猪Gasp1和Gasp2基因对C2C12细胞分化的影响
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摘要
为研究猪Gasp1、Gasp2对成肌细胞分化的影响,采用RT-PCR技术从猪肌肉组织中扩增Gasp1和Gasp2基因片段,将其克隆入phi C31载体,构建重组质粒phi C31-Gasp1和phi C31-Gasp2。将重组质粒转染至C2C12小鼠成肌细胞,经嘌呤霉素筛选在荧光显微镜下观察,获得稳定表达Gasp1和Gasp2基因的细胞系后,应用CCK-8法检测细胞增殖能力。分别在细胞分化1、2、3、4、5 d时收集细胞,采用实时荧光定量PCR检测MyoD、MyoG、P21及MHC肌细胞分化相关基因的表达变化,进而分析Gasp1和Gasp2基因对肌肉分化的影响。结果显示,成功克隆了猪Gasp1和Gasp2基因,构建了phi C31-Gasp1和phi C31-Gasp2重组质粒,荧光显微镜下可见phi C31载体和Gasp1、Gasp2基因共转染的C2C12细胞表达强烈的绿色荧光,经CCK-8检测发现,过表达Gasp1、Gasp2基因对细胞的生长增殖影响为初期促进、后期抑制;实时荧光定量PCR检测结果表明,猪Gasp1和Gasp2可以促进细胞分化因子MyoD、MyoG、P21、MHC基因的表达。以上结果表明,Gasp1、Gasp2基因可以促进肌肉分化。
In order to study the effects of porcine Gasp1 and Gasp2 on the differentiation of C2C12 cells,Gasp1 and Gasp2 gene fragments were amplified by RT-PCR from porcine muscle tissue,and phi C31 vector was used as skeleton to construct recombinant plasmid phi C31-Gasp1 and phi C31-Gasp2. The recombinant plasmids were transfected into C2C12 cells and the cell lines stably expressing Gasp1 and Gasp2 genes were screened by puromycin and observed under the fluorescence microscope. Then the effect of the stable expression of Gasp1 and Gasp2 on the cells proliferation was detected by CCK-8 method. At last,the cells were harvested at 1,2,3,4 and 5 d,and the expression of MyoD,MyoG,P21 and MHC myoblast differentiation genes was detected by quantitative real-time PCR to analyze the influence of Gasp1 and Gasp2 genes on mouse myoblasts differentiation. The results showed that the eukaryotic expression vectors phi C31-Gasp1 and phi C31-Gasp2 were successfully constructed. The strong green fluorescence expression vectors phi C31 and phi C31-Gasp1 and phi C31-Gasp2 were transfected into C2C12 cells under the fluorescence microscope; the detection of CCK-8 showed that overexpression of Gasp1 and Gasp2 genes promotedthe growth and proliferation of cells in the early stage and inhibited them in the later stage; finally quantitative real-time PCR detection of MyoD,MyoG,P21,MHC confirmed that the Gasp1 and Gasp2 genes could promote the differentiation of C2C12 cells. In conclusion,the Gasp1 and Gasp2 genes could promote muscle differentiation.
In order to study the effects of porcine Gasp1 and Gasp2 on the differentiation of C2C12 cells,Gasp1 and Gasp2 gene fragments were amplified by RT-PCR from porcine muscle tissue,and phi C31 vector was used as skeleton to construct recombinant plasmid phi C31-Gasp1 and phi C31-Gasp2. The recombinant plasmids were transfected into C2C12 cells and the cell lines stably expressing Gasp1 and Gasp2 genes were screened by puromycin and observed under the fluorescence microscope. Then the effect of the stable expression of Gasp1 and Gasp2 on the cells proliferation was detected by CCK-8 method. At last,the cells were harvested at 1,2,3,4 and 5 d,and the expression of MyoD,MyoG,P21 and MHC myoblast differentiation genes was detected by quantitative real-time PCR to analyze the influence of Gasp1 and Gasp2 genes on mouse myoblasts differentiation. The results showed that the eukaryotic expression vectors phi C31-Gasp1 and phi C31-Gasp2 were successfully constructed. The strong green fluorescence expression vectors phi C31 and phi C31-Gasp1 and phi C31-Gasp2 were transfected into C2C12 cells under the fluorescence microscope; the detection of CCK-8 showed that overexpression of Gasp1 and Gasp2 genes promotedthe growth and proliferation of cells in the early stage and inhibited them in the later stage; finally quantitative real-time PCR detection of MyoD,MyoG,P21,MHC confirmed that the Gasp1 and Gasp2 genes could promote the differentiation of C2C12 cells. In conclusion,the Gasp1 and Gasp2 genes could promote muscle differentiation.
