Dissertation
Dissertation > Medicine, health > Basic Medical > Human morphology > Human histology

Functional Study of miR-26a in Influencing the Differentiation Capacity of Bone Marrow Mesenchymal Stem Cells Into Osteoblasts and Its Mechanism in Regulating Osteoporosis

Author FanLongKun
Tutor HuaZeQuan
School Liaoning Medical
Course Clinical Stomatology
Keywords miRNA-26a bone marrow mesenchymal stem cells osteoblastdifferentiation osteoporosis
CLC R329
Type Master's thesis
Year 2012
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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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