补肾活血汤水提物调控Cbfal/RUNX2基因沉默骨髓间充质干细胞SP7/Osterix及碱性磷酸酶的表达
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摘要
背景:补肾活血汤可以促进骨髓间充质干细胞成骨分化,探究分子机制利于推向临床。目的:探讨补肾活血汤水提物对Cbfal/RUNX2基因沉默的骨髓间充质干细胞Osterix、碱性磷酸酶表达的影响。方法:骨髓贴壁筛选法分离和培养大鼠骨髓间充质干细胞,Cbfal/RUNX2基因沉默慢病毒转染骨髓间充质干细胞。取第3代骨髓间充质干细胞(空白对照组)、慢病毒沉默骨髓间充质干细胞(沉默组)和阴性病毒载体预处理骨髓间充质干细胞(阴性对照组),以及上述3组骨髓间充质干细胞加入100 mg/L补肾活血汤水提物干预3 d后,检测RUNX2、Osterix蛋白和mRNA表达以及碱性磷酸酶活性。结果与结论:(1)Cbfal/RUNX2基因沉默慢病毒转染效率达约90%;(2)与空白对照组和阴性对照组比较,沉默组骨髓间充质干细胞RUNX2和Osterix的蛋白及mRNA表达均下降,碱性磷酸酶活性也明显下降,差异有显著性意义(P<0.01);(3)补肾活血汤水提物干预后,沉默组骨髓间充质干细胞RUNX2、Osterix的表达和碱性磷酸酶活性明显增加,差异有显著性意义(P<0.01);(4)结果表明,补肾活血汤水提物可以促进骨髓间充质干细胞的RUNX2、Osterix表达,亦增加了碱性磷酸酶活性,从而促进骨髓间充质干细胞成骨分化。
BACKGROUND: Bushen Huoxue Decoction(BSHXD) can promote osteogenesis of bone marrow mesenchymal stem cells(BMSCs) in vitro. Exploring the molecular mechanisms involved is of clinical benefits. OBJECTIVE: To discuss the changes in the expression of SP7/Osterix and alkaline phosphatase(ALP) in BMSCs with Cbfal/RUNX2 gene silencing regulated by the water extracts from BSHXD. METHODS: BMSCs were isolated and cultured by the bone marrow adherent method, and BMSCs at passage 3 were used in the assay. BMSCs were transfected with nothing(blank control group), Cbfal/RUNX2 gene silencing lentivirus(silencing group), and negative viral vector(negative control group), respectively. Then, the cells were cultured in 100 mg/L BSHXD water extract, and 3 days later, the protein and mRNA expression of RUNX2 and Osterix was detected by western blot and qPCR, respectively. Activity of ALP in the BMSCs was also detected in each group. RESULTS AND CONCLUSION: The transfection efficiency of Cbfal/RUNX2 gene silencing lentivirus was about 90%. The protein and mRNA expressions of RUNX2 and Osterix were significantly decreased in the BMSCs transfected with Cbfal/RUNX2 gene silencing lentivirus as compared with the other two groups, and so was the ALP activity(P < 0.01). After treated with the water extracts from BSHXD, the expression of RUNX2 and Osterix as well as the ALP activity in the BMSCs transfected with Cbfal/RUNX2 gene silencing lentivirus increased significantly(P < 0.01). To conclude, the water extract from the BXHXD can up-regulate the expression of RUNX2 and Osterix and the activity of ALP, thus promoting BMSCs osteogenic differentiation.