引文
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[16]Haidet A M,Rizo L,Handy C,et al.Long-term enhancement of skeletal muscle mass and strength by single gene administration of myostatin inhibitors[J].Proc Natl Acad Sci USA,2008,105(11):4318-4322.
[17]Monestier O,Brun C,Heu K,et al.Ubiquitous Gasp1overexpression in mice leads mainly to a hypermuscular phenotype[J].BMC Genomics,2012,13:541.
[18]Barberi L,Dobrowolny G,Pelosi L,et al.Muscle involvement and IGF-1 signaling in genetic disorders:New therapeutic approaches[J].Endocr Dev,2009,14:29-37.
[19]Guardiola O,Lafuste P,Brunelli S,et al.Cripto regulates skeletal muscle regeneration and modulates satellite cell determination by antagonizing myostatin[J].Proc Natl Acad Sci U S A,2012,109(47):3231-3240.
[20]Minetti G C,Colussi C,Adami R,et al.Functional and morphological recovery of dystrophic muscles in mice treated with deacetylase inhibitors[J].Nat Med,2006,12:1147-1150.
[21]Kota J,Handy C R,Haidet A M,et al.Follistatin gene delivery enhances muscle growth and strength in nonhuman primates[J].Sci Transl Med,2009,1:6-15.
[22]Lee S J,Mc Pherron A C.Regulation of myostatin activity and muscle growth[J].Proc Natl Acad Sci USA,2001,98:9306-9311.
[23]Kondás K,Szláma G,Trexler M,et al.Both WFIKKN1and WFIKKN2 have high affinity for growth and differentiation factors 8 and 11[J].J Biol Chem,2008,283:23677-23684.
[24]Trexler M,Bányai L,Patthy L.Distinct expression pattern of two related human proteins containing multiple types of protease-inhibitory modules[J].Biol Chem,2002,383:223-228.
[25]Sarkar A,Sim C,Hong Y S,et al.Molecular evolutionary analysis of the widespread piggy Bac transposon family and related“domesticated”sequences[J].Mol Genet Genom,2003,270(2):173-180.
[26]Calos M P.The phi C31 integrase system for gene therapy[J].Curr Gene Ther,2006,6(6):633-645.
[27]Olson E N,Brennan T J,Chakraborty T,et al.Molecular control of myogenesis:Antagonism between growth and differentiation[J].Mol Cell Biochem,1991,104(1/2):7-13.
[2]Buckingham M,Tajbakhsh S.Expression of Myo Genic factors in the mouse:myf-5,the first member of the Myo D gene family to be transcribed during skeletal Myo Genesis[J].C R Acad SciⅢ,1993,316(9):1032-1046.
[3]Lassar A B,Skapek S X,Novitch B.Regulatory mechanisms that coordinate skeletal muscle differentiation and cell cycle withdrawal[J].Curr Opin Cell Biol,1994,6(6):788-794.
[4]Miyake M,Hayashi S,Iwasaki S,et al.TIEG1 negatively controls the myoblast pool indispensable for fusion during Myo Genic differentiation of C2C12 cells[J].J Cell Physiol,2011,226(4):1128-1136.
[5]Muscat G E,Rea S,Downes M.Identification of a regulatory function for an orphan receptor in muscle:COUP-TFⅡaffects the expression of the Myo D gene family during Myo Genesis[J].Nucleic Acids Res,1995,23(8):1311-1318.
[6]Davis R L,Weintraub H,Lassar A B.Expression of a single transfected c DNA converts fibroblasts to myoblasts[J].Cell,1987,51(6):987-1000.
[7]Wyzykowski J C,Winata T I,Mitin N,et al.Identification of novel Myo D gene targets in proliferating myogenic stem cells[J].Mol Cell Biol,2002,22(17):6199-6208.
