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Abstract：
BACKGROUND: Carbonic anhydrase 1 (CA1) has been found to be involved in osteogenesis and osteoclast in various human diseases, but the molecular mechanisms are not completely understood. In this study, we aim to use siRNA and lentivirus to reduce or increase the expression of CA1 in Dental follicle stem cells (DFSCs), in order to further elucidate the role and mechanism of CA1 in osteogenesis, and provide better osteogenic growth factors and stem cell selection for the application of bone tissue engineering in alveolar bone fracture transplantation.
METHODS: The study used RNA interference and lentiviral vectors to manipulate the expression of the CA1 gene in DFSCs during in vitro osteogenic induction. The expression of osteogenic marker genes was evaluated and changes in CA1, alkaline phosphatase (ALP), Runt-related transcription factor 2 (RUNX2), and Bone morphogenetic proteins (BMP2) were measured using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting (WB). The osteogenic effect was assessed through Alizarin Red staining.
RESULTS: The mRNA and protein expression levels of CA1, ALP, RUNX2, and BMP2 decreased distinctly in the si-CA1 group than other groups (p < 0.05). In the Lentivirus-CA1 (LV-CA1) group, the mRNA and protein expressions of CA1, ALP, RUNX2, and BMP2 were amplified to varying degrees than other groups (p < 0.05). Apart from CA1, BMP2 (43.01%) and ALP (36.69%) showed significant upregulation (p < 0.05). Alizarin red staining indicated that the LV-CA1 group produced more calcified nodules than other groups, with a higher optical density (p < 0.05), and the osteogenic effect was superior.
CONCLUSIONS: CA1 can impact osteogenic differentiation via BMP related signaling pathways, positioning itself upstream in osteogenic signaling pathways, and closely linked to osteoblast calcification and ossification processes.
METHODS: The study used RNA interference and lentiviral vectors to manipulate the expression of the CA1 gene in DFSCs during in vitro osteogenic induction. The expression of osteogenic marker genes was evaluated and changes in CA1, alkaline phosphatase (ALP), Runt-related transcription factor 2 (RUNX2), and Bone morphogenetic proteins (BMP2) were measured using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting (WB). The osteogenic effect was assessed through Alizarin Red staining.
RESULTS: The mRNA and protein expression levels of CA1, ALP, RUNX2, and BMP2 decreased distinctly in the si-CA1 group than other groups (p < 0.05). In the Lentivirus-CA1 (LV-CA1) group, the mRNA and protein expressions of CA1, ALP, RUNX2, and BMP2 were amplified to varying degrees than other groups (p < 0.05). Apart from CA1, BMP2 (43.01%) and ALP (36.69%) showed significant upregulation (p < 0.05). Alizarin red staining indicated that the LV-CA1 group produced more calcified nodules than other groups, with a higher optical density (p < 0.05), and the osteogenic effect was superior.
CONCLUSIONS: CA1 can impact osteogenic differentiation via BMP related signaling pathways, positioning itself upstream in osteogenic signaling pathways, and closely linked to osteoblast calcification and ossification processes.
摘要：
背景：已发现碳酸酐酶1（CA1）参与各种人类疾病的成骨和破骨细胞，但是分子机制还不完全清楚。在这项研究中,我们的目标是使用siRNA和慢病毒来降低或增加CA1在牙卵泡干细胞（DFSCs）中的表达，为了进一步阐明CA1在成骨过程中的作用和机制,为骨组织工程在牙槽骨骨折移植中的应用提供更好的成骨生长因子和干细胞选择。
方法：该研究使用RNA干扰和慢病毒载体在体外成骨诱导过程中操纵DFSCs中CA1基因的表达。评价成骨标记基因的表达及CA1、碱性磷酸酶(ALP)、Runt相关转录因子2(RUNX2),使用定量实时聚合酶链反应（qRT-PCR）和蛋白质印迹（WB）测量骨形态发生蛋白（BMP2）。通过茜素红染色评估成骨作用。
结果：CA1、ALP、si-CA1组的RUNX2和BMP2明显低于其他组（p<0.05）。在慢病毒CA1（LV-CA1）组中，CA1，ALP，ALP的mRNA和蛋白表达，RUNX2、BMP2均较其他组不同程度扩增(p<0.05)。除CA1外,BMP2(43.01%)和ALP(36.69%)均表现出显著上调(p<0.05)。茜素红染色显示LV-CA1组比其他组产生更多的钙化结节，具有较高的光密度（p<0.05），成骨效果优越。
结论：CA1可通过BMP相关信号通路影响成骨分化，定位在成骨信号通路的上游，与成骨细胞钙化和骨化过程密切相关。
方法：该研究使用RNA干扰和慢病毒载体在体外成骨诱导过程中操纵DFSCs中CA1基因的表达。评价成骨标记基因的表达及CA1、碱性磷酸酶(ALP)、Runt相关转录因子2(RUNX2),使用定量实时聚合酶链反应（qRT-PCR）和蛋白质印迹（WB）测量骨形态发生蛋白（BMP2）。通过茜素红染色评估成骨作用。
结果：CA1、ALP、si-CA1组的RUNX2和BMP2明显低于其他组（p<0.05）。在慢病毒CA1（LV-CA1）组中，CA1，ALP，ALP的mRNA和蛋白表达，RUNX2、BMP2均较其他组不同程度扩增(p<0.05)。除CA1外,BMP2(43.01%)和ALP(36.69%)均表现出显著上调(p<0.05)。茜素红染色显示LV-CA1组比其他组产生更多的钙化结节，具有较高的光密度（p<0.05），成骨效果优越。
结论：CA1可通过BMP相关信号通路影响成骨分化，定位在成骨信号通路的上游，与成骨细胞钙化和骨化过程密切相关。
