心脏转录因子Nkx2.5重组蛋白对人骨髓间充质干细胞向心肌样细胞转分化的作用
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摘要
背景:大多数研究通过使用小分子物质或病毒等方式诱导获取心肌样细胞,而关于重组蛋白用于细胞重编程获得心肌样细胞的报道较少。目的:探讨Nkx2.5重组蛋白对人骨髓间充质干细胞向心肌样细胞转分化的作用。方法:构建Nkx2.5原核表达载体,不同时间点诱导Nkx2.5重组蛋白表达和分离纯化。使用纳米转导试剂包裹Nkx2.5形成纳米-蛋白复合体,转染人骨髓间充质干细胞,观察蛋白转导效率;蛋白转导成功后,检测心肌细胞特异性标志MHC的表达,观察人骨髓间充质干细胞向心肌样细胞转化情况。结果与结论:(1)28℃ IPTG诱导培养4 h获取的蛋白表达量更高;(2)纳米-Nkx2.5复合体可成功转染人骨髓间充质干细胞并诱导其向心肌样细胞转化;(3)结果表明,重组蛋白能被高效转导到细胞内,并能促使人骨髓间充质干细胞重编程为心肌样细胞,但细胞转分化效率尚待提高。
BACKGROUND:Most studies mainly focus on using small molecules or viruses to obtain cardiomyocytes, but reports about the use of recombinant proteins to reprogram cells to obtain cardiomyocytes are rare.OBJECTIVE:To explore the effect of recombinant Nkx2.5 protein on the transdifferentiation of human bone marrow mesenchymal stem cells into cardiomyocytes.METHODS:The prokaryotic expression vector of Nkx2.5 was constructed. Nkx2.5 protein was induced to express at different time points, and then was isolated and purified. Nkx2.5 was encapsulated with nano-transducers to form nano-protein complexes, which were transfected into human bone marrow mesenchymal stem cells. The protein transfection efficiency was then detected. After successful protein transduction, the expression of myosin heavy chain, a specific marker of cardiomyocytes, was detected and the transformation of human bone marrow mesenchymal stem cells into cardiomyocytes was observed.RESULTS AND CONCLUSION:The protein expression was higher when the cells were induced with isopropyl-β-d-thiogalactoside at 28 ℃ for 4 hours. To conclude, the nano-Nkx2.5 complexes can be efficiently transfected into human bone marrow mesenchymal stem cells and promote the reprogramming of bone marrow mesenchymal stem cells into cardiomyocytes, but future studies are needed to improve the efficiency of the transdifferentiation.
BACKGROUND:Most studies mainly focus on using small molecules or viruses to obtain cardiomyocytes, but reports about the use of recombinant proteins to reprogram cells to obtain cardiomyocytes are rare.OBJECTIVE:To explore the effect of recombinant Nkx2.5 protein on the transdifferentiation of human bone marrow mesenchymal stem cells into cardiomyocytes.METHODS:The prokaryotic expression vector of Nkx2.5 was constructed. Nkx2.5 protein was induced to express at different time points, and then was isolated and purified. Nkx2.5 was encapsulated with nano-transducers to form nano-protein complexes, which were transfected into human bone marrow mesenchymal stem cells. The protein transfection efficiency was then detected. After successful protein transduction, the expression of myosin heavy chain, a specific marker of cardiomyocytes, was detected and the transformation of human bone marrow mesenchymal stem cells into cardiomyocytes was observed.RESULTS AND CONCLUSION:The protein expression was higher when the cells were induced with isopropyl-β-d-thiogalactoside at 28 ℃ for 4 hours. To conclude, the nano-Nkx2.5 complexes can be efficiently transfected into human bone marrow mesenchymal stem cells and promote the reprogramming of bone marrow mesenchymal stem cells into cardiomyocytes, but future studies are needed to improve the efficiency of the transdifferentiation.
引文
[1]王振华.心肌再生的研究进展[J].中国循环杂志,2011,26(6):480-482.
[2]蔡明清.心肌细胞再生和腔室重塑[J].中国分子心脏病学杂志,2002,2(1):49-50.
[3]Ye L,Chang YH,Xiong Q,et al.Cardiac repair in a porcine model of acute myocardial infarction with human induced pluripotent stem cell-derived cardiovascular cells.Cell Stem Cell.2014;15(6):750-761.
[4]Cahill TJ,Choudhury RP,Riley PR.Heart regeneration and repair after myocardial infarction:translational opportunities for novel therapeutics.Nat Rev Drug Discov.2017;16(10):699-717.
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[6]田胜男,王博,李琦,等.诱导多能干细胞的安全性及应用研究进展[J].中国组织工程研究,2017,21(5):815-818.
