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中华细胞与干细胞杂志(电子版) ›› 2021, Vol. 11 ›› Issue (05) : 292 -297. doi: 10.3877/cma.j.issn.2095-1221.2021.05.006

论著

大豆异黄酮上调Wnt-1基因表达促进骨髓间充质干细胞增殖和成骨分化的研究
刘一栋1, 贾玉凤2, 王燕3, 刘扬4, 宁金月5,()   
  1. 1. 054000 邢台,河北省邢台市第五医院糖尿病消化科
    2. 054000 邢台,河北省邢台市医学高等专科学校第二附属医院超声科
    3. 054000 邢台,河北省邢台市医学高等专科学校第二附属医院消化内科
    4. 054000 邢台,河北省邢台市医学高等专科学校第二附属医院内分泌科
    5. 054000 邢台,河北省邢台市医学高等专科学校第一附属医院神经内科
  • 收稿日期:2021-03-27 出版日期:2021-10-01
  • 通信作者: 宁金月
  • 基金资助:
    邢台市重点研发计划自筹项目(2020ZC236)

Upregulation of Wnt-1 gene expression by soybean isoflavones to promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells

Yidong Liu1, Yufeng Jia2, Yan Wang3, Yang Liu4, Jinyue Ning5,()   

  1. 1. Department of Diabetes Metabolism, the Fifth Hospital of Xingtai, Xingtai 054000, China
    2. Department of Ultrasound, the Second Affiliated Hospital of Xingtai Medical College, Xingtai 054000, China
    3. Department of Gastroenterology, the Second Affiliated Hospital of Xingtai Medical College, Xingtai 054000, China
    4. Department of Endocrinology, the Second Affiliated Hospital of Xingtai Medical College, Xingtai 054000, China
    5. Department of Neurology, the First Affiliated Hospital of Xingtai Medical College, Xingtai 054000, China
  • Received:2021-03-27 Published:2021-10-01
  • Corresponding author: Jinyue Ning
引用本文:

刘一栋, 贾玉凤, 王燕, 刘扬, 宁金月. 大豆异黄酮上调Wnt-1基因表达促进骨髓间充质干细胞增殖和成骨分化的研究[J/OL]. 中华细胞与干细胞杂志(电子版), 2021, 11(05): 292-297.

Yidong Liu, Yufeng Jia, Yan Wang, Yang Liu, Jinyue Ning. Upregulation of Wnt-1 gene expression by soybean isoflavones to promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells[J/OL]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2021, 11(05): 292-297.

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、80 μg/ 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±0.06)比(2.14±0.21)]及蛋白[(0.21±0.02)比(0.62±0.06)]的表达量均呈逐渐升高趋势,差异有统计学意义(P均< 0.05)。与ISO+si-NC组比较,ISO+si-Wnt-1组细胞活力[24 h:(0.25±0.03)比(0.19±0.02);48 h:(0.56±0.05)比(0.31±0.03);72 h:(0.82±0.08)比(0.49±0.04)]、ALP mRNA[(3.14±0.31)比(1.67±0.16)]及蛋白[(0.87±0.08)比(0.51±0.05)]、OPN mRNA[(2.79±0.26)比(1.43±0.13)]及蛋白[(0.79±0.08)比(0.43± 0.04)]、Runx2 mRNA[(2.36±0.24)比(1.35±0.13)]及蛋白[(0.68±0.07)比(0.34±0.03)]的表达量均呈逐渐降低趋势,差异有统计学意义(P均< 0.05)。

结论

ISO通过调控Wnt-1基因表达而增强骨髓间充质干细胞增殖及成骨分化功能。

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 viability of the pcDNA-Wnt-1 group [24 h: (0.18±0.02) vs (0.24±0.02) ; 48h: (0.25±0.03) vs (0.51±0.05) ; 72h: (0.36±0.03) vs (0.72±0.06) ], ALP mRNA [ (1.01±0.08) vs (2.89±0.27) ] and protein [ (0.40±0.04) vs (0.79±0.07) ], OPN mRNA [ (0.99±0.07) vs (2.53±0.25) ] and protein [ (0.33±0.03) to (0.71± 0.06) ], Runx2 mRNA [ (1.00±0.06) vs (2.14±0.21) ] and protein [ (0.21±0.02) vs (0.62±0.06) ] expression levels all showed a gradual increase trend, and the difference was statistically significant (all P < 0.05) . Compared with the ISO+si-NC group, the cell viability of the ISO+si-Wnt-1 group [24 h: (0.25±0.03) vs (0.19±0.02) ; 48 h: (0.56±0.05) vs (0.31±0.03) ; 72 h: (0.82±0.08) vs (0.49±0.04) ], ALP mRNA [ (3.14±0.31) vs (1.67±0.16) ] and protein [ (0.87±0.08) vs (0.51±0.05) ], OPN mRNA [ (2.79±0.26) vs (1.43±0.13) ]and protein [ ( 0.79±0.08) vs (0.43±0.04) ], Runx2 mRNA [ (2.36±0.24) vs (1.35±0.13) ]and protein [ (0.68±0.07) vs (0.34±0.03) ] expression levels all showed a gradual decrease trend, and the differences were statistically significant (all P < 0.05) .

Conclusion

ISO can enhance the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells by regulating the expression of Wnt-1.

表1 基因引物序列
表2 大豆异黄酮对骨髓间充质干细胞增殖的影响( ± s
图1 大豆异黄酮对骨髓间充质干细胞中ALP、OPN和Runx2表达的影响
表3 大豆异黄酮对骨髓间充质干细胞中ALP、OPN和Runx2表达的影响( ± s
图2 大豆异黄酮对骨髓间充质干细胞中Wnt-1表达的影响
表4 大豆异黄酮对骨髓间充质干细胞中Wnt-1表达的影响( ± s
图3 Wnt-1过表达对骨髓间充质干细胞Wnt-1和ALP、OPN、Runx2蛋白表达的影响
表5 Wnt-1过表达对骨髓间充质干细胞增殖的影响( ± s
表6 Wnt-1过表达对骨髓间充质干细胞分化的影响( ± s
图4 抑制Wnt-1表达逆转了大豆异黄酮对骨髓间充质干细胞Wnt-1和ALP、OPN、Runx2蛋白表达的作用
表7 抑制Wnt-1表达逆转了大豆异黄酮对骨髓间充质干细胞增殖的作用( ± s
表8 抑制Wnt-1表达逆转了大豆异黄酮对骨髓间充质干细胞分化的作用( ± s
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