| Backgroud: Osteoporosis is one of the most common metabolic bone diseases, mainly characterized by pain, prone to fracture, even respiratory dysfunction, seriously impacting on health and quality of life of patients. In that case, the treatment of osteoporosis has got more and more concern. Parathyroid hormone is an important hormone secreted by the chief parathyroid cells regulating bone metabolism and maintaining the body's calcium balance. Whereas, its regulation of bone metabolism has a dual role: the promotion of low-dose intermittent PTH stimulates bone formation andwhilehigh-dose continuous PTH stimulates bone resorption. However, its specific mechanism of action has not been confirmed. Therefore, PTH could not be clinically applied on the treatment of osteoporosis in patients. Further clarifying of the mechanism of PTH on bone formation will provide more theoretical basis for the its clinical application.ClC-3 voltage-gated chloride channels exist in the osteoblastic lineage cells, and have much in common with PTH on the effect on osteoblasts. Previous studies found that PTH stimulation significantly increase ClC-3 expression in osteoblast. And PTH could regulate the expression of the downstream genes related to osteogenesis via ClC-3, but the molecular mechanism remains unclear. Some studies show that PTH and TGF?1 may influence each other on the membranal receptor level, regulating bone resorption and bone formation together, but its mechanism also remains unclear. In our previous research we found that ClC-3 is also involved in the regulation of TGF-?1 signal in the osteoblasts, TGF-?1 could regulate the proliferation and differentiation of osteoblasts, among which TGF-?1 regulates the expression of Runx2, the core binding protein factor, via Smad2/3, thus accelerating bone formation. For this reason, the interaction between ClC-3, TGF-?1 and PTH stimuli requires further study. This study is mianly focused on the interaction between ClC-3 chloride channel, TGF-?1 and downstream signaling molecules under intermittent PTH stimulation, providing new ideas for further research of osteogenic differentiation mechanism of PTH.Objective: This study was designed to observe the mechanism of ClC-3 chloride channel in PTH regulating osteogenic differentiation, by different expressing levels of ClC-3 chloride channel and TGF-?1.Contents:(1) Under intermittent promotion of 1×10-9 M PTH on MC3T3-E1 cells every 6 hours,studying the interaction between TGF-?1 and ClC-3 expression when the gene expression of TGF-?1 and ClC-3 were downregulated.(2) Under intermittent promotion of 1×10-9 M PTH on MC3T3-E1 cells every 6 hours,studying osteogenesis-related and osteoclastgenesis-related genes expression, when the gene expression of both TGF-?1 and ClC-3 were downregulated.(3) Research the role of ClC-3 chloride channel and TGF-?1 on the promotion of 1×10-9 M PTH in the osteogenesis of osteoblasts.Methods:(1) We detected the gene expression levels of TGF- ?1 and ClC-3 in MC3T3-E1 cells, and osteogenic/osteoblastic genes(Runx2?Alp?Bsp?Oc?Opg and Rankl) with Real-Time PCR.(2) We detected the expression of TGF-?1 and ClC-3 in MC3T3-E1 cells under PTH stimulation by using Western-blot Method and immunofluorescence method.(3) We used Alizarin red S staining to observe the mineralization of MC3T3-E1 cells when ClC-3 or(and) TGF-?1 expression is interrupted in MC3T3-E1 cells under PTH stimulation.Results:(1) Under intermittent promotion of 1×10-9 M PTH, when ClC-3 expression is interrupted in MC3T3-E1 cells, the level of gene and protein expression of TGF-?1 is strengther than the control group. On the other hand, when TGF-?1 expression is interrupted in MC3T3-E1 cells, the level of gene and protein expression of ClC-3 is strengther than the control group.(2) Under intermittent promotion of 1×10-9 M PTH 10-9 M, when ClC-3 or(and) TGF-?1 expression is interrupted in MC3T3-E1 cells, the level of gene expression of osteogenic/osteoblastic genes(Runx2?Alp?Bsp?Oc and Rankl) lower than the control group.(3) Under intermittent promotion of 1×10-9 M PTH 10-9 M, when ClC-3 or(and) TGF-?1 expression is interrupted in MC3T3-E1 cells, the level of gene expression of osteogenic/osteoblastic genes(Runx2?Alp?Bsp?Oc and Rankl) had no significant difference than the control group.(4) Under intermittent promotion of 1×10-9 M PTH 10-9 M, when ClC-3 or TGF- ?1 expression is interrupted in MC3T3-E1 cells, the mineralization of MC3T3-E1 cells is lower than the control group. But, when ClC-3 and TGF- ?1 expression is interrupted in MC3T3-E1 cells, the mineralization of MC3T3-E1 cells had no significant difference with the control group.Conclusions:(1) When PTH promotes the osteogenic differentiation in osteoblasts, the signal of ClC-3 and TGF-?1 can affect each other.(2) Both of ClC-3 chloride channel and TGF-?1 can be responsed to PTH stimulation. And that, they can chang the expression of osteogenic/osteoblasticgenes(Runx2?Alp?Bsp?Ocp?Opg and Rankl).(3) Different levels of ClC-3 chloride channel and TGF-?1 can affect the mineralization of MC3T3-E1 cells. |
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