BACKGROUND: Bushen Huoxue Decoction(BSHXD) can promote osteogenesis of bone marrow mesenchymal stem cells(BMSCs) in vitro. Exploring the molecular mechanisms involved is of clinical benefits. OBJECTIVE: To discuss the changes in the expression of SP7/Osterix and alkaline phosphatase(ALP) in BMSCs with Cbfal/RUNX2 gene silencing regulated by the water extracts from BSHXD. METHODS: BMSCs were isolated and cultured by the bone marrow adherent method, and BMSCs at passage 3 were used in the assay. BMSCs were transfected with nothing(blank control group), Cbfal/RUNX2 gene silencing lentivirus(silencing group), and negative viral vector(negative control group), respectively. Then, the cells were cultured in 100 mg/L BSHXD water extract, and 3 days later, the protein and mRNA expression of RUNX2 and Osterix was detected by western blot and qPCR, respectively. Activity of ALP in the BMSCs was also detected in each group. RESULTS AND CONCLUSION: The transfection efficiency of Cbfal/RUNX2 gene silencing lentivirus was about 90%. The protein and mRNA expressions of RUNX2 and Osterix were significantly decreased in the BMSCs transfected with Cbfal/RUNX2 gene silencing lentivirus as compared with the other two groups, and so was the ALP activity(P < 0.01). After treated with the water extracts from BSHXD, the expression of RUNX2 and Osterix as well as the ALP activity in the BMSCs transfected with Cbfal/RUNX2 gene silencing lentivirus increased significantly(P < 0.01). To conclude, the water extract from the BXHXD can up-regulate the expression of RUNX2 and Osterix and the activity of ALP, thus promoting BMSCs osteogenic differentiation.
引文
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[3]Nakashima K,Zhou X,Kunkel G,et al.The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation.Cell.2002;108(1):17-29.
[4]Prockop DJ.Marrow stromal cells as stem cells for nonhematopoietic tissues.Science.1997;276(5309):71-74.
[5]Syed-Picard FN,Shah GA,Costello BJ,et al.Regeneration of periosteum by human bone marrow stromal cell sheets.J Oral Maxillofac Surg.2014;72(6):1078-1083.
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[7]Wu T,Liu Y,Wang B,et al.The Use of Cocultured Mesenchymal Stem Cells with Tendon-Derived Stem Cells as a Better Cell Source for Tendon Repair.Tissue Eng Part A.2016;22(19-20):1229-1240.
[8]黄永铨,罗毅文,王斌,等.补肾活血汤治疗老年桡骨远端骨折的临床疗效观察[J].中国中医骨伤科杂志,2015,23(3):5-8.
[9]黄永铨,罗毅文,王斌,等.补肾活血汤提取物促进大鼠骨髓间充质干细胞增殖的研究[J].广州中医药大学学报,2015,32(1):86-91.
[10]罗毅文,王斌,吴志方,等.补肾活血汤提取物促进大鼠骨髓间充质干细胞体外迁移及CXCR4表达的研究[J].中药新药与临床药理,2016,27(3):356-361.
[11]刘亚梅,王斌,罗毅文,等.补肾活血法调控大鼠骨髓间充质干细胞增殖活性的实验研究[J].中国中医骨伤科杂志,2015,23(3):1-4.
[12]姜宇,牛鹏飞,徐又佳.锌指结构Osterix对骨骼发育影响的研究进展[J].中国骨质疏松杂志,2016,22(3):381-386.
[13]Sinha KM,Zhou X.Genetic and molecular control of osterix in skeletal formation.J Cell Biochem.2013;114(5):975-984.
[14]Fang J,Hall BK.Chondrogenic cell differentiation from membrane bone periostea.Anat Embryol(Berl).1997;196(5):349-362.
[15]Renn J,Winkler C.Osterix-m Cherry transgenic medaka for in vivo imaging of bone formation.Dev Dyn.2009;238(1):241-248.
[16]Smits P,Li P,Mandel J,et al.The transcription factors L-Sox5and Sox6 are essential for cartilage formation.Dev Cell.2001;1(2):277-290.
[17]De Lise AM,Fischer L,Tuan RS.Cellular interactions and signaling in cartilage development.Osteoarthritis Cartilage.2000;8(5):309-334.
[18]Olsen BR,Reginato AM,Wang W.Bone development.Annu Rev Cell Dev Biol.2000;16:191-220.
[19]Tickle C.Vertebrate limb development.Curr Opin Genet Dev.1995;5(4):478-484.