[7]宋维文,安铁洙,王春生.micro RNAs在多能干细胞向心肌细胞分化中的作用[J].中国生物化学与分子生物学报,2015,31(3):244-250.
[8]曾春雨.诱导多能干细胞和心脏干/祖∶细胞对心肌修复作用的研究进展[J].解放军医药杂志,2012,24(7):1-4.
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[10]尹成果,刘学刚.NKX2.5基因在先天性心脏病中的研究进展[J].蚌埠医学院学报,2015,40(11):1608-1611.
[11]许耘红,何穗镕,李丽青.心脏发育相关基因NKX2.5和GATA4突变与先天性心脏病的相关性[J].中国临床药理学杂志,2016,32(22):2111-2113.
[12]丁建东,方翔,李开如,等.转录因子Nkx2.5和GATA4的相互作用[J].中国组织工程研究,2012,16(11):1933-1936.
[13]Pradhan L,Gopal S,Li S,et al.Intermolecular Interactions of Cardiac Transcription Factors NKX2.5 and TBX5.Biochemistry.2016;55(12):1702-1710.
[14]Anderson DJ,Kaplan DI,Bell KM,et al.NKX2-5 regulates human cardiomyogenesis via a HEY2 dependent transcriptional network.Nat Commun.2018;9(1):1373.
[15]蔡花,付四清.Nkx2.5基因与先天性心脏病[J].中国优生与遗传杂志,2014,22(4):1-3.
[16]陈炜,张雷,尹青,等.外源性Nkx2.5、GATA4促进大鼠骨髓间充质干细胞的心肌分化[J].中国民康医学,2015,27(14):161.
[17]Li P,Zhang L.Exogenous Nkx2.5-or GATA-4-transfected rabbit bone marrow mesenchymal stem cells and myocardial cell co-culture on the treatment of myocardial infarction in rabbits.Mol Med Rep.2015;12(2):2607-2621.
[18]姜霖,吴岳恒,李晓红,等.聚乙烯亚胺-蛋白纳米复合体转染蛋白进入骨髓间充质干细胞的应用研究[J].中国病理生理杂志,2015,31(6):1057-1063.
[19]牛玉宏,史剑慧,胡昕婴,等.骨髓基质干细胞心肌化诱导过程中转录因子NKx2.5的表达[J].中国临床医学,2004,11(3):300-302.
[20]林钊,周春燕.转录因子Nkx2.5和GATA4在胚胎心肌发育早期的作用[J].北京大学学报(医学版),2002,34(6):732-734.
[21]Akazawa H,Komuro I.Cardiac transcription factor Csx/Nkx2-5:Its role in cardiac development and diseases.Pharmacol Ther.2005;107(2):252-268.
[22]Maury P,Gandjbakhch E,Baruteau AE,et al.Cardiac Phenotype and Long-Term Follow-Up of Patients With Mutations in NKX2-5 Gene.J Am Coll Cardiol.2016;68(21):2389-2390.
[23]Nimura K,Yamamoto M,Takeichi M,et al.Regulation of alternative polyadenylation by Nkx2-5 and Xrn2 during mouse heart development.Elife.2016;5.pii:e16030.
[24]Tong YF.Mutations of NKX2.5 and GATA4 genes in the development of congenital heart disease.Gene.2016;588(1):86-94.
[25]Ruan Z,Zhu L,Yin Y,et al.Overexpressing NKx2.5 increases the differentiation of human umbilical cord drived mesenchymal stem cells into cardiomyocyte-like cells.Biomed Pharmacother.2016;78:110-115.
[26]Deutsch MA,Doppler SA,Li X,et al.Reactivation of the Nkx2.5 cardiac enhancer after myocardial infarction does not presage myogenesis.Cardiovasc Res.2018;114(8):1098-1114.
[27]Kinnunen SM,T?lli M,V?lim?ki MJ,et al.Cardiac Actions of a Small Molecule Inhibitor Targeting GATA4-NKX2-5 Interaction.Sci Rep.2018;8(1):4611.
[28]Ruan ZB,Chen GC,Ren Y,et al.Expression profile of long non-coding RNAs during the differentiation of human umbilical cord derived mesenchymal stem cells into cardiomyocyte-like cells.Cytotechnology.2018;70(4):1247-1260.
[29]Colombo S,de Sena-Tomás C,George V,et al.Nkx genes establish second heart field cardiomyocyte progenitors at the arterial pole and pattern the venous pole through Isl1repression.Development.2018;145(3):dev161497.
[30]Dorn T,Goedel A,Lam JT,et al.Direct nkx2-5 transcriptional repression of isl1 controls cardiomyocyte subtype identity.Stem Cells.2015;33(4):1113-1129.
[31]Han SS,Wang G,Jin Y,et al.Investigating the Mechanism of Hyperglycemia-Induced Fetal Cardiac Hypertrophy.PLoSOne.2015;10(9):e0139141.
