Based On The Theory Of "The Kidney Generating Marrow And Dominating Bone " To Study The Effect And Mechanism Of ICA Regulation Of MiR-122-5p Which From Osteoblasts-derived Exosomes On Osteogenesis And Migration Of BMSCs

Part I Extraction,identification and culture of rabbit bone marrow mesenchymal stem cellsObjective: To isolate and extract bone marrow mesenchymal stem cells(BMSCs)from the bones of the limbs of adult rabbits and to subculture them.At the same time,to identify bone marrow mesenchymal stem cells and provide experimental cells for subsequent experiments.Methods: BMSCs were extracted and expanded by Percoll density gradient separation method,and BMSCs were identified by flow cytometry,osteogenic and adipogenic differentiation induction medium.Results: The primary and passaged BMSCs were in good condition.The identification of flow cytometry,osteogenic and adipogenic differentiation showed that the isolated and extracted BMSCs had the unique characteristics of BMSCs,indicating that the isolation and extraction were successful.Conclusion: The cells isolated from the bones of rabbits isolated from the experiment were identified as BMSCs,which can be used for subsequent experimental research.Part II Extraction,identification and culture of rabbit osteoblastsObjective: To isolate and extract osteoblasts(OB)from the periosteum of the limbs of adult rabbits,subculture them,and identify OBs at the same time to provide experimental cells for subsequent experiments.Methods: The periosteal-derived osteoblasts were extracted and cultured.The OBs were identified by Gissam staining,alizarin red staining,scanning electron microscope.Results: The primary and passaged OB cells were in good condition.Gissam staining,alizarin red staining,scanning electron microscopy were used to identify OB cells.The results showed that the isolated OB cells had corresponding characteristics,indicating that the isolation and extraction were successful.Conclusion: The periosteal cells of rabbit limbs isolated from the experiment were identified as OB cells,which can be used for subsequent experimental research.Part III Optimum Concentration and Time Rule of Icariin on Bone Marrow Mesenchymal Stem Cells Osteogenesis and MigrationObjective: It is verified that Icariin(ICA)can promote the osteogenic differentiation and migration of BMSCs,find the optimal concentration and time law of its function,and the signal pathway of function.Method: The experimental process is roughly divided into five parts,which are described as follows:(1)The CCK-8 kit was used to detect the effects of different concentrations of ICA on the activity and proliferation of BMSCs.(2)Observe the mineralized nodules formed by BMSCs with different concentrations of ICA.(3)Observe the m RNA and protein expression of osteogenic differentiation and migration after BMSCs treated by ICA on 3d and 7d.(4)Observe the time pattern of osteogenesis and migration expression of ICA on BMSCs.(5)si-Runx2 and AMD3100 were used to verify the signal pathway of ICA to promote osteogenic differentiation and migration of BMSCs.Results:(1)The ICA concentrations of 1×10-7M and 1×10-8M can promote cell proliferation.(2)The mineralization effect of the ICA concentration of 1 × 10-7M was the best.(3)The concentration of 1×10-7M ICA on BMSCs promoted the osteogenesis and migration of m RNA and protein expression.(4)BMSCs under the action of ICA reached the first peak of osteogenesis and migration expression at 3-4d,and reached the second peak at7 d.(5)Both si-Runx2 and AMD3100 can effectively inhibit the role of ICA in promoting osteogenic differentiation and migration.Conclusion:ICA can clearly promote the osteogenic differentiation and migration of BMSCs,which are carried out together.The optimal concentration of ICA to function is 1×10-7M,which exerts osteogenesis and migration through the Runx2 signal axis and CXCR4 signal axis,respectively.ICA promotes the occurrence of osteogenic differentiation and migration of BMSCs,which is regular in time.Part IV Extraction and identification of osteoblast-derived exosomesObjective: Frozen stored OB cells were passaged and expanded and cultured.OB-derived exosomes were extracted and related identification was provided to provide materials for subsequent experiments.Methods: The recovered osteoblasts were routinely cultured,followed by expansion culture,and the cell supernatant was collected.The exosomes of OB cells were extracted by two methods(one is a kit and the other is ultracentrifugation),and identified by transmission electron microscopy and Western blotting.Results: The expanded and cultured OB primary and passage cells were in good condition.The typical morphology of exosomes was observed by transmission electron microscopy.Western blotting was performed on OB-derived exosomes to detect Tsg101,Hsp70,and CD63.The expression levels of the above-mentioned index proteins in the ultra-isolation group and the kit group were significantly higher than those in the blank group.Conclusion: Exosomes were successfully isolated from OB cell supernatants,ultracentrifugation and kits.The OB-derived exosomes extracted by these two methods can be used for subsequent experimental research.Part V Effect of icariin combined with osteoblast-derived exosomes on bone formation and migration of bone marrow mesenchymal stem cellsObjective: It was verified that ICA combined with osteoblast-derived exosomes(OB-exosome,OB-exo)has a clear role in promoting osteogenic differentiation and migration of BMSCs.Methods: It was divided into four groups,namely the blank group,the ICA group,the OB-exo group,and the ICA + OB-exo group for continuous culture.On the 3rd day,BMSCs were collected,and the expression of each target m RNA and protein was detected in each group.Conclusion: Through q PCR and Western blotting,it was found that the ICA group,OB-exo group,and ICA + OB-exo group were significantly higher than the blank group,and the ICA + OB-exo group was the highest.Part VI High-throughput sequencing and miRNAs analysis of osteoblast-derived exosomesObjective: Identification of osteoblast-derived exosomes,prediction of target miRNAs,and reference for subsequent experimental verification.Methods: The recovered osteoblasts were routinely cultured and expanded,and the cell supernatants were collected,exosomes were extracted by ultracentrifugation,and high-throughput sequencing was performed.Use relevant software to analyze the test results.Results:The total amount of mi RNA in exosomes accounted for17.47% of all components.The target genes of mi RNA were predicted by software,and the relevant target mi RNA was found as mi R-122-5p.Conclusion: It is predicted that mi R-122-5p is closely related to biological effects such as osteogenic differentiation and migration,and mi R-122-5p will be used as a reference for subsequent research.Part VII Icariin regulates osteoblast-derived exosomes mi R-122-5p on bone formation and migration of bone marrow mesenchymal stem cellsObjective: It was verified that the target mi RNA of ICA regulation of OB-exo on BMSCs is mi R-122-5p.Methods: The experimental process is roughly divided into two parts,which are described as follows:(1)Four groups(mi R-122-5p minic NC group,mi R-122-5p minic group,mi R-122-5p minic NC + ICA group,mi R-122-5p minic + ICA group),acting on BMSCs,continuously culturing for 3 days,and detecting the osteogenesis and migration expression of BMSCs.(2)Four groups(mi R-122-5p inhibitor NC group,mi R-122-5p inhibitor group,mi R-122-5p inhibitor NC + ICA group,mi R-122-5p inhibitor + ICA group),acting on BMSCs,BMSCs were cultured continuously for3 days to detect the osteogenesis and migration expression of BMSCs.Results:(1)The mi R-122-5p minic + ICA group had the highest expression of osteogenic,migrating m RNA and protein.(2)The expression of mi R-122-5p inhibitor + ICA group was reduced in various osteogenesis and migration m RNAs and proteins.Conclusion: ICA works by regulating mi R-122-5p in OB-exo.