目的:探究双调蛋白(amphiregulin,Areg)对ATDC5细胞增殖和成骨分化的影响.方法:实时荧光定量逆转录聚合酶链反应(quantitative real time polymerase chain reaction,qRT-PCR)检测 ATDC5 细胞诱导成骨分化过程中Areg的表达;免疫组织化学染色分析胫骨生长板Areg表达量.Areg过表达或敲低慢病毒感染ATDC5细胞,CCK-8检测Areg对ATDC5细胞增殖的影响;qRT-PCR和Western blot分别从mRNA和蛋白水平检测Areg对ATDC5细胞诱导成骨分化7 d或14 d相关标志物Sox9、Ⅱ型胶原蛋白(collagentype Ⅱ alpha1chain,Col2α1)、X型胶原蛋白(collagentype X alpha1chain,ColX α1)、骨桥蛋白(osteopontin,OPN)和 Runt 相关转录因子 2(Runt-related transcription factor 2,Runx2)的影响;甲苯胺蓝、阿尔新蓝和茜素红染色检测细胞外基质形成.Western blot和染色检测ATDC5细胞诱导成骨分化7 d及加入p-蛋白激酶B(p-protein kinase B,p-AKT)抑制剂MK2206后磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)/AKT通路的磷酸化水平和细胞外基质变化.结果:Ar-eg 在ATDC5细胞成骨分化过程中始终表达,并于7 d达到峰值;胫骨生长板可见软骨细胞高表达Areg;过表达Areg后细胞活力,细胞成骨分化能力和细胞外基质形成显著增加;敲低Areg后则表现为相反的结果;Areg过表达激活了 PI3K/AKT通路,p-AKT抑制剂部分逆转了 PI3K/AKT通路的激活和Areg过表达对软骨细胞外基质生成的促进作用.结论:Areg促进ATDC5细胞增殖,且部分经PI3K/AKT通路促进ATDC5细胞成骨分化.
Amphiregulin Regulates Proliferation and Differentiation of ATDC5 Cells through PI3K/AKT Signaling Pathway
Objective:To explore the impact of amphiregulin(Areg)on the proliferation and osteogenic differenti-ation of ATDC5 cells.Methods:Quantitative real time polymerase chain reaction(qRT-PCR)was used to assess Areg expression in ATDC5 cells.The expression of Areg in tibial growth plates underwent analysis via immunohistochemical staining.Lentivirus-mediated Areg overexpression or knockdown was conducted in ATDC5 cells,and the impact on cell proliferation was evaluated using CCK-8 assay.qRT-PCR and Western blot analyses evaluated the impact of Areg on osteogenic differentiation in ATDC5 cells on markers associated with 7-day,such as Sox9 and collagentype Ⅱ alpha1chain(Col2α1)or 14-day,such as collagentype X alpha1chain(Col X α1),osteopontin(OPN),and Runt-related transcription factor 2(Runx2).Cartilage matrix formation was assessed u-sing staining techniques.Western blot analysis was conducted 7 days after initiating ATDC5 cells osteogenic differ-entiation to assess the phosphorylation of the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)path-way.The phosphorylation of PI3K/AKT was attenuated following the administration of the p-AKT inhibitor MK2206.Results:Areg expression persisted throughout the osteogenic differentiation of ATDC5 cells,peaking on day 7.Areg was highly expressed in chondrocytes of tibial growth plates.Furthermore,Areg overexpression significantly enhanced cell viability,chondrogenic differentia-tion and extracellular matrix formation.While the opposite effect was observed when Areg was knockdown.Areg over-expression activated PI3K/AKT pathway,and the addition of p-AKT inhibitor MK2206 reversed the activation.Conclusion:Areg promotes the proliferation of ATDC5 cells,and partially stimulates the osteogenic differentiation of ATDC5 cells via the PI3K/AKT pathway.
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