[32]Ketharnathan S,Koshy T,Sethuratnam R,et al.Investigation of NKX2.5 gene mutations in congenital heart defects in an Indian population.Genet Test Mol Biomarkers.2015;19(10):579-583.
[33]Zheng J,Li F,Liu J,et al.Investigation of Somatic NKX2-5Mutations in Chinese Children with Congenital Heart Disease.Int J Med Sci.2015;12(7):538-543.
[34]Bouveret R,Waardenberg AJ,Schonrock N,et al.NKX2-5mutations causative for congenital heart disease retain functionality and are directed to hundreds of targets.Elife.2015;4:e06942.
[35]Wingfield PT.Overview of the purification of recombinant proteins.Curr Protoc Protein Sci.2015;80:1-35.
[36]Ferrer-Miralles N,Saccardo P,Corchero JL,et al.General introduction:recombinant protein production and purification of insoluble proteins.Methods Mol Biol.2015;1258:1-24.
[37]Mahalik S,Sharma AK,Mukherjee KJ.Genome engineering for improved recombinant protein expression in Escherichia coli.Microb Cell Fact.2014;13:177.
[38]Di Baldassarre A,Cimetta E,Bollini S,et al.Human-Induced Pluripotent Stem Cell Technology and Cardiomyocyte Generation:Progress and Clinical Applications.Cells.2018;7(6):E48.
[39]Qin H,Zhao A,Fu X.Small molecules for reprogramming and transdifferentiation.Cell Mol Life Sci.2017;74(19):3553-3575.
[40]Li XH,Li Q,Jiang L,et al.Generation of Functional Human Cardiac Progenitor Cells by High-Efficiency Protein Transduction.Stem Cells Transl Med.2015;4(12):1415-1424.
[2]蔡明清.心肌细胞再生和腔室重塑[J].中国分子心脏病学杂志,2002,2(1):49-50.
[3]Ye L,Chang YH,Xiong Q,et al.Cardiac repair in a porcine model of acute myocardial infarction with human induced pluripotent stem cell-derived cardiovascular cells.Cell Stem Cell.2014;15(6):750-761.
[4]Cahill TJ,Choudhury RP,Riley PR.Heart regeneration and repair after myocardial infarction:translational opportunities for novel therapeutics.Nat Rev Drug Discov.2017;16(10):699-717.
[5]杨梁,张晓刚,魏新伟.维生素C体外诱导多能干细胞向心肌细胞的分化[J].中国组织工程研究与临床康复,2011,15(23):4229-4232.
[6]田胜男,王博,李琦,等.诱导多能干细胞的安全性及应用研究进展[J].中国组织工程研究,2017,21(5):815-818.
[7]宋维文,安铁洙,王春生.micro RNAs在多能干细胞向心肌细胞分化中的作用[J].中国生物化学与分子生物学报,2015,31(3):244-250.
[8]曾春雨.诱导多能干细胞和心脏干/祖∶细胞对心肌修复作用的研究进展[J].解放军医药杂志,2012,24(7):1-4.
[9]Romagnuolo R,Laflamme MA.Programming cells for cardiac repair.Curr Opin Biotechnol.2017;47:43-50.
[10]尹成果,刘学刚.NKX2.5基因在先天性心脏病中的研究进展[J].蚌埠医学院学报,2015,40(11):1608-1611.
[11]许耘红,何穗镕,李丽青.心脏发育相关基因NKX2.5和GATA4突变与先天性心脏病的相关性[J].中国临床药理学杂志,2016,32(22):2111-2113.
[12]丁建东,方翔,李开如,等.转录因子Nkx2.5和GATA4的相互作用[J].中国组织工程研究,2012,16(11):1933-1936.
[13]Pradhan L,Gopal S,Li S,et al.Intermolecular Interactions of Cardiac Transcription Factors NKX2.5 and TBX5.Biochemistry.2016;55(12):1702-1710.
[14]Anderson DJ,Kaplan DI,Bell KM,et al.NKX2-5 regulates human cardiomyogenesis via a HEY2 dependent transcriptional network.Nat Commun.2018;9(1):1373.
[15]蔡花,付四清.Nkx2.5基因与先天性心脏病[J].中国优生与遗传杂志,2014,22(4):1-3.
[16]陈炜,张雷,尹青,等.外源性Nkx2.5、GATA4促进大鼠骨髓间充质干细胞的心肌分化[J].中国民康医学,2015,27(14):161.
[17]Li P,Zhang L.Exogenous Nkx2.5-or GATA-4-transfected rabbit bone marrow mesenchymal stem cells and myocardial cell co-culture on the treatment of myocardial infarction in rabbits.Mol Med Rep.2015;12(2):2607-2621.
