| The bone marrow mesenchymal stem cells (BMSCs) have multi-directional differentiation potential, and they are successfully induced into osteoblasts by adding 10-8M dexamethasone, 50mg/L Vitamin C andβ-glycerophosphate sodium 10Mm in culture system. These osteoblasts induced from BMSCs provide the best in vitro experimental model to study the function of osteoblasts and its role in bone formation. In recent years, many researches have successfully separated the BMSCs of human, rat, mouse, rabbit, dog, and so on. Even if there are different methods to separate BMSCs in different literature, there are at present commonly used four kinds methods: entire marrow, density gradient centrifuge process, flow cytometry and immunomagnetic beads method.We used density gradient centrifugation and adherence screening method in this research to separate BMSCs from rat bone marrow. We identified the BMSCs by observation of histological anatomy and detected BMSCs cell surface antigen by using flow cytometry. In order to make differentiation of inducing BMSCs to osteoblast we add 10-8M dexamethasone, 50mg/L antiscorbic acid and 10 mMβ-sodium glycerophosphate in culture system. The differentiated cells were identified by observation used inverted phase contrast microscope, ALP staining, and the ability of forming mineralization nodus. The results of identification were that the appearance of BMSCs was correct, the positive expression and negative expression of cell surface antigen were coincidence with rat BMSCs. These proved that the index of cells we had obtained was coincidence with characteristic of rat BMSCs. The cells induced from BMSCs had the characteristic of osteoblast appearance, positive ALP staining, the ability of forming mineralization nodus and positive mineralization nodus staining. It means the cells we had obtained could be used as osteoblast in our research.New epithelia calcium channels TRPV5 and TRPV6 unique physiology function have the very vital role to the calcium ion metabolism and bone metabolism. And it is very clearly that they are regulated by 1,25(OH)2D3, PTH, the diet calcium, the estrogen and the medicine. At present, many researches focus on calmodulin (CaM), S100A10- annexin-2 compound, 80K-H accessory protein an so on. But there are no reports on the expressions of TRPV5 and TRPV6 in osteoblast originated from BMSCs. And it need further discuss in many sphere such as TRPV5 and TRPV6 to bone metabolism influence, the characteristic of TRPV5 and TRPV6 expression in osteocyte, mechanism of medicine adjust TRPV5 and TRPV6, the relation between TRPV5/ TRPV6 expression and bone tumor and drug resistance, the relation between TRPV5/TRPV6 protein deactivation and osteoporosis, and so on.To research the expression of TRPV5/TRPV6 in BMSCs and osteoblasts and explore the significance of TRPV5/TRPV6 in osteoblasts, we used the methods of immunohistochemistry and RT-PCR technology to detect the expressions of TRPV5/TRPV6 in different induction period MSCs and those in osteoblasts. We found that the expressing of TRPV5/TRPV6 were both positive in osteoblasts during and after the induction period, but the expression of TRPV5 and TRPV6 were downregulated quickly as adding inductor during induction period. Along with the induction proceeding, BMSCs differentiate and multiplies to osteoblast, and the positive indexes of TRPV5 were increased from 21.67±4.04 to 93.0±4.58 (F=147.785, v=3,8, P<0.001), the expression of TRPV5 mRNA were increased from 0.229±0.035 to1.095±0.119 (F=52.19,v=3,8,P<0.001), while the positive indexes of TRPV6 were increased from 21.00±5.57 to 94.67±2.52 (F=154.615, v=3,8, P<0.001), the expression of TRPV6 mRNA were increased from 0.168±0.053 to1.022±0.074 (F=100.55, v=3,8, P<0.001). The expressions of TRPV5/ TRPV6 of osteoblasts are strong positive, and the positive index was 93.0±4.58 and 94.67±2.52 respectively. According to this study results, the expression of TRPV5/TRPV6 calcium ion channels are present during the induction differentiation process from rats marrow mesenchyma stem cells to osteoblasts. And with the mature of osteoblasts, the