摘要
目的探讨大豆异黄酮(ISO)调控Wnt-1基因表达影响骨髓间充质干细胞增殖及成骨分化的机制。方法原代分离培养骨髓间充质干细胞,分别加入不同浓度(40、60、80μg/μL)的ISO处理(ISO-L组、ISO-M组、ISO-H组),用脂质体转染法将pcDNA、pcDNA-Wnt-1分别转染至骨髓间充质干细胞(pcDNA组、pcDNA-Wnt-1组),将si-NC、si-Wnt-1分别转染至骨髓间充质干细胞后用ISO处理(ISO+si-NC组、ISO+si-Wnt-1组);甲基噻唑基四唑(MTT)检测细胞增殖;通过实时荧光定量聚合酶链反应(RT-qPCR)与蛋白免疫印迹法(Western blot)分别检测ISO对Wnt-1表达的影响;RT-qPCR与Western blot分别检测碱性磷酸酶(ALP)、骨桥蛋白(OPN)、Runt相关转录因子2(Runx2)的表达量。两组间比较采用独立样本t检验,多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验。结果与对照组比较,ISO-L组、ISO-M组、ISO-H组细胞活力[24 h:(0.17±0.02)比(0.21±0.02)、(0.25±0.02)、(0.29±0.03);48 h:(0.26±0.03)比(0.39±0.03)、(0.48±0.04)、(0.53±0.05);72 h:(0.37±0.03)比(0.54±0.04)、(0.65±0.06)、(0.78±0.07)],骨髓间充质干细胞中ALP mRNA(1.00±0.06比1.74±0.16、2.53±0.21、3.06±0.29)及蛋白(0.41±0.04比0.62±0.06、0.74±0.05、0.83±0.07)、OPN mRNA(1.01±0.08比1.52±0.15、2.11±0.19、2.74±0.25)及蛋白(0.34±0.03比0.51±0.05、0.63±0.06、0.76±0.07)、Runx2 mRNA(1.01±0.08比1.52±0.15、2.11±0.19、2.74±0.25)及蛋白(0.34±0.03比0.51±0.05、0.63±0.06、0.76±0.07)、Wnt-1 mRNA(1.01±0.06比1.48±0.14、1.83±0.17、2.53±0.24)及蛋白(0.21±0.02比0.36±0.03、0.49±0.04、0.62±0.06)均呈逐渐升高趋势,差异有统计学意义(P均<0.05)。与pcDNA组比较,pcDNA-Wnt-1组细胞活力[24 h:(0.18±0.02)比(0.24±0.02);48 h:(0.25±0.03)比(0.51±0.05);72 h:(0.36±0.03)比(0.72±0.06)]、ALP mRNA[(1.01±0.08)比(2.89±0.27)]及蛋白[(0.40±0.04)比(0.79±0.07)]、OPN mRNA[(0.99±0.07)比(2.53±0.25)]及蛋白[(0.33±0.03)比(0.71±0.06)]、Runx2 mRNA[(1.00±
Objective To investigate the mechanism of the effect of soybean isoflavone (ISO)on the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells by regulating Wnt-1 gene expression.Methods Primary marrow mesenchymal stem cells were isolated and cultured,and treated with ISO at different concentrations (40,60,80 μg/ μL) (ISO-L group,ISO-M group,ISO-H group).Liposome-based transfection technology was used to transfect pcDNA and pcDNA-Wnt-1 into bone marrow mesenchymal stem cells (pcDNA group and pcDNA-Wnt-1 group),respectively,and to transfect si-NC and si-Wnt-1 into bone marrow mesenchymal stem cells,followed by the treatment with ISO (ISO+si-NC group and ISO+si-Wnt-1 group).MTT was used to detect cell proliferation.The effects of ISO on Wnt-1 expression were detected by RT-qPCR and Western blot,respectively.RT-qPCR and Western blot were used to detect the expression levels of ALP,OPN,and Runx2,respectively.The independent sample t test was used for the comparison between two groups,and the one-way analysis of variance and pair LSD-t test were used for the comparison between multiple groups.Results Compared with the control group,the cell viability of ISO-L group,ISO-M group and ISO-H group was significantly increased[24 h:(0.17±0.02)vs(0.21±0.02),(0.25±0.02),(0.29±0.03),48 h:(0.26±0.03)vs(0.39±0.03),(0.48±0.04),(0.53±0.05);72 h:(0.37±0.03)vs(0.54±0.04),(0.65±0.06),(0.78±0.07)],mRNA and protein levels of ALP,OPN,Runx2 and Wnt-1 in bone marrow mesenchymal stem cells were also significantly increased[ALP mRNA:(1.00±0.06)vs(1.74±0.16),(2.53±0.21),(3.06±0.29);ALP protein:(0.41±0.04)vs(0.62±0.06),(0.74±0.05),(0.83±0.07).OPN mRNA:(1.01±0.08) vs(1.52±0.15),(2.11±0.19),(2.74±0.25);OPN protein:(0.34±0.03)vs(0.51±0.05),(0.63±0.06),(0.76±0.07).Runx2 mRNA:(1.01±0.08)vs(1.52±0.15),(2.11±0.19),(2.74±0.25);Runx2 protein:(0.34±0.03)vs(0.51±0.05),(0.63±0.06),(0.76±0.07)],and they all showed a gradually increasing trend (P < 0.05).Compared with the pcDNA group,the cell
作者
刘一栋
贾玉凤
王燕
刘扬
宁金月
Liu Yidong;Jia Yufeng;Wang Yan;Liu Yang;Ning Jinyue(Department of Diabetes Metabolism,the Fifth Hospital of Xingtai,Xingtai 054000,China;Department of Ultrasound,the Second Affiliated Hospital ofXingtai Medical College,Xingtai 054000,China;Department of Gastroenterology,the Second Affiliated Hospital of Xingtai Medical College,Xingtai 054000,China;Department of Endocrinology,the Second Affiliated Hospital of Xingtai Medical College,Xingtai 054000,China;Department of Neurology,the First Affiliated Hospital of Xingtai Medical College,Xingtai 054000,China)
出处
《中华细胞与干细胞杂志（电子版）》
2021年第5期292-297,共6页
Chinese Journal of Cell and Stem Cell（Electronic Edition）
基金
邢台市重点研发计划自筹项目(2020ZC236)。
关键词
大豆异黄酮
WNT-1
骨髓间充质干细胞
增殖
成骨分化
Soy bean isoflavone
Wnt-1
Bone marrow mesenchymal stem cells
Proliferation
Osteogenic differentiation
