| Objective: Mesenchymal stem cells(MSC)possess high self-renewal ability,multidirectional differentiation potential and immunomodulatory function.These advantages make them an attractive research and clinical tool.Mesenchymal stem cells(MSCs)are of great significance in the treatment of bone-related diseases.In addition,mesenchymal stem cells(MSCs)can differentiate into a variety of cell types,including adipocytes and osteoblasts,under the transcriptional regulation of various factors and intracellular signals.However,its specific mechanism in osteogenic differentiation is not completely clear.Calcium voltage-gated 3 Subunit(Cacnb3)is a Subunit of the Voltage Gated Channel and is related to the regulation of Calcium ions.Calcium ions are indispensable to the formation of bone.The study of Cacnb3 on osteogenic differentiation of mesenchymal stem cells has not been reported,so it is of great significance to explore the role of Cacnb3 in the osteogenic differentiation of mesenchymal stem cells.This study focused on how Cacnb3 affects the osteogenic differentiation of mesenchymal stem cells and the mechanism of action.Methods:(1)The gene expression profiles of MC3T3-E1 preosteoblasts treated with HDACi was downloaded from the GEO database,and were assessed by microarray analysis with DECenter and then combined with the osteoblast gene data set to screen the osteoblast related genes.(2)Mesenchymal stem cells were isolated from mouse fat by collagenase digestion,and cultured in vitro.Cell surface markers were detected by flow cytometry.(3)Mouse adipose mesenchymal stem cells(ADSCs)were cultured in vitro,and the osteogenic induction medium was the experimental group and the normal medium was the control group.After 14 days of osteogenic induction,alkaline phosphatase(ALP)staining were performed to test the activity of ALP.After 14 and 21 days,the calcification deposition was determined by Alizarin Red S(ARS)staining and were used to determine whether the mouse ADSCs had the potential of multidirectional differentiation and the stemness characteristics of mesenchymal stem cells.(4)Osteogenic induction and quantitative analysis of mouse adipose mesenchymal stem cell(ADSCs)were conducted to detect osteogenic genes and screen genes expression.MC3T3-E1 preosteoblasts and human bone marrow mesenchymal stem cells(hBMSCs)were given the same experiments to verify the consistency of gene expression.In addition,the same alkaline phosphatase and alizarin red staining analysis was performed on the MC3T3-E1 preosteoblasts for 14 days to verify the success of osteoblastic induction.(5)By using siRNA to knock down the expression of Cacnb3 in MC3T3-E1,the effect of knockdown of Cacnb3 on the expression of osteogenic related genes was detected.Meanwhile,Fluo-4 AM was used to detect the effect of knockdown Cacnb3 on intracellular calcium ion concentration.Results:(1)Biological information technology was used to analyze the expression profile data to select 22 genes,which were up-regulated in osteogenic differentiation and down-regulated in adipose differentiation,and strictly regulated by deacetylase inhibitors.Through functional analysis,6 genes(Cacnb3,Lpcat2,etc.)were found to be related to the regulation of calcium ions.(2)Using enzyme digestion method successfully cultivate adipose tissue-derived stromal cells(ADSC)in mice,extend the stable and proliferation ability strong,using flow cytometry instrument testing has confirmed adipose tissue-derived stromal cells(ADSC)surface markers specific expression(CD45,CD44,CD29,Sca1),shows that we have separated cultivate adipose tissue-derived stromal cells(ADSC)was of high purity,can be used for follow-up study.(3)After 14 days and 21 days of osteogenic induction of ADSCs,alkaline phosphatase staining and Alizarin Red S(ARS)red staining showed that the ADSCs isolated by us had osteogenic differentiation potential and stem cell specific stemness.(4)The expression levels of osteogenic related genes(Runx2,Alp,Col1a1,Opn)and selected genes Cacnb3 and Lpcat2 in mouse ADSCs induced by osteogenic induction were significantly higher in the experimental group than in the control group(P< 0.05),which further verified that similar results were found in the osteogenic induction of MC3T3-E1 cell line and human bone marrow mesenchymal stem cells.(5)Compared with siRNA-NC group,the mRNA expression of Cacnb3 gene in siCacnb3 cells was down-regulated,and the mRNA expression level was reduced by 70%(P<0.001).The mRNA expression of osteogenic related genes(ALP,Runx2)in siCacnb3 group was decreased after 5 days of silencing compared with that in siRNA-NC group.In addition,the calcium ion concentration after knockdown of Cacnb3 was significantly lower than that of the siRNA-NC group.Conclusion: Cacnb3 plays an important role in the osteogenic differentiation of ADSCs,and can effectively promote the osteogenic differentiation of ADSCs in vitro.Knockdown of Cacnb3 will reduce the intracellular calcium ion concentration,which lays a foundation for further exploring the osteogenic differentiation mechanism of mesenchymal stem cells. |
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