Effect Of CKIP-1 On Osteogenic Differentiation Ability Of Bone Marrow Mesenchymal Stem Cells In Osteoporosis Rats

Objective:A model of osteoporosis induced by retinoic acid in rat was established.The osteogenic ability of BMSCs?bone marrow mesenchymal stem cell?and the dynamic expression of CKIP-1?casein kinase 2-interaction protein-1?were studied both in osteoporosis rat and normal rat.The expression of CKIP-1in osteoporosis BMSCs was reduced by using RNAi technique.The objective of this research was to illustrate the function of CKIP-1 in the regulation the osteogenic ability of BMSCs and provide a molecule theoretical direction of osteoporosis alveolar bone defect remediation using targeting gene therapy.Method:?1?.Establishment and validation of rat osteoporosis model:Twenty of 12-week-old SD rats were divided into two groups randomly.An osteoporosis model in rat was induced by using retinoic acid gavage.The normal group was feeding by an equal volume of 0.85%NaCl solution.After two weeks of growth,the BMD of these rats was determined by using a densimeter.The bilateral proximal femoral condylar bone tissues were taken for HE staining.The differences of bone trabecula between two groups were studied under microscopic observation to verify the success of osteoporosis model establishment.?2?.Isolation,culture and identification of BMSCs:The limbs of rats were separated and the bone marrow was excised.The BMSCs were cultured by whole bone marrow adherence method.During the incubation,the morphology of primary cell was observed and the P3 generation cells were stained by Giemsa.A cell proliferation experiment was conducted by using logarithmic growth phase cells.The growth curve of cell was used to studied the multiplication capacity of BMSCs.The surface markers?CD90,CD44 and CD34?of BMSCs were detected by flow cytometry.BMSCs were stained by alizarin red in 21d^thh after osteogenic induction to observe the formation of calcium nodules.Besides,BMSCs were stained by oil red“O”in 28d^thh after adipogenic induction.The absorbance of oil red“O”was measured to determine the quantities of lipid droplet formation.The formations of calcium nodules and lipid droplet will demonstrate the multipotential differentiation potential of BMSCs.?3?.Comparison of BMSCs osteogenic ability between normal and osteoporosis rats:Osteogenic induction was happened to both of the normal and osteoporosis BMSCs.After 21d,BMSCs were stained by alizarin red to determine the quantity and scale of calcium nodules.ALP?Alkaline phosphatase?activity of BMSCs was measured using a microplate reader in 7d^thh and 14d^thh for quantitive analysis,and BMSCs was stained by ALP in 14d^thh for qualitative analysis.The dynamic expression of CKIP-1 was analyzed via RT-PCR after osteogenic induction for 0,3,7,10 and 14 d at two group BMSCs.Furthermore,OPN?Osteopontin?and Runx2?Runt-related transcription factor 2?were determined by using RT-PCR in 10d^th.?4?Effect of Silencing CKIP-1 Gene on Osteogenic Ability of BMSCs in Osteoporosis Rats:The expression of CKIP-1in osteoporosis BMSCs regulated by RNAi technique,and three treatments were set up,as follow,1)OP+NC;2)OP+CKIP-1^-;3)Normal+NC.Transfection was performed by lentivirus transfection for 72h,and transfection efficiency was determined via RT-PCR.After transfection,the two groups cell in logarithmic growth phase was selected to conducted cell proliferation experiment.All treatments'cells after transfection were used for osteogenic induction.After 21d,the cells were stained by alizarin red,and the number and scale of calcium nodules of BMSCs were observed under a microscope.ALP activity of BMSCs was measured using a microplate reader in 14d^thh after osteogenic induction.Furthermore,OPN and Runx2 were determined by using RT-PCR in 10d^th.Results:?1?Success of osteoporosis model establishment in rat:The BMD of normal rats was 0.46±0.06 g/cm²,which was significantly different from the osteoporosis rats?0.41±0.03 g/cm²?under p<0.05 level.The results of HE staining of the proximal femoral condyle in rats showed that there was more bone mass in normal group.?2?Successful isolation,culture and identification of BMSCs:In the 5d^thh of BMSCs incubation,the cells were mostly star-shaped,polygonal,and colony-like,and the cells of P3 generation were short-spotted.There was no dramatic difference of cell proliferation but a significant difference in 9 days between osteoporosis and normal group?p<0.05?.And osteoporosis group was faster.A positive expression of CD90 and CD44 as well as a negative expression of CD34 were observed in BMSCs of both groups.Besides,the formations of calcium nodules and lipid droplet were clear and distinct after osteogenic and adipogenic induction.The OD value of oil red O was higher in osteogenic group.?3?Osteogenic ability test results of BMSCs in osteoporosis group:Compared with the normal group,BMSCs in the osteoporosis group had smaller quantity and area of calcium nodules in 21d^thh after osteogenic induction,and the ALP staining granules were more than the osteoporosis group in 14d^thh after osteogenic induction,the ALP activity was also stronger than that of the osteoporosis group?P<0.05?.Compared with the normal group,CKIP-1 dynamic expression showed that the higher expression level of CKIP-1 in the osteoporosis group.Furthermore,the osteogenesis-related gene OPN and Runx2 mRNA in the osteoporosis group were lower than those in the normal group?P<0.05?.?4?The results of osteogenic ability in the Osteoporosis+silencing group after silencing CKIP-1 gene:After lentivirus transfected,the mRNA expression of CKIP-1 in the OP+CKIP-1^-was significantly lower than that in the OP+NC?P<0.05?.The growth curve showed that lentivirus had no significant effect on cell proliferation.After osteogenic induction for 21d^th,the formation of calcium nodules in the OP+CKIP-1^-and the Normal+NC was largeher than that in the osteoporosis+empty group,and the ALP staining and ALP activity tests supported this result.In 10d^thh of osteogenic induction,Compared with OP+NC,the osteogenesis-related genes OPN and Runx2 were significantly increased in the OP+CKIP-1^-?P<0.05?,but osteogenesis-related genes OPN and Runx2 in the OP+CKIP-1^-still lower than in the Normal+NC.Conclusion:?1?Retinoic acid-induced osteoporosis model BMSCs have enhanced proliferative capacity,decreased osteogenic capacity,and increased adipogenic capacity compared with the normal group.?2?Silencing CKIP-1 gene can partially improve the osteogenic differentiation capacity of osteoporosis BMSCs.