[18]姜霖,吴岳恒,李晓红,等.聚乙烯亚胺-蛋白纳米复合体转染蛋白进入骨髓间充质干细胞的应用研究[J].中国病理生理杂志,2015,31(6):1057-1063.
[19]牛玉宏,史剑慧,胡昕婴,等.骨髓基质干细胞心肌化诱导过程中转录因子NKx2.5的表达[J].中国临床医学,2004,11(3):300-302.
[20]林钊,周春燕.转录因子Nkx2.5和GATA4在胚胎心肌发育早期的作用[J].北京大学学报(医学版),2002,34(6):732-734.
[21]Akazawa H,Komuro I.Cardiac transcription factor Csx/Nkx2-5:Its role in cardiac development and diseases.Pharmacol Ther.2005;107(2):252-268.
[22]Maury P,Gandjbakhch E,Baruteau AE,et al.Cardiac Phenotype and Long-Term Follow-Up of Patients With Mutations in NKX2-5 Gene.J Am Coll Cardiol.2016;68(21):2389-2390.
[23]Nimura K,Yamamoto M,Takeichi M,et al.Regulation of alternative polyadenylation by Nkx2-5 and Xrn2 during mouse heart development.Elife.2016;5.pii:e16030.
[24]Tong YF.Mutations of NKX2.5 and GATA4 genes in the development of congenital heart disease.Gene.2016;588(1):86-94.
[25]Ruan Z,Zhu L,Yin Y,et al.Overexpressing NKx2.5 increases the differentiation of human umbilical cord drived mesenchymal stem cells into cardiomyocyte-like cells.Biomed Pharmacother.2016;78:110-115.
[26]Deutsch MA,Doppler SA,Li X,et al.Reactivation of the Nkx2.5 cardiac enhancer after myocardial infarction does not presage myogenesis.Cardiovasc Res.2018;114(8):1098-1114.
[27]Kinnunen SM,T?lli M,V?lim?ki MJ,et al.Cardiac Actions of a Small Molecule Inhibitor Targeting GATA4-NKX2-5 Interaction.Sci Rep.2018;8(1):4611.
[28]Ruan ZB,Chen GC,Ren Y,et al.Expression profile of long non-coding RNAs during the differentiation of human umbilical cord derived mesenchymal stem cells into cardiomyocyte-like cells.Cytotechnology.2018;70(4):1247-1260.
[29]Colombo S,de Sena-Tomás C,George V,et al.Nkx genes establish second heart field cardiomyocyte progenitors at the arterial pole and pattern the venous pole through Isl1repression.Development.2018;145(3):dev161497.
[30]Dorn T,Goedel A,Lam JT,et al.Direct nkx2-5 transcriptional repression of isl1 controls cardiomyocyte subtype identity.Stem Cells.2015;33(4):1113-1129.
[31]Han SS,Wang G,Jin Y,et al.Investigating the Mechanism of Hyperglycemia-Induced Fetal Cardiac Hypertrophy.PLoSOne.2015;10(9):e0139141.
[32]Ketharnathan S,Koshy T,Sethuratnam R,et al.Investigation of NKX2.5 gene mutations in congenital heart defects in an Indian population.Genet Test Mol Biomarkers.2015;19(10):579-583.
[33]Zheng J,Li F,Liu J,et al.Investigation of Somatic NKX2-5Mutations in Chinese Children with Congenital Heart Disease.Int J Med Sci.2015;12(7):538-543.
[34]Bouveret R,Waardenberg AJ,Schonrock N,et al.NKX2-5mutations causative for congenital heart disease retain functionality and are directed to hundreds of targets.Elife.2015;4:e06942.
[35]Wingfield PT.Overview of the purification of recombinant proteins.Curr Protoc Protein Sci.2015;80:1-35.
[36]Ferrer-Miralles N,Saccardo P,Corchero JL,et al.General introduction:recombinant protein production and purification of insoluble proteins.Methods Mol Biol.2015;1258:1-24.
[37]Mahalik S,Sharma AK,Mukherjee KJ.Genome engineering for improved recombinant protein expression in Escherichia coli.Microb Cell Fact.2014;13:177.
[38]Di Baldassarre A,Cimetta E,Bollini S,et al.Human-Induced Pluripotent Stem Cell Technology and Cardiomyocyte Generation:Progress and Clinical Applications.Cells.2018;7(6):E48.
[39]Qin H,Zhao A,Fu X.Small molecules for reprogramming and transdifferentiation.Cell Mol Life Sci.2017;74(19):3553-3575.
[40]Li XH,Li Q,Jiang L,et al.Generation of Functional Human Cardiac Progenitor Cells by High-Efficiency Protein Transduction.Stem Cells Transl Med.2015;4(12):1415-1424.