[20]Johnson RL,Tabin CJ.Molecular models for vertebrate limb development.Cell.1997;90(6):979-990.
[21]余向前,张柳.Osterix对成骨细胞分化影响的研究进展[J].华北煤炭医学院学报,2007,9(1):44-45.
[22]张驰.成骨细胞特异性转录因子Osterix对骨形成作用的分子机制(英文)[J].北京大学学报:医学版,2012,44(5):659-665.
[23]陈思旭,宗兆文,贾敏,等.Osterix基因敲除及过表达对小鼠脊椎骨量的影响[J].第三军医大学学报,2013,35(21):2306-2309.
[24]Tu Q,Valverde P,Li S,et al.Osterix overexpression in mesenchymal stem cells stimulates healing of critical-sized defects in murine calvarial bone.Tissue Eng.2007;13(10):2431-2440.
[25]Tu Q,Valverde P,Chen J.Osterix enhances proliferation and osteogenic potential of bone marrow stromal cells.Biochem Biophys Res Commun.2006;341(4):1257-1265.
[26]Xu B,Zhang J,Brewer E,et al.Osterix enhances BMSC-associated osseointegration of implants.J Dent Res.2009;88(11):1003-1007.
[27]Kaback LA,Soung do Y,Naik A,et al.Osterix/Sp7 regulates mesenchymal stem cell mediated endochondral ossification.JCell Physiol.2008;214(1):173-182.
[28]Komori T.Requisite roles of Runx2 and Cbfb in skeletal development.J Bone Miner Metab.2003;21(4):193-197.
[29]Lee MH,Kwon TG,Park HS,et al.BMP-2-induced Osterix expression is mediated by Dlx5 but is independent of Runx2.Biochem Biophys Res Commun.2003;309(3):689-694.
[30]王度,戴燚,范彦博,等.中药杜仲补骨脂对去势鼠成骨细胞增殖以及MMP3/OPN通路蛋白表达的作用[J].中国医院药学杂志,2016,36(8):620-624.
[31]杨丽,张荣华,朱晓峰,等.淫羊藿苷对大鼠间充质干细胞骨向分化过程中转化生长因子β1、骨形态发生蛋白2表达的影响[J].中国组织工程研究与临床康复,2010,14(19):3518-3522.
[32]侯秋科,吴静,易香华,等.龟板提取物上调维生素D受体表达促骨髓间充质干细胞向成骨分化[J].中草药,2010,41(4):607-612.
[33]桑红灵,周安方,喻小明,等.沉默Cx43基因对左归丸调控MC3T3-E1细胞Runx2、Osterix表达的影响[J].中华中医药杂志,2017,32(11):5045-5049.
[34]张玉卓,任辉,余翔,等.金匮肾气丸调控FNDC5、BMP2对BMSCs成骨分化的作用[J].中国骨质疏松杂志,2017,23(10):1346-1352.
[35]张颖,张蕾蕾,孙瑞波,等.补肾活血法对人骨髓间充质干细胞成骨能力的影响及mi R-93-5p对其的抑制作用[J].中华中医药杂志,2018,33(2):667-671.
[36]柳诗意,张宁,孟祥飞,等.补肾活血方通过调节BMP-2/Runx2/Osterix信号通路抑制慢性肾衰竭大鼠血管钙化的实验研究[J].中国中西医结合杂志,2016,36(3):327-332.
[37]许应星,吴承亮,吴岩,等.Wnt/β-catenin信号通路相关蛋白在补肾活血颗粒含药血清影响成骨细胞过程中的表达及意义[J].中国中西医结合杂志,2011,31(4):537-541.
[38]许兵,王萧枫,杨益宇,等.补肾活血含药血清对成骨细胞的经典Wnt/β-Catenin信号通路的影响研究[C].杭州:2012年浙江省骨科学术年会论文集,2012.
[39]孙正平,梁灿德,吴少鹏,等.补肾活血方对人成骨细胞的增殖和分化中p38MAPK及PI3K/Akt信号转导通路的影响[J].世界中西医结合杂志,2015,10(11):1589-1592.
