| ObjectiveBased on the BMSCs micro-array results in our laboratory, gene, proteinand in vivo level changes are detected by using mouse BMSCs ossifydifferentiation model, in order to discuss the expression change and regulatoryfunction of miR-26in BMSCs, thereafter to further explore the mechanism ofmiR-26a against osteoporosis.Methods(1) Successful OPM mouse model is established by ovariectomy.Osteoporosis mice BMSCs (O-BMSCs), sham mice BMSCs (S-BMSCs) andnormal mice BMSCs (N-BMSCs) are isolated by using the whole bone marrowseparation method. Through the determination of growth curve, calculation ofcolony forming rate and inspection of surface molecules, the stem-cellphenotype of BMSCs is identified;(2) Inducted BMSCs for14d by osteogeneticsolution, real-time quantitative polymerase chain reaction (RT-PCR) isconducted to examine the expression of miR-26and osteogenetic genes(Runx-2, OCN, Col-1) in3d,7d,10d and14d after induction. Western Blot isused to further validate the RT-PCR results;(3) Using siPORTTMNeoFXTMagentto transfect miR-RiboTMmicroRNA-26a mimics, miR-RiboTMmicroRNA-26ainhibitor and its negative control microRNA-26a mimics NC into BMSCs3dbefore osteogenetic solution is added to the culture medium. After7d or14d, cell phenotype, alkaline phosphatase dyeing, calcium salt nodules (alizarin reddyeing) are detected. MiR-26a and osteogenetic genes (Runx-2, OCN, Col-1)are examined10d after induction via RT-PCR;(4) BMSCs are extracted andcultured from OVX and Sham group, the expression of miR-26a andosteogenetic genes (Runx-2, OCN, Col-1) in OVX group are detected byRT-PCR and Western Blot;(5) MiR-RiboTMmicroRNA-26a mimics combineswith HA/TACP material are subcutaneouly implanted into nude mice or into therenal capsule of SD rats. Two months after the implantation, materials aredislodged and H&E staining is conducted to detect osteogenesis.Results(1) Separation and identification of stem cells: N-BMSCs, O-BMSCs andS-BMSCs are successfully cultured. Growth curve shows proliferation capacityof O-BMSCs is lower than S-BMSCs, colony forming assay shows the cloningformation rate of O-BMSCs is lower than the S-BMSCs, after osteogeneticinduction, ALP dyeing, alizarin red staining and osteogenetic gene expression ofO-BMSCs is significantly lower than S-BMSCs;(2) MiR-26a and osteogeneticdifferentiation of BMSCs: RT-PCR detection shows the expression of miR-26arise of about25times during the differentiation process of BMSCs to osteoblast,with the increase of expression of osteogenetic genes gradually (P<0.05).7dafter miR-26a mimics transfection, BMSCs gradually change from a spindleshape into a polygonal deformation which is similar with the negative controlgroup; ALP staining reveals that negative control group are tested to be positiveafter7d culture, while the experimental group is strongly positive; alizarin redstaining shows there are more number of calcium salt deposits nodules thannegative control group after14d culture. The expression of osteogenetic genesin miR-26a mimics group are higher than those in control group (P <0.05),Western Blot has the same results with RT-PCR.(3) Expression of miR-26a andosteogenetic genes in OVX group: RT-PCR results show that expression of miR-26a in OVX group is4-fold lower than sham group, while the osteogeneticgenes are also lower than Sham groups (P<0.05). Western Blot results areaccordance to RT-PCR.(4) H&E sections show that the osteogenetic ability ofmiR-26a mimics group is significantly better than the control group.Conclusions(1) The proliferation activity and capacity in OPM BMSCs are reduced;(2)This experiment first verifies the relationship between miR-26a andosteogenesis of BMSCs, which confirmed the osteogenesis promoting functionof miR-26a against BMSCs;(3) MiR-26a may have reversed the osteogeneticability of O-BMSCs, which may be a new target for treatment of OPM. Thus thiswork could lead the research of the pathogenesis of bone metabolic diseasessuch as osteoporosis further into stem cell and miRNAs level. |
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