expressions of TRPV5 and TRPV6 are strengthened gradually. These hint us that the calcium ion channel of TRPV5 and TRPV6 are possible candidate channels to adjusts the multiply and differentiation of osteoblasts. However, the role of calcium ion channels TRPV5 and TRPV6 in osteoblasts need to study further.As the second messenger, the calcium ion plays vital role in the signal conduction, and it is key position in each signal conduction way to directly or indirectly affect DNA replication and RNA expresses. It transmits stimulus from external to inner of cell to complete the biological signal the cross membrane transmission. And it activates the different transcription factors to adjust cell growth, the differentiation and activity. Researches confirmed that the processes of the bone absorption and the bone formation need calcium ion transportation across cell in bone metabolism. While the calcium channel is a specified protein associated with calcium transport, which locates on cellular membrane, and it is changed by opening or the off-position through the conformation to control calcium ion flowing and display calcium ion mediating cell biology function. In the TRP superfamily, TRPV5 and TRPV6 are at present only known calcium ion high selective channel, therefore, the discovery of new calcium ion channel TRPV5 and TRPV6 enables us to study the relation between TRPV5/TRPV6 and the bone metabolisms under molecular level.In order to observe the influence of TRPV5 and TRPV6 to the calcium ion of osteoblast, MAPK and the RANKL signaling system, we discussed the relation between TRPV5/TRPV6 and the calcium ion of osteoblast, MAPK and RANKL signaling system. In this research, we used 1,25(OH)2D3 and CuCl2 to change the status of TRPV5 and TRPV6, then observed calcium ion change in the osteoblasts with the laser confocal microscopy. We conducted the semi-quantitative analysis of TRPV5, TRPV6, ERK1 and RANKL with the RT-PCR. This research indicated that 1,25 (OH) 2D3 increased the expression of TRPV5 and TRPV6 truly, and the expression of TRPV5 mRNA were 1.458±0.064 and 1.654±0.079 (P<0.01) respectively before and after induced. The expression of TRPV6 mRNA were 1.461±0.082 and 1.815±0.037 (P<0.01) respectively before and after induced. The fluorescence intensity of calcium ion in the osteoblasts was increased from 1013.19±194.25 to 1348.37±104.64 (P<0.05), with the increase of TRPV5 and TRPV6 expression quantity, at the same time, the expression of ERK1 and the RANKL in the osteoblasts also increased. We blocked TRPV5 and the TRPV6 channels by blocking agents, the fluorescence intensity of calcium ion in the osteoblasts was decreased significantly, with the expression of ERK1 and RANKL also obviously declined. These phenomena indicated that calcium ion in the osteoblasts was influenced by TRPV5 and TRPV6, along with the more TRPV5 and TRPV6 expression, the more calcium ion increased in the osteoblasts obviously. When TRPV5 and TRPV6 were blocked, the extracellular calcium ion is unable to enter into the cells, while the calcium ion inner cells still passed through NCX1 and Ca2+-ATPase 1b (PMCA1b) to the extracellular extension, which caused calcium ion decreased inner cells. Then the changes of calcium ion inner cells affected ERK1 and RANKL expression. When the density of calcium ion increased inner cells, the expression of ERK1 and RANKL were increased correspondingly, while the expression of ERK1 and RANKL were decreased followed with the calcium ion reduced.In summary, this research we found that TRPV5 and TRPV6 were positive expression during the process of rats marrow mesenchyma stem cells induced into the osteoblasts. It hinted that TRPV5 and TRPV6 might possibly as candidate channel to mediate the osteoblasts proliferation. The relationship between TRPV5/TRPV6 and MAPK, RANK signal pathway indicates that TRPV5 and TRPV6 adjust MAPK and the RANKL signal conduction by regulating calcium ion in osteoblasts. In the end they accommodate the proliferation of osteoblast and the function of bone metabolism.
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