| Osteoporosis is a systemic metabolic bone disease characterized by decreased bone mass and damaged bone tissue microstructure,resulting in increased bone fragility and increased fracture risk.The fundamental reason for its occurrence is that the bone resorption of osteoclasts is greater than the bone formation of osteoblasts.However,BMSCs present in bone marrow are a type of pluripotent adult stem cells that can differentiate not only into osteoblasts but also into adipocytes.It has been found that BMSCs are more divided into osteoblasts and less into adipocytes during childhood and adolescence.On the contrary,BMSCs were more differentiated into adipocytes and less into osteoblasts in old age.These studies suggest that the direction of BMSCs differentiation is closely related to the occurrence of osteoporosis.Therefore,if the osteogenic differentiation of BMSCs can be effectively promoted or the adipogenic differentiation of BMSCs can be effectively inhibited,the effect of bone formation can be effectively promoted,thus achieving the effect of osteoporosis treatment.A large number of studies have found that drugs can promote the bone formation of osteoblasts through the directional induction of osteogenic differentiation of BMSCs,thus achieving the effect of reducing osteoporosis.At present,many traditional Chinese medicines and the ir monomer components have been found to have significant effects on the treatment of osteoporosis and the increase of peak bone mass.It has been proved in our laboratory that a variety of traditional Chinese medicine monomers,including icariin,isopalen and isopalen,have significant effects on the activity of osteoblasts,the ability of mineralization and the increase of peak bone mass in rats.Amylolactone is a sesquiterpene lactone extracted from Amylolactone,which has anti-inflammatory,anti-cancer,anti-fungal and other pharmacological activities.Many reports have shown that Amylolactone has antioxidant and anti-inflammatory properties and inhibits RANKL-induced osteoclast differentiation of HSCs in a dose-dependent manner.It has been reported that Amylolide can effectively reduce the inflammatory signaling pathway NF-κB and activate the Wnt/β-catenin signaling pathway.Therefore,it was considered whether Amylolactone could promote the differentiation of BMSCs into osteoblasts.In this study,Amylolactone was used as an inducer to detect the induction effect of Amylolactone on osteogenic differentiation of BMSCs into osteoblasts in vitro,to study the effect of Amylolactone on osteogenic differentiation of rat bone marrow mesenchymal stem cells,and to explore the application value of Amylolactone in treatment and prevention of osteoporosis.The paper is reported as follows:Main research methods used in this thesis:1.The whole bone marrow adherent culture method between the extraction and isolati on and culture of rat bone marrow mesenchymal stem cells,through the cell morphological observation,induction training after 9 d into osseous organization chemical staining and ALP osseous induction training after 12 d calcified nodules histochemical sta ining establish rBMSCs in vitro cultivation system and induced into osseous system;2.By comparing the effects of different concentrations(1×10-8mol/L～1×10-5mol/L)of Amylolide on ALP activity after 9d of osteogenic induction culture of rBMSCs,the optimal concentration of Amylolide for osteogenic induction of rBMSCs was determined and used for subsequent experiments;3.By detecting the best concentration of combination of hydrocarbon lactones of rBMSCs into osseous induction training after 9 d ALP posit ive clone formation,the influence of osseous induction training after 12 d calcified nodules positive clone formation,the influence of osseous induction training after 24 h,48 h and 96 h osteogenesis related gene（Collagen1,RUNX2,OSX,BMP-2）m RNA expression,the influence of osseous induction training after 48 h and 96 h osteogenesis related protein（Collagen1,RUNX2,OSX,BMP-2）protein expression and the effect of To determine the effect of costinolide on osteogenic differentiation of rBMSCs;4.The effects of optimal concentration of Amylolides on lipid droplet formation after osteogenic induction culture of rBMSCs at 9d and the m RNA expression of adipose marker genes（PPARγ,C/EBPα,C/EBPβ,Adiponectin）after osteogenic induction culture at 24h,48h and 96h were determined to determine the effects of Amylolides on adipogenic differentiation of rBMSCs.5.Wnt10b,β-catenin,OPG and RANKL,key factors of Wnt/β-catenin and OPG/RANKL/RANK signaling pathway,were detected by detecting the optimal concentration of Amylolactone osteogenetic induction culture of rBMSCs for 24h,48h and 96h The expression of Wnt/β-catenin and key factors of OPG/RANKL/RANK signaling pathway Wnt10b,β-catenin,OPG and RANKL after 48h and 96h of osteogenic induction culture were investigated to determine the mechanism of Amylolactone promoting osteogenic differentiation of rat bone marrow mesenchymal stem cells.The main experimental results obtained in this paper are as follows:1.1×10-6mol/L Amylolactone has the best activity of promoting the osteogenic differentiation of rBMSCs;2.The formation of ALP positive clones in Amylolide group was significantly higher than that in control group（P<0.01）;3.The positive clones of calcification nodules in costolide group were signific antly higher than those in control group（P<0.01）;4.The m RNA expression levels of Collagen1,Runx2,OSX and BMP-2 in Amylolide group were significantly higher than those in control group after 24h,48h and 96h treatment of BMSCs.5.The expression levels of Collagen1,Runx2,OSX and BMP-2 in Amylolide group were also significantly higher than those in control group after treatment of BMSCs for 48h and 96h.6.The lipid droplets in costolide group were significantly lower than t hose in control group（P<0.01）;7.The m RNA expression levels of PPARγ,C/EBPα,C/EBPβand Adiponectin in Amylolide group were lower than those in control group after 24h,48h and 96h treatment of BMSCs.8.The m RNA expression levels of Wnt10B,β-catenin and OPG in costunolide group were higher than those in control group after 24h,48h and 96h treatment of BMSCs（P<0.01）,and the m RNA expression level of RANKL in costunolide group was significantly lower than that in control group（P<0.01）.9.The protein expression levels of Wnt10B,β-catenin and OPG in Amylolide group were significantly higher than those in control group after trea tment of BMSCs for 48h and96h.Meanwhile,RANKL protein expression level was significantly lower than the control group.The above results indicated that rBMSCs were successfully isolated and cultured in this study,and 1×10-6mol/L Amylolide was selected as the optimal drug concentration to induce osteogenic differentiation of rBMSCs.The results showed that Amylolides could not only promote the osteogenic activity of rBMSCs,but also inhibit the adipogenic differentiation of rBMSCs.Amylolides also significantly increased the activity of Wnt/β-catenin signaling pathway,β-catenin and OPG/RANKL ratio.Based on the above results,we hypothesized that abiolac tone may promote the osteogenic differentiation of rBMSCs through the combined influence of Wnt/β-catenin signaling pathway and OPG/RANKL/RANK signaling pathway. |
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