Effect Of Compound Kidney-Invigorating Granule On The Expression Of MiR-545-3P In BMSCs-Exosomes From Senile Osteoporosis Patients And A Preliminary Study On The Regulation Of Osteogenic Or Adipogenic Differentiation Of BMSCs Via Wnt Signaling Pathway

Objective:1.To observe the difference in the expression of mi R-545-3p in exosomes(Exo)derived from bone marrow mesenchymal stem cells(h BMSCs)between senile osteoporosis(SOP)population and non-SOP population;2.Investigating the effect of Compound Kidney-Invigorating Granule(CKG)-containing serum on the expression of h BMSCs-Exo-mi R-545-3p in SOP population;3.Preliminary analysis of the effect of Compound Kidney-Invigorating Granule on h BMSC osteogenic or adipogenic differentiation in SOP population by regulating Wnt pathway through h BMSCs-Exo pathway.Methods:1.Isolation and culture of h BMSCs;extraction and identification of Exo from h BMSCs sources;detection of differences in the expression of Exo-mi R-545-3p between SOP and non-SOP populations.2.Preparation of CKG-containing serum and group culture of cells;extraction of Exo from h BMSCs in different groups;detection of Exo-mi R-545-3p expression in h BMSCs of different groups.3.Determination of indicators related to osteogenic or adipogenic differentiation in different groups of h BMSCs;Detection of indicators related to Wnt signaling pathway.Result:1.HBMSCs were successfully isolated and Exo extracted from them:Electron microscopic observation of h BMSCs-derived Exo conforms to the morphological characteristics of exosomes;NTA detects their particle size within their standard division;WB detected positive expression of the marker protein of Exo;RT-qPCR results showed that the expression level of h BMSCs-Exo-mi R-545-3p was higher in the SOP group than in the non-SOP group(p<0.05).2.RT-qPCR results showed that the level of h BMSCs-Exo-mi R-545-3p in the blank group was higher than that in the drug-administered group after the CKG-containing serum intervention(P<0.01).3.(1)The results of alkaline phosphatase staining and alizarin red staining showed that the dark blue stained area and the size of red-stained calcium nodules in the experimental h BMSCs were higher than those in the control group;the results of oil red O staining showed that the number and size of oil droplets in the experimental h BMSCs were significantly lower than those in the control group.(2)RT-qPCR results showed that the expression levels of osteogenesis-related genes(RUNX2,OSX)in h BMSCs were increased and the expression levels of lipogenesis-related genes(PPARγ,C/EBPα)were decreased in the experimental group after 14 days of CKG-containing serum intervention compared with the control group.The differences were all statistically significant(P<0.05).(3)WB results showed that the expression levels of osteogenic-associated proteins(RUNX2,OSX)were increased and lipogenic-associated proteins(PPARγ,C/EBPα)were decreased in h BMSCs after 14 days of drug-containing serum intervention,and the expression levels of key Wnt pathway proteins(β-catenin,LRP5)were increased in the experimental group compared with the control group.Conclusion:The expression level of h BMSCs-derived Exo-mi R-545-3p was increased in SOP population compared to non-SOP population;CKG-containing serum reduced the expression of h BMSCs-derived Exo-mi R-545-3p in SOP population;h BMSCs-derived Exo in non-SOP population promoted osteogenic differentiation and inhibited lipogenic differentiation of h BMSCs in SOP population,and the mechanism may be related to Wnt signaling pathway.