Effect Of CKIP-1 KO On The Proliferation And Osteogenesis Differentiation Of Different Parts Of Mesenchymal Stem Cells

Osteoporosis(Osteoporosis,OP)is a typical disease of bone metabolic disease.OP seriously affect the health of women and the elderly and is characterized by decreased bone mass,damaged bone microstructure,and reduced number of bone trabecular,which lead to increased osteopsathyrosis and occurrence of fragility fractures.Thus,in medical it is a hot topic to enhance bone mass and improve osteoporosis.Normally,the bone metabolism is in a state of balance between bone formation and bone resorption,which lead to unchanged bone mass.If bone formation is greater than bone resorption for some reasons,which could lead to bone mass increase,and could be used to improve the symptoms of OP.Recently,to treatment OP,the key molecules to regulate the bone formation have been paid more attention in the clinical research.CKIP-1(Casein Kinase-2 Interaction Protein-1)is founded as a negative regulator of bone formation in recent years.The CKIP-1-deficient mice can show the symptoms of bone sclerosis.CKIP-1 gene could provide a new way for the treatment of OP,thus the knockout(KO)mice was used to make some study.Study showed that CKIP-1 KO mice have higher bone mass and optimized structure than Wildtype(WT)ones,and that CKIP-1 KO has more significant influence on mandible bone mass relative to femur and vertebrae.Mesenchymal stem cells(MSCs)have multi-directional differentiation potential,and can differentiate into osteoblasts,fibroblasts,and chondrocyte under certain conditions.Study shown CKIP-1 influence bone formation through negative regulates the proliferation and osteogenesis differentiation of BMMSCs.Objective: We speculate that CKIP-1 have varies effect on different part of bone through influencing the proliferation and osteogenesis differentiation of MSCs.To detect the hypothesis,we use the model of CKIP-1 KO mice and study the biological characteristics of different parts of MSCs from in vivo and in vitro aspects.Methods:1 The OMSCs and BMMSCs of CKIP-1 KO and WT mice were isolated and cultivated in vitro,and the morphology feature of them were observed use microscope.Flow cytometry was used to detect the expression of surface marker molecules to ensure that the cells cultivated were stem cells coming from the mesenchymal tissue.2 MTT and tablet cloning experiments were used to detect the proliferation ability of OMSCs and BMMSCs when CKIP-1 deficiency.Flow cytometry was used to detect the apoptosis of cells to sure whether CKIP-1 deficiency had effects on biological characteristics of OMSCs and BMMSCs.3 The multi-directional differentiation of cells were detected through osteogenesis and adipogenic induced,so that we could detect their difference in differentiate toward bone and fat.The alkaline phosphatase activity(ALP)and alizarin red staining were conducted after osteogenesis induced,to explore whether CKIP-1 deficiency had effects on early and late markers of osteogenesis differentiation of OMSCs and BMMSCs.After induction toward osteogenesis,real-time quantitative polymerase chain reaction(RT-PCR)were used to detect osteogenesis related molecular changes in the mRNA expression level,to explore which molecular CKIP-1 affected during the osteogenesis differentiation process of OMSCs and BMMSCs.4 About 2 x 107 MSCs of varies group were collected and inoculated on HA/?-TCP,respectively,and then subcutaneously implanted into nude mice.After transplant about 2 months,HE staining and Masson trichromatic dyeing were used to evaluate the condition of new bone formation.Further verify the effect of CKIP-1 deficiency on the osteogenesis differentiation of OMSCs and BMMSCs in vivo.Results:1 After digestion with collagenase II,we cultivated the OMSCs and BMMSCs successfully.And the flow cytometry results showed that the cells cultured expressed CD29,CD44 and CD90,which were expressed in cells coming from mesenchymal tissue,and they didn't express CD31 or CD34,which were expressed in cells coming from hematopoietic system.Besides,those CDs expressed in OMSCs and BMMSCs have no significant difference between WT and KO(P>0.05).2 MTT and tablet cloning experiments were used to explore the proliferation ability of cells.Results shown the KO group had a faster speed in proliferation than WT ones(P<0.05),and the proliferation ability of OMSCs group increased significantly than the BMMSCs after CKIP-1 deficiency(P<0.05).However,no significant difference was found in apopotosis ability between OMSCs and BMMSCs under CKIP-1 KO(P>0.05).3 After osteogenesis induced,the ALP staining and the index of osteogenesis differentiation(Runx2,ALP)detected by RT-PCR were used to explore the ability of osteogenesis differentiation of MSCs.Results shown MSCs in KO group have stronger osteogenesis differentiation ability in vitro,and its ALP straining and Runx2,ALP expression higher than that of WT group(P<0.05).Besides,the osteogenesis differentiation ability of OMSCs group increased faster than the BMMSCs after CKIP-1 deficiency(P<0.05).Similarly,we use the index of osteogenesis(COL I,OCN)detected by RT-PCR and the alizarin red staining to study the osteogenesis ability of MSCs.The results shown that the osteogenesis ability of OMSCs increased siginificantly after CKIP-1 deficiency(P<0.05).4 For determination of whether in vitro differences are recapitulated in vivo,MSCs were seeded on scaffold and then was implanted subcutaneously into nude mice for 2 months.The gross observation shown fibrous tissue were formed in the four groups,but the KO group is bigger and especially the OMSCs ones.The Masson's results shown that a lot of blue fibrous tissue and a certain amount of bone were formed in the four groups,and that there have some difference between them.Results shown that the KO group had more bone formed than WT ones(P<0.05),and the new bone formed much more in OMSCs group after CKIP-1 deficiency(P<0.05).Conclusion:1 Digestion with collagenase II,we can cultivate the OMSCs and BMMSCs successfully.CKIP-1 and different bone didn't influence the expression of stem cell surface markers and molecular.2 CKIP-1 KO can significantly enhance the proliferation capacity of OMSCs and BMMSCs,and OMSCs is more sensitive to it.3 CKIP-1 KO can significantly enhance the osteogenesis differentiation ability of OMSCs and BMMSCs,and the osteogenesis differentiation ability of OMSCs increased faster after CKIP-1 deficiency.4 Similar to the in vitro results,once again the in vivo experiments verify that CKIP-1 KO can significantly enhance the osteogenesis differentiation ability of OMSCs and BMMSCs,and OMSCs is more sensitive to it.In conclusion,CKIP-1 KO has more significant influence on mandible bone mass relative to femur,and the reason is the proliferation and the osteogenesis differentiation ability of OMSCs increased faster after CKIP-1 deficiency.