellsObjective: To isolate and identify the bone marrow mesenchymal stem cells in mice and to investigate the effect of s FRP5 on bone marrow mesenchymal differentiation in mice.Methods: Separation of 4 weeks male mouse femoral bone marrow mesenchymal stem cells,we use flow cytometry to detect cell surface molecular markers,and adopt oil red O staining to detect lipid drops;ALP and alizarin red detected osteogenic conditions.We divived the cell into RFP + osteogenesis medium,s FRP5 + osteogenesis medium,RFP +ordinary culture medium,the s FRP5 + common medium.After treated,the four group were stained by ALP staining,oil red O staining,alizarin red staining.Four groups of RNA and proteins were extracted,respectively by PCR and Western blot to detect the osteogenic lipid factor and WNT signaling pathway.Results: The expression of adiponectin,PPAR-r,and the m RNA of osteogenic transcription factor runx2 were detected in the four groups,and the expression of adipogenic factors in the Ad-s FRP5 group was increased,and the expression of osteogenic factors was decreased.Conclusion: s FRP5 can regulate the differentiation of bone marrow mesenchymal stem cells through WNT signaling pathway to inhibit its osteogenic differentiation and promote the dipogenic differentiation.Part III :The role of autophagy in high fat-induced obese miceObjective: To explore the levels of bone autophagy in obese mice induced by high-fat diet and the changes of autophagy during differentiation of bone marrow mesenchymal stem cells.Methods: The expressions of Beclin-1 and LC3,autophagy-related factors in high-fat obese mice,were detected by PCR.The expression of Beclin-1 in bone was detected by histochemistry.The proposed bone marrow mesenchymal stem cells were divided into adipogenic medium,osteogenic medium and common medium for treatment,and autophagosomes were observed under electron microscope.After the cells were transfected with the LC3 double plasmid,three groups of treatments were performed and observed under the laser confocal microscope.Three groups of proteins were extracted and western blot was used to detect the expression of three autophagy-related proteins.After three groups of cells were treated with 3MA(autophagy inhibitor),oil red and alizarin red staining were used to observe the changes of their adipogenic capacity.Results: The expression of autophagy-related genes was detected after high-fat diet.The expression of Beclin-1 and LC3 in bone was increased after high-fat diet.The results of histochemistry showed that the expression of Beclin-1 in bone increased.After induction of electron microscopy,theautophagy bodies were increased after induction of adipogenesis in three groups of cells.Western blot showed that autophagy-associated protein increased after induction of adipogenic culture.Osteogenic medium also increased after induction.After 3MA inhibition,oil red staining and alizarin red staining were reduced.Conclusion: High-fat diet induced increased autophagy in bone tissue.Autophagy was significantly increased in the adipogenic differentiation of bone marrow mesenchymal stem cells.Inhibition of autophagy inhibits osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells. |
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