[40]张淼,张荣华,沈家珍.补肾活血复方对成骨细胞谷氨酸NMDA(N-methyl-D-aspartate)受体信号通路的影响[J].中国中医基础医学杂志,2012,18(6):629-632.
[2]Ohyama Y,Nifuji A,Maeda Y,et al.Spaciotemporal association and bone morphogenetic protein regulation of sclerostin and osterix expression during embryonic osteogenesis.Endocrinology.2004;145(10):4685-4692.
[3]Nakashima K,Zhou X,Kunkel G,et al.The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation.Cell.2002;108(1):17-29.
[4]Prockop DJ.Marrow stromal cells as stem cells for nonhematopoietic tissues.Science.1997;276(5309):71-74.
[5]Syed-Picard FN,Shah GA,Costello BJ,et al.Regeneration of periosteum by human bone marrow stromal cell sheets.J Oral Maxillofac Surg.2014;72(6):1078-1083.
[6]Zhao X,Hwang NS,Bichara DA,et al.Chondrogenesis by bone marrow-derived mesenchymal stem cells grown in chondrocyte-conditioned medium for auricular reconstruction.JTissue Eng Regen Med.2017;11(10):2763-2773.
[7]Wu T,Liu Y,Wang B,et al.The Use of Cocultured Mesenchymal Stem Cells with Tendon-Derived Stem Cells as a Better Cell Source for Tendon Repair.Tissue Eng Part A.2016;22(19-20):1229-1240.
[8]黄永铨,罗毅文,王斌,等.补肾活血汤治疗老年桡骨远端骨折的临床疗效观察[J].中国中医骨伤科杂志,2015,23(3):5-8.
[9]黄永铨,罗毅文,王斌,等.补肾活血汤提取物促进大鼠骨髓间充质干细胞增殖的研究[J].广州中医药大学学报,2015,32(1):86-91.
[10]罗毅文,王斌,吴志方,等.补肾活血汤提取物促进大鼠骨髓间充质干细胞体外迁移及CXCR4表达的研究[J].中药新药与临床药理,2016,27(3):356-361.
[11]刘亚梅,王斌,罗毅文,等.补肾活血法调控大鼠骨髓间充质干细胞增殖活性的实验研究[J].中国中医骨伤科杂志,2015,23(3):1-4.
[12]姜宇,牛鹏飞,徐又佳.锌指结构Osterix对骨骼发育影响的研究进展[J].中国骨质疏松杂志,2016,22(3):381-386.
[13]Sinha KM,Zhou X.Genetic and molecular control of osterix in skeletal formation.J Cell Biochem.2013;114(5):975-984.
[14]Fang J,Hall BK.Chondrogenic cell differentiation from membrane bone periostea.Anat Embryol(Berl).1997;196(5):349-362.
[15]Renn J,Winkler C.Osterix-m Cherry transgenic medaka for in vivo imaging of bone formation.Dev Dyn.2009;238(1):241-248.
[16]Smits P,Li P,Mandel J,et al.The transcription factors L-Sox5and Sox6 are essential for cartilage formation.Dev Cell.2001;1(2):277-290.
[17]De Lise AM,Fischer L,Tuan RS.Cellular interactions and signaling in cartilage development.Osteoarthritis Cartilage.2000;8(5):309-334.
[18]Olsen BR,Reginato AM,Wang W.Bone development.Annu Rev Cell Dev Biol.2000;16:191-220.
[19]Tickle C.Vertebrate limb development.Curr Opin Genet Dev.1995;5(4):478-484.
[20]Johnson RL,Tabin CJ.Molecular models for vertebrate limb development.Cell.1997;90(6):979-990.
[21]余向前,张柳.Osterix对成骨细胞分化影响的研究进展[J].华北煤炭医学院学报,2007,9(1):44-45.
