| [Background and Objective]Osteoporosis is disease of bone loss.This is mainly because the balance between bone absorption and bone formation is broken, that leads to more bone absorption than bone formation. Generally, the enhanced activity of osteoclast causes the increasing of bone absorption, which is the important reason for the formation of osteoporosis.Bone marrow mesenchymal stem cells (BMSCs) can differentiate all kinds of cells under specific conditions, including the osteoblast, nerve cells, myocardium cartilage cells, fat cells and so on. In view of their potential of osteoblast differentiation, BMSCs become the main source for artificial bone material. The study for BMSCs causes more and more people’s attention.The increase of alkaline phosphatase activity is one of the important signs for osteoblast’s polarization. ALP is also involved in the calcification of bone cells. Bone morphogenetic protein (BMP) is the multi-functional growth factor, which belongs to transformation growth factor-β(TGF-β) family. It is conservative and functional protein having a group of similar structures. It can induce the formation of bone and cartilage in vitro, and also play an important role in body growth, endochondral ossification, fracture ossification in early cartilage repairing, the embryonic development renewable repairing and so on. Currently,17kinds of cDNA of BMP have been cloned. BMP, especially the BMP2/3/4/5/7can effectively stimulate osteoblast differentiation and induce the formation of bone. BMP-7is a member of the BMP family, mainly expresses in the tissues of bone and kidney, highly expresses in bone growth and bone fracture healing process. Studies found that BMP-7in autogenous bone surface can generate articular cartilage surface, still can repair cartilage, tendons and ligaments in some situations. Both domestic and foreign researches show that giving exogenous BMP-7can promote the ossification of BMSCs. Overseas research suggests that the cure rate of bone damage in the group using BMP-7is higher than control group. So BMP-7as one of important bone morphogenetic proteins factors in bone formation, causes the attention of more and more people.Osterix(OSX), is a zinc finger a structure of transcription factors recently found by Nakashima et al, belonging to the Sp/XKLF family, which is a protein of428amino acids. Research shows that OSX protein distribution in the nucleus, which is closely related to the differentiation and maturation of the organization. In OSX gene knockout mouse embryos, membranous bones of the dense mesenchymal and cartilage within the periosteum of the bone and mesenchymal, the level of expression of various markers of bone cell differentiation is severely reduced or absent, so osteoblast differentiation was completely blocked. OSX knockout cells expressing the characteristic markers of cartilage cells, so OSX may have the function to inhibit bone/cartilage progenitor cells into chondrocytes.Strontium is a new anti-osteoporosis drugs, which can promote bone formation and inhibit bone absorption effect. It also can protect bone loss in ovariectomized rats, and increase the expression the activity of ALP in preosteoblast and osteoblast of mice and rats. Given strontium salt treatment can improve the expression of osteoblast transcription factors. At the molecular level, bone formation mechanism of strontium promotion research is causing more and more scholars’attention. In this study, through the observing the activity of ALP, the expression of BMP-7, calcium nodules and OSX in transformation process of BMSCs, we explore how Sr promotes bone formation. After adding noggin, the blocker of BMP-7, we observed the changes of ALP activity, BMP-7, calcium nodules, OSX gene expression in bone formation, and explored the role of BMP-7in Sr promoting BMSCs to osteoblasts. We hope this study can provide novel experimental evidence for the clinical application of Sr for preventing osteoporosis and other diseases.[Methods]1. The culture of SD rat bone marrow mesenchymal stem cells. In this experiment, BMSCs were isolated from the bone marrow using whole bone marrow adherent method with relatively simple steps. BMSCs were cultured and expanded in vitro, and the proliferation and growth characteristics were observed using inverted microscope. So a stable, efficient and reproducible method was established in vitro using for the separation, culture and amplification of BMSCs. The well grown of3-4generations BMSCs were used for the experiments.2. Using alkaline phosphatase standard, Alizarin red staining, immunoblotting (Western blotting) and reverse transcription-polymerase chain reaction (RT-PCR) to detect the activity of alkaline phosphatase (ALP), the expression of calcium nodules, the level of BMP-7and expression of OSX a specific transcription factor of osteogenesis under different concentration of Sr and the same concentration of Sr with different times.3. The cells were pretreated with noggin a blocker of BMP-7for2h before exposure to Sr. Using alkaline phosphatase standard, Alizarin red staining, Western blotting and RT-PCR to detect the activity of ALP, the expression of calcium nodules, the level of BMP-7and expression of OSX.4. Statistical Analysis All measurement data were expressed as mean±S. Statistical analyses were conducted with SPSS17.0for Windows, comparing continuous variables of groups used One-way ANOVA, LSD method was used as multiple comparison for homoscedasticity and Dunnet-t was used for unhomoscedasticity. Significance was defined as P<0.05.