Study Of Naringin On Promoting Osteogenic Differentiation And Migration Of MSCs Under The Guidance Of Bushen Huoxue Therapy

Part ? Isolation,Culture and Identification of Bone Marrow Mesenchymal Stem CellsObjective:Isolation and culture of Mesenchymal Stem Cells(MSCs)to provide sufficient seed cells for subsequent experiments.Methods:MSCs were isolated and cultured by Percoll density gradient separation method.The three-line differentiation of mesenchymal stem cells was induced by osteogenic differentiation induction medium,chondrogenic differentiation induction medium and adipogenic differentiation induction medium.Results:The MSCs were in good condition.The three lines were identified to show that the extracted MSCs could differentiate into osteoblasts,chondrocytes and adipocytes.Conclusion:The isolated cultured cells are MSCs and can be used in subsequent experiments.Part ? Effect of different oxyg en concentrations on proliferation,osteogenesis and migration of MSCsObjective:To study the changes of proliferation,osteogenic differentiation and migration of mesenchymal stem cells(MSCs)under different oxygen concentrations,and to observe the osteogenic differentiation and migration ability of MSCs at different durations in 1%hypoxic environment.In order to explore hypoxic environment caused by blood stasis and the mechanism of action of oxygen environment on the repair ability of congenital MSCs.Methods:The MSCs were cultured in an incubator with 20%,5%,and 1%oxygen concentrations to simulate normal oxygen,mild hypoxia,and extreme hypoxia.The morphological changes of cells at different time points were observed at 3,5,and 7 days.The cell growth curve was drawn by CCK-8 method.The expression of alkaline phosphatase(ALP)was detected at different time points of 3,5 and 7 days.The formation of mineralized nodules was detected 2 weeks after osteogenic induction.The cell migration ability was detected by Tranwell test.The MSCs were cultured in 1%oxygen concentration,and the mRNA expressions of key genes BMP-2,Runx2 and SDF-1? and CXCR4 were detected by qPCR at Oh,24h,48h and 72h.Results:At 20%and 5%oxygen concentration,the morphology of MSCs cells was good,and the morphology of cells changed at 1%oxygen concentration;the number of cells increased at 5%oxygen concentration,and the number of cells decreased at 1%oxygen concentration(P<0.05);The expression of ALP was significantly lower than that of 20%oxygen concentration at%and 1%oxygen concentration(P<0.05).The formation of mineralized nodules was significantly lower than that of 20%oxygen concentration at 5%and 1%oxygen concentration(P<0.05).The cell migration number of 5%and 1%oxygen concentration was significantly lower than that of 20%oxygen concentration;the mRNA expression of BMP-2,Runx2,SDF-1? and CXCR4 decreased with time at 1%oxygen concentration(P<0.05).Conclusion:Mild hypoxia can promote the proliferation of MSCs.Extreme hypoxia inhibits the growth of MSCs.Hypoxia can reduce the osteogenic differentiation and migration of MSCs.Therefore,the repair capacity of MSCs is severely affected by the hypoxic environment caused by blood stasis.Part III Determination of the optimal concentration of naringin under normoxia and its mechanism of affecting the expression of osteogenic signal axisObjective:To determine the optimal naringin concentration for MSCs and to examine its effect on the expression of BMP-2/Smads/Runx2/Osterix in MSCs.Methods:Concentration gradient naringin culture medium was used.qPCR was used to detect the mRNA expression of key morphogenetic genes BMP-2,Runx2 and migration key genes SDF-1? and CXCR4 by different concentrations of naringin.CCK-8 method was used to detect the proliferation of MSCs by concentration gradient naringin,and the optimal concentration of naringin was determined.MSCs were treated with optimal concentration of naringin,negative control SiRNA and Runx2 specific SiRNA.After 3 days,Western blotting was used to detect the protein expression level of osteogenic signal axis BMP-2/Smads/Runx2/Osterix,and the expression level of ALP was detected 1 week later.Results:Different concentrations of naringin could promote osteogenic differentiation of MSCs(P<0.05),but high concentration of naringin inhibited migration and proliferation of MSCs(P<0.05).Low concentration of naringin had no effect on migration and proliferation.(P>0.05),1×10-4mol/L was determined as the best concentration;Western blotting showed that the optimal concentration of naringin could promote the expression of BMP-2/Smads/Runx2/Osterix(P>0.05),SiRNA treatment Reduced osteogenic expression of posterior osteogenesis.Conclusion:The optimal concentration of naringin is 1 × 10-4mol/L.Naringin can promote osteogenic differentiation of MSCs through BMP-2/Smads/Runx2/Osterix axis;and naringin has no effect on the migration and proliferation of MSCs;Part IV Mechanism of osteogenesis and migration of MSCs by naringin combined with SDF-la adenovirus in extremely low oxygenObjective:According to the principle of Bushen Huoxue,MSCs with only osteogenesis and serotonin-promoting SDF-1 a adenovirus were combined and placed in a culture environment of 1%oxygen concentration to verify extreme hypoxia.Whether the combination of the two can still promote the osteogenic differentiation and migration ability of MSCs.Methods:Simulating extreme hypoxic conditions with 1%oxygen concentration and treating MSCs with optimal concentrations of naringin,Ad-GFP and Ad-SDF-laAfter 3 days,the expression of BMP-2/Smads/Runx2/Osterix and CXCR4 was detected by qPCR and Western blotting.The expression of CXCR4 protein was detected by immunofluorescence.The expression of SDF-1? was detected by Transwell assay,and the migration ability of MSCs was detected by Trans well assay.Mineralized nodule formation was detected after 2 weeks.Results:Naringin,Ad-SDF-1 a and naringin+Ad-SDF-1? significantly promoted the expression of BMP-2/Smads/Runx2/Osterix and increased osteogenic differentiation ability(P<0.05).Ad-SDF-1? and naringin+Ad-SDF-1? can promote the expression of SDF-1?/CXCR4 and improve the migration ability of MSCs(P<0.05).Conclusion:Naringin can synergize with Ad-SDF-1? in extremely low oxygen environment to improve the osteogenic differentiation and migration of MSCs.Moreover,Ad-SDF-1? can promote osteogenic differentiation of MSCs under extreme hypoxic conditions,but it has no significant effect on late osteogenic differentiation,and naringin can promote the whole process of osteogenic differentiation of MSCs..