氧化苦参碱对牙周膜干细胞干性标志物表达和成骨分化的作用

Effect of oxymatrine on expression of stem markers and osteogenic differentiation of periodontal ligament stem cells

背景:人牙周膜干细胞是牙周再生组织工程潜在的功能细胞,然而长期体外培养会导致牙周膜干细胞干性减低并发生复制性衰老,从而影响其治疗效果。目的:探讨氧化苦参碱在体外对牙周膜干细胞干性维持和骨向分化的影响,并寻找潜在的影响机制。方法:采用组织块酶消化法从人牙周膜组织中分离、培养得到牙周膜干细胞,并使用流式细胞仪进行间充质细胞表面标志物鉴定。用0,2.5,5,10μg/mL氧化苦参碱孵育牙周膜干细胞,通过CCK8实验检测氧化苦参碱对牙周膜干细胞增殖活性的影响,筛选后续实验合适的药物质量浓度,采用Western blot检测牙周膜干细胞中干细胞非特异性蛋白SOX2和OCT4的表达,采用qRT-PCR、Western blot检测牙周膜干细胞中成骨相关基因和蛋白表达水平。结果与结论:①CCK8实验结果显示2.5μg/mL氧化苦参碱对牙周膜干细胞增殖活性有显著增强作用,后续实验选用2.5μg/mL氧化苦参碱进行干预;②与空白对照组相比,氧化苦参碱组牙周膜干细胞的干性标志物SOX2蛋白表达水平变化不明显(P>0.05),OCT4蛋白表达明显上调(P<0.05);③与成骨诱导组相比,氧化苦参碱+成骨诱导组牙周膜干细胞成骨相关基因ALP、RUNX2 mRNA表达及成骨相关蛋白ALP蛋白表达明显下调(P<0.05);④氧化苦参碱上调牙周膜干细胞干性标志物表达,抑制牙周膜干细胞骨向分化,高通量测序结果表明可能与WNT2、WNT16、COMP、BMP6有关。

BACKGROUND:Human periodontal ligament stem cells are potential functional cells for periodontal tissue engineering.However,long-term in vitro culture may lead to reduced stemness and replicative senescence of periodontal ligament stem cells,which may impair the therapeutic effect of human periodontal ligament stem cells.OBJECTIVE:To investigate the effect of oxymatrine on the stemness maintenance and osteogenic differentiation of periodontal ligament stem cells in vitro,and to explore the potential mechanism.METHODS:Periodontal ligament stem cells were isolated from human periodontal ligament tissues by tissue explant enzyme digestion and cultured.The surface markers of mesenchymal cells were identified by flow cytometry.Periodontal ligament stem cells were incubated with 0,2.5,5,and 10μg/mL oxymatrine.The effect of oxymatrine on the proliferation activity of periodontal ligament stem cells was detected by CCK8 assay.The appropriate drug concentration for subsequent experiments was screened.Western blot assay was used to detect the expression of stem cell non-specific proteins SOX2 and OCT4 in periodontal ligament stem cells.qRT-PCR and western blot assay were used to detect the expression levels of related osteogenic genes and proteins in periodontal ligament stem cells.RESULTS AND CONCLUSION:(1)The results of CCK8 assay showed that 2.5μg/mL oxymatrine significantly enhanced the proliferative activity of periodontal stem cells,and the subsequent experiment selected 2.5μg/mL oxymatrine to intervene.(2)Compared with the blank control group,the protein expression level of SOX2,a stem marker of periodontal ligament stem cells in the oxymatrine group did not change significantly(P>0.05),and the expression of OCT4 was significantly up-regulated(P<0.05).(3)Compared with the osteogenic induction group,the osteogenic genes ALP,RUNX2 mRNA expression and their osteogenic associated protein ALP protein expression of periodontal ligament stem cells were significantly down-regulated in the oxymatrine+osteogenic induction group(P<0.05).(4)The oxymatrine up-regulated the expression of stemness markers of periodontal ligament stem cells and inhibited the bone differentiation of periodontal ligament stem cells,and the results of high-throughput sequencing showed that it may be associated with WNT2,WNT16,COMP,and BMP6.