[22]张驰.成骨细胞特异性转录因子Osterix对骨形成作用的分子机制(英文)[J].北京大学学报:医学版,2012,44(5):659-665.
[23]陈思旭,宗兆文,贾敏,等.Osterix基因敲除及过表达对小鼠脊椎骨量的影响[J].第三军医大学学报,2013,35(21):2306-2309.
[24]Tu Q,Valverde P,Li S,et al.Osterix overexpression in mesenchymal stem cells stimulates healing of critical-sized defects in murine calvarial bone.Tissue Eng.2007;13(10):2431-2440.
[25]Tu Q,Valverde P,Chen J.Osterix enhances proliferation and osteogenic potential of bone marrow stromal cells.Biochem Biophys Res Commun.2006;341(4):1257-1265.
[26]Xu B,Zhang J,Brewer E,et al.Osterix enhances BMSC-associated osseointegration of implants.J Dent Res.2009;88(11):1003-1007.
[27]Kaback LA,Soung do Y,Naik A,et al.Osterix/Sp7 regulates mesenchymal stem cell mediated endochondral ossification.JCell Physiol.2008;214(1):173-182.
[28]Komori T.Requisite roles of Runx2 and Cbfb in skeletal development.J Bone Miner Metab.2003;21(4):193-197.
[29]Lee MH,Kwon TG,Park HS,et al.BMP-2-induced Osterix expression is mediated by Dlx5 but is independent of Runx2.Biochem Biophys Res Commun.2003;309(3):689-694.
[30]王度,戴燚,范彦博,等.中药杜仲补骨脂对去势鼠成骨细胞增殖以及MMP3/OPN通路蛋白表达的作用[J].中国医院药学杂志,2016,36(8):620-624.
[31]杨丽,张荣华,朱晓峰,等.淫羊藿苷对大鼠间充质干细胞骨向分化过程中转化生长因子β1、骨形态发生蛋白2表达的影响[J].中国组织工程研究与临床康复,2010,14(19):3518-3522.
[32]侯秋科,吴静,易香华,等.龟板提取物上调维生素D受体表达促骨髓间充质干细胞向成骨分化[J].中草药,2010,41(4):607-612.
[33]桑红灵,周安方,喻小明,等.沉默Cx43基因对左归丸调控MC3T3-E1细胞Runx2、Osterix表达的影响[J].中华中医药杂志,2017,32(11):5045-5049.
[34]张玉卓,任辉,余翔,等.金匮肾气丸调控FNDC5、BMP2对BMSCs成骨分化的作用[J].中国骨质疏松杂志,2017,23(10):1346-1352.
[35]张颖,张蕾蕾,孙瑞波,等.补肾活血法对人骨髓间充质干细胞成骨能力的影响及mi R-93-5p对其的抑制作用[J].中华中医药杂志,2018,33(2):667-671.
[36]柳诗意,张宁,孟祥飞,等.补肾活血方通过调节BMP-2/Runx2/Osterix信号通路抑制慢性肾衰竭大鼠血管钙化的实验研究[J].中国中西医结合杂志,2016,36(3):327-332.
[37]许应星,吴承亮,吴岩,等.Wnt/β-catenin信号通路相关蛋白在补肾活血颗粒含药血清影响成骨细胞过程中的表达及意义[J].中国中西医结合杂志,2011,31(4):537-541.
[38]许兵,王萧枫,杨益宇,等.补肾活血含药血清对成骨细胞的经典Wnt/β-Catenin信号通路的影响研究[C].杭州:2012年浙江省骨科学术年会论文集,2012.
[39]孙正平,梁灿德,吴少鹏,等.补肾活血方对人成骨细胞的增殖和分化中p38MAPK及PI3K/Akt信号转导通路的影响[J].世界中西医结合杂志,2015,10(11):1589-1592.
[40]张淼,张荣华,沈家珍.补肾活血复方对成骨细胞谷氨酸NMDA(N-methyl-D-aspartate)受体信号通路的影响[J].中国中医基础医学杂志,2012,18(6):629-632.