Mechanism Of Wnt Signaling Pathway Promote BMSCs Differentiation Into Osteoblasts On The Surface Of Titanium

Part one:Effects of Wnt signaling pathway promote BMSCs differentiation into osteoblasts on the surface of titaniumIn part one of this study, BMSCs were isolated form bone marrow of SD rats. By means of gradient centrifugation combined with adherent culture, the adherent spindle-shaped BMSCs population was cultured for3passages and used in this study. BMSCs were cultured on the surface of titanium, in an incubator with osteogenic medium. After7days and14days, we tested the osteogenic index which proved the ability of BMSCs differentiation into osteoblasts.The results indicated that the BMSCs were of highly purity. The analysis of alizard red, ALP activity and OC release showed BMSCs could differentiation into osteoblasts.In part two of this study, BMSCs were cultured on the surface of titanium. After24h in culture, medium of BMSCs of experimental group were removed and replaced into medium supplemented with0.1umol/l of DXM,10mmol/l b-sodium glycerophosphate, and50ug/ml ascorbic acid. BMSCs of control group were cultured in an incubator with L-DMEM medium, supplemented with10%fetal bovine serum (FBS) and antibiotics.7days later, DNA of BMSCs were extracted by trizol and tested by microarray. Form the gene analysis we found the dynamic changes of gene expression of Wnt signaling pathway.Gene Pathway analysis showed that Wnt signaling pathway and TGF-β signaling pathway, MAPK signaling pathway were involved in the differentiation process. Moreover, three ways of Wnt singaling pathway were not working alone, there was a cross talk between them. Gene analysis showed that among all89genes of Wnt signaling pathway low-density-lipoprotein receptor-related protein5(LRP5), Wnt5b were significantly changed, suggesting that they might play dominant roles in the process of osteogenic differentiation. However, the expression of (3-catenin did not changed significantly, but we found its especial position in the Wnt signaling pathway which might leads to important discovery, so we added it into our study group.Part two:Mechanism of Wnt canonical signaling pathway promote BMSCs differentiation into osteoblasts on the surface of titaniumIn part one of this study, the dual expression lentivirus vector carrying siRNA of LRP5(LV3-LRP5) was constructed and transduced into BMSCs. We constructed four lentivirus vector carriers (2723,2724,2725,2726). RT-PCR and western blot revealed that interference RNA of LRP5(2725,2726) turned to be better. After infected with LV3-LRP5, we tested both gene expression and protein accumulation of LRP5, β-catenin, BMP2. Furthermore, we investigated the ability of BMSCs differentiation into osteoblasts by the assays of ALP, OC and Alizard red.RT-PCR and western blot revealed that interference RNA of LRP5(2725,2726) decreased the accumulation of LRP5, β-catenin, BMP2significant, and subsequently interrupted the differentiation process of BMSCs on the surface of titanium. This result showed that LRP5played an important role in the Wnt canonical signaling pathway. The interference of LRP5blocked the process of BMSCs differentiation into osteoblasts. Furthermore, BMP signaling pathway was involved in the process.In part two of this study, the dual expression lentivirus vector carrying siRNA of P-catenin (LV3β-catenin) was constructed and transduced into BMSCs. We constructed four lentivirus vector carriers (3081、3082、3083、3084). RT-PCR and western blot revealed that interference RNA of LRP5(3083) turned to be better. After infected with LV3-β-catenin, we tested both gene expression and protein accumulation of LRP5, β-catenin, BMP2. Furthermore, we investigated the ability of BMSCs differentiation into osteoblasts by the assays of ALP, OC and Alizard red.RT-PCR and western blot revealed that interference RNA of LRP5(3083) decreased the accumulation of LRP5, β-catenin, BMP2significant, and subsequently interrupted the differentiation process of BMSCs on the surface of titanium. These results showed that β-catenin played an important role in the Wnt canonical signaling pathway. The interference ofβ-catenin blocked the process of BMSCs differentiation into osteoblasts. Furthermore, BMP signaling pathway was involved in the process. Part three:Mechanism of Wnt noncanonical signaling pathway promote BMSCs differentiation into osteoblasts on the surface of titaniumIn this study, the dual expression lentivirus vector carrying siRNA of Wnt5b (LV3-Wnt5b and LV5-Wnt5b) was constructed and transduced into BMSCs. We constructed four interfering lentivirus vector carriers (484、918、975、1579) and an overexpressed lentivirus vector carrier (v5053). RT-PCR and western blot revealed that interference RNA of LRP5(1579) turned to be better. After infected with LV3-LRP5, we tested both gene expression and protein accumulation of LRP5, p-catenin, BMP2. Furthermore, we investigated the ability of BMSCs differentiation into osteoblasts by the assays of ALP, OC and Alizard red.RT-PCR and western blot revealed that interference RNA of LRP5(2725,2726) decreased the accumulation of LRP5, β-catenin, BMP2significant, and subsequently interrupted the differentiation process of BMSCs on the surface of titanium. BMSCs infected with LV5-Wnt5b increased the accumulation of LRP5, β-catenin, BMP2significant, and promoted the process of BMSCs differentiation into osteoblasts. Above all, we considered Wnt noncanonical signaling pathway also participated in the process of differentiation, and cooperated with Wnt canonical signaling patyway.Conclusion:1, By means of gradient centrifugation combined with adherent culture, we collected BMSCs of highly purity.2, Wnt signaling pathway promotes BMSCs differentiation into osteoblasts on the surface of titanium.3, Interference of LRP5blocked the transduction of Wnt/p-catenin signaling pathway, postponed the process of BMSCs differentiation into osteoblasts. 4, Interference of β-catenin blocked the transduction of Wnt/β-catenin signaling pathway, postponed the process of BMSCs differentiation into osteoblasts.5,β-catenin and BMP2were both involved in the process of BMSCs differentiation into osteoblasts on the surface of titanium.6, Noncanonical Wnt/Wnt5b signaling pathway regulated the process of BMSCs differentiation into osteoblasts on the surface of titanium.