[8]Wright W E,Sassoon D A,Lin V K.Myo Genin,a factor regulating myogenesis,has a domain homologous to Myo D[J].Cell,1989,56(4):607-617.
[9]Weintraub H,Davis R,Tapscott S,et al.The Myo D gene family:Nodal point during specification of the muscle cell lineage[J].Science,1991,251(4995):761-766.
[10]Ott M O,Bober E,Lyons G,et al.Early expression of the myogenic regulatory gene,myf-5,in precursor cells of skeletal muscle in the mouse embryo[J].Development,1991,111(4):1097-1107.
[11]Gartel A L,Radhakrishnan S K.Lost in transcription:P21 repression,mechanisms,and consequences[J].Cancer Res,2005,65(10):3980-3985.
[12]Spiller M P,Kambadur R,Jeanplong F,et al.The myostatin gene is a downstream target gene of basic helixloop-helix transcription factor Myo D[J].Mol Cell Biol,2002,22(20):7066-7082.
[13]Tsuchida K,Arai K Y,Kuramoto Y,et al.Identification and characterization of a novel follistatin-like protein as a binding protein for the TGF-beta family[J].J Biol Chem,2000,275(52):40788-40796.
[14]Hill J J,Qiu Y,Hewick R M,et al.Regulation of myostatin in vivo by growth and differentiation factor-associated serum protein-1:A novel protein with protease inhibitor and follistatin domains[J].Mol Endocrinol,2003,17(6):1144-1154.
[15]Szlama G,Kondas K,Trexler M,et al.WFIKKN1 and WFIKKN2 bind growth factors TGFbeta1,BMP2 and BMP4 but do not inhibit their signalling activity[J].FEBS J,2010,277(24):5040-5050.
[16]Haidet A M,Rizo L,Handy C,et al.Long-term enhancement of skeletal muscle mass and strength by single gene administration of myostatin inhibitors[J].Proc Natl Acad Sci USA,2008,105(11):4318-4322.
[17]Monestier O,Brun C,Heu K,et al.Ubiquitous Gasp1overexpression in mice leads mainly to a hypermuscular phenotype[J].BMC Genomics,2012,13:541.
[18]Barberi L,Dobrowolny G,Pelosi L,et al.Muscle involvement and IGF-1 signaling in genetic disorders:New therapeutic approaches[J].Endocr Dev,2009,14:29-37.
[19]Guardiola O,Lafuste P,Brunelli S,et al.Cripto regulates skeletal muscle regeneration and modulates satellite cell determination by antagonizing myostatin[J].Proc Natl Acad Sci U S A,2012,109(47):3231-3240.
[20]Minetti G C,Colussi C,Adami R,et al.Functional and morphological recovery of dystrophic muscles in mice treated with deacetylase inhibitors[J].Nat Med,2006,12:1147-1150.
[21]Kota J,Handy C R,Haidet A M,et al.Follistatin gene delivery enhances muscle growth and strength in nonhuman primates[J].Sci Transl Med,2009,1:6-15.
[22]Lee S J,Mc Pherron A C.Regulation of myostatin activity and muscle growth[J].Proc Natl Acad Sci USA,2001,98:9306-9311.
[23]Kondás K,Szláma G,Trexler M,et al.Both WFIKKN1and WFIKKN2 have high affinity for growth and differentiation factors 8 and 11[J].J Biol Chem,2008,283:23677-23684.
[24]Trexler M,Bányai L,Patthy L.Distinct expression pattern of two related human proteins containing multiple types of protease-inhibitory modules[J].Biol Chem,2002,383:223-228.
[25]Sarkar A,Sim C,Hong Y S,et al.Molecular evolutionary analysis of the widespread piggy Bac transposon family and related“domesticated”sequences[J].Mol Genet Genom,2003,270(2):173-180.
[26]Calos M P.The phi C31 integrase system for gene therapy[J].Curr Gene Ther,2006,6(6):633-645.
[27]Olson E N,Brennan T J,Chakraborty T,et al.Molecular control of myogenesis:Antagonism between growth and differentiation[J].Mol Cell Biochem,1991,104(1/2):7-13.