[Results]1. Cytology morphologyThe cells were immediately observed using inverted microscope after inoculation, There are a lot of different size rounded cells, most of them are hematopoietic cells. After the original generation BMSC were vaccinated for24h, there are a few of adherent cells, similar to round and short spindle.48h later more cells adhered gradually, Cells gradient length. The culture medium was changed after days. Remove most of the suspended cells, we can found scattered BMSCs colony, radially grew. The cell colony increased with culture time, with long spindle shape. When the cells were cultured for9or10days, the cell sets dropped and fused with each other, showing the vortex-like growth.2.Effect of Sr on activity of ALP in BMSCsBMSCs were treated with different ratios of Sr(0.1,1,3,5and7mmol/L). Differentiation of BMSCs were quantified by activity of ALP after7days. We can found that the ALP activity was significantly enhanced.At range from0.1-3mmol/L Sr dose-dependently increased ALP activity,peaking at3mmol/L.3.Effect of Sr on mineralization in BMSCs.Both the experimental group with Sr of3mmol/L and the control group without Sr were cultured for21days. Using Alizarin red staining to detect changes in mineralization. The results showed the expression of calcium nodules significantly higher than those in the control group, which shows that Sr significantly promote the transformation of BMSCs.4.Effects of Sr on expression of BMP-7in BMSCs.At range from0.1-5mmol/L Sr increased the expression of BMP-7during the ossification of the BMSCs. At range from0.1-3mmol/L concentration, Sr dose-dependently enhanced expression of BMP-7, peaking at3mmol/L, At5mmol/L, the promotion declined, but compared with the control group, the difference is still statistically significant (P<0.05). Within seven days,Sr time-dependent promoted the expression of BMP-7under the same concentration,peaking at seventh day, compared with the control group, difference was statistically significant (P<0.001). Compared to the seventh day, BMP-7expression in10day was significantly reduced, but there was significant difference between the case and control groups.5. Effects of Sr on expression of OSX in BMSCs.Within the3mmol/L, Sr can raised the expression of OSX, highest at lmmol/L. At the concentration of5mmol/L, Sr can reduce the expression of OSX, compared with the control group, there was a statistically significant difference(P<0.001). The BMSCs were cultured with Sr with1mmol/L for different times (30min-14d), Sr time-dependent promoted the expression of OSX, peaking at7day, compared with the control group, difference was statistically significant (P<0.001). Compared to the7day, the expression of OSX at14day was significantly reduced, but there was significant difference between the case and control groups(P<0.001).6. Effect of inhibitor of BMP-7noggin on Sr-induced BMP-7.At3mmol/L, Sr can promote BMP-7expression in BMSCs, the difference was statistically significant (P<0.001) Give pretreatment of noggin(100ng/ml) for2h, followed by the treatment of Sr. BMP-7expression was decreased, compared with the Sr group, the difference was statistically significant (P<0.001).7. Effect of inhibitor of BMP-7noggin on Sr-induced the activity of ALP.The activity of ALP in Sr group was significantly higher than the control group, the difference was statistically significant (P<0.01). The BMSCs were pretreated with noggin(100ng/ml), for2hours, followed by the treatment of Sr. ALP activity significantly reduced, compared with Sr group, the difference was statistically significant (P<0.001). Compared with the control group noggin and PBS had no significant effect for the activity of ALP, and the difference was not statistically significant (P>0.05).8. Effect of BMP-7inhibitor noggin on Sr-induced mineralization of BMSCs.Compared with the control group, calcium nodules were significantly increased in Sr (3mmol/L) group. The BMSCs were pretreated with noggin(100ng/ml), for2hour, followed by the treatment of Sr, which leads to the significantly reduction of calcium nodules. Compared with Sr group the difference was statistically significant (P<0.001). Compared with the control group noggin and PBS had no significant effect for the expression of calcium nodules, the difference was not statistically significant (P>0.05).9. Effect of BMP-7inhibitor noggin on Sr-induced the expression of OSX in BMSCsCompared with the control group, the expression of OSX were significantly increased in Sr(1mmol/L) group. The BMSCs were pretreated with noggin (100ng/ml), for2hour, followed by the treatment of Sr, which leads to the significantly reduction of expression of OSX. Compared with Sr group the difference was statistically significant (P<0.001). Compared with the control group noggin and PBS had no significant effect for the expression of calcium nodules, the difference was not statistically significant (P>0.05).[Conclusions]Our experiment confirmed that in osteogenesis process of BMSCs. Sr can stimulate the osteogenic signal expression, including the activity of ALP, calcium nodules, BMP-7and osteogenesis gene (OSX), which further proof that Sr can promote the ossification of BMSCs. Sr dose-dependently enhanced expression of ALP activity, BMP-7and OSX in differentiation of mesenchymal cells to osteoblasts. To explore the relationship between the Sr in promoting BMSCs osteoblast differentiation and expression of BMP-7. In this experiment we giving noggin an antagonist of BMP-7before giving Sr. The result is that noggin can obviously block the expression of BMP-7raised by Sr and reduce the expression of the activity of ALP, calcium nodules and OSX gene. Therefore, we conclude that Sr promote BMSCs to the osteoblast conversion by the BMP pathway, and it correlated with the expression of BMP-7. |
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