Osteogenesis Of Mouse Bone Marrow Mesenchymal Stem Cells And The Impact Of Adrenocorticotropic Hormone

ObjectiveFuther study the effect of ascorbic acid and β-glycerophosphate and determinethe signal pathway during the osteogenic differentiation, plus evaluateAdrenocorticotropic Hormone’s influence on osteogenic differentiation of rat BoneMarrow Mesenchymal Stem Cells(BMMSCs).Method1.Use mouse bone marrow mesenchymal stem cell line (BMMSC)D1fromATCC as experimental materials.2.Divide this experiment into two sections:The first section:Group1: control group1, BMSCs were cultured in DMEM supplemented withFBS, and100IU/ml penicillinG and100ug streptomycin.Group2: BMMSCs were cultured in DMEM supplemented with FBS, and10ug/ml Ascorbic acid, and100IU/ml penicillin G and100ug streptomycin.Group3: BMMSCs were cultured in DMEM supplemented with FBS, and50ug/ml Ascorbic acid, and100IU/ml penicillin G and100ug streptomycin. Group4: BMMSCs were cultured in DMEM supplemented with FBS, and10mMβ-glycerophosphate, and100IU/ml penicillin G and100ug streptomycin.Group5: BMMSCs were cultured in DMEM supplemented with FBS, and1mMβ-glycerophosphate, and100IU/ml penicillin G and100ug streptomycin.Group6: BMMSCs were cultured in DMEM supplemented with FBS, and10ug/ml Ascorbic acid, and10mMβ-glycerophosphate, and100IU/ml penicillin Gand100ug streptomycin.Group7: BMMSCs were cultured in DMEM supplemented with FBS, and50ug/ml Ascorbic acid, and10mMβ-glycerophosphate, and100IU/ml penicillin Gand100ug streptomycin.Group:8BMMSCs were cultured in DMEM supplemented with FBS, and10ug/ml Ascorbic acid, and1mMβ-glycerophosphate, and100IU/ml penicillin G and100ug streptomycin.Group:9BMMSCs were cultured in DMEM supplemented with FBS, and50ug/ml Ascorbic acid, and1mMβ-glycerophosphate, and100IU/ml penicillin G and100ug streptomycin.Group10: control group2, BMMSCs were cultured in DMEM supplementedwith FBS, and50ug/ml Ascorbic Acid, and100IU/ml penicillin G and100ugstreptomycin, Basal Medium.Group11: BMMSCs were cultured in DMEM supplemented with FBS, and50ug/ml Ascorbic Acid, and10mMβ-glycerophosphate, and100IU/ml penicillin Gand100ug streptomycin, Osteogenic Medium.Group12: BMMSCs were cultured in Basic medium with p38MAPK inhibitor.Group13: BMMSCs were cultured in Osteogenic medium with p38MAPKinhibitorGroup14: BMMSCs were cultured in Basic medium with JNK inhibitorGroup15: BMMSCs were cultured in Osteogenic medium with JNK inhibitorGroup16: BMMSCs were cultured in Basic medium with AKT inhibitorGroup17: BMMSCs were cultured in Osteogenic medium with AKT inhibitor.The second section:Group1: control group, BMMSCs were cultured in DMEM supplemented with FBS, and50ug/ml Ascorbic Acid, and100IU/ml penicillin G and100ug streptomycin,Basal Medium.Group2: BMMSCs were cultured in Basic medium with ACTH10-9M.Group3: BMMSCs were cultured in Basic medium with ACTH10-8M.Group4: BMMSCs were cultured in DMEM supplemented with FBS, and50ug/ml Ascorbic Acid, and10mMβ-glycerophosphate, and100IU/ml penicillin Gand100ug streptomycin, Osteogenic Medium.Group5: BMMSCs were cultured in Osteogenic medium with ACTH10-9M.Group6: BMMSCs were cultured in Osteogenic medium with ACTH10-8M.3.Mineralization: BMMSCs were stained with Alizarin Red at day6-day7,followed by quantification of dye retained in cells.4.Gene expression analysis: Harvest the total RNA from each group at day1,day3and day6, and measure the yield of RNA,then synthesize the cDNA fromtotal RNA and carry out the real-time PCR by using the cDNA synthesis kit and theiQ TM SYBR Green Supermix kit, respectively.5.Western blotting analysis: Harvest the cellular protein from each group at15minutes,1hour, day3and day6with lysis buffer, and measure the concentration ofprotein by the BCA method. Then20ug aliquots of cell lysates were applied to12%SDS-PAGE gel and the proteins n the gel were transferred to NCmembranes.Usingthe particular antibody probing each target protein to assess protein, proteinexpression were assayed for different groups.Results1.The osteogenic differentiation in mouse bone marrow mesenchymal stem cells:By Alizarin Red staining the group6with50ug/ml Ascorbic Acid and10mMβ-glycerophosphate present greater mineralization than the group5with10ug/mlAscorbic Acid and10mM β-glycerophosphate, plus group4without AA but with10mM β-glycerophosphate. Meanwhile, the group6exhibits much staining than thegroup7without AA and the group9with50ug/ml with the lower concentration1mMβ-glycerophosphate; the group10through group17indicated that both JNK inhibitor and AKT inhibitor enhanced osteogenesis, however P38inhibitor decreased theexpression of proteins of Runx2,BSP and p-p38apparently increased with treatementtime.2.The effects of Adrenocorticotropic hormone （ACTH） on osteogenicdifferentiation in BMMSCs: Alizarin Red staining showed that ACTH10-8M couldsuppress the mineralization of BMMSCs. For relevant gene expression, theexpression of Runx2, ALP and VEGFa were downregulated by ACTH10-8M, whichwas statistically significant (P<0.05). However the ACTH10-9M have no effect onosteogenesis in BMMSCs；The protein level at day3and day6varied similarly togene expression, i.e, levels of proteins Runx2,p-p38,p-AKTand BSP decreased aftertreatment of ACTH10-8M.Conclusion1.Ascorbic Acid and β-glycerophosphate can enhance the osteogenicdifferentiation of BMMSCs in a dose dependent manner.2.Singnal pathways of p-p38, JNK and AKT are involved in the osteogenicdifferentiation of BMMSCs.3.Adrenocorticotropic Hormone（ACTH）can suppress osteogenic differentiationof BMMSCs.