wnt5a诱导心肌祖细胞向心肌细胞分化的作用研究
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摘要
目的探讨wnt5a诱导心肌祖细胞CP15-5a向心肌细胞分化的作用。方法利用HEK293细胞扩增重组腺病毒Ad-wnt5a和Ad-GFP;转染CP15-5a细胞后,设置wnt5a组、GFP组和空白对照组。采用流式细胞技术检测Ad-wnt5a和Ad-GFP的转染效率;1周后,通过实时定量PCR(q RT-PCR)检测经Ad-wnt5a诱导的CP15-5a细胞中心肌组织特异性转录因子GATA结合蛋白4(GATA4)、心肌增强因子2C(MEF2C)的表达;2周后,通过Western blotting及免疫荧光技术检测经Ad-wnt5a诱导的CP15-5a细胞中心肌连接因子43(CX43)、心肌肌钙蛋白T(cTnT)的表达;全细胞膜片钳技术检测钠电流(I_(Na))表达。结果 Ad-wnt5a和Ad-GFP转染效率分别为42.8%、44.3%。腺病毒转染CP15-5a细胞1周后,wnt5a组GATA4和MEF2C基因的表达量(分别为1.717±0.220、1.847±0.190)较GFP组和空白对照组(GATA4:1.003±0.087、0.961±0.063,P<0.05;MEF2C:0.456±0.042、0.500±0.095,P<0.05)明显升高,而GFP组与空白对照组比较差异无统计学意义;腺病毒转染2周后,wnt5a组CX43和c Tn T蛋白的表达量(分别为1.597±0.267、0.727±0.100)较GFP组和空白对照组(CX43:0.723±0.047、0.783±0.1333,P<0.05;cTnT:0.217±0.021和0.253±0.102,P<0.01)明显升高,而GFP组与空白对照组比较差异无统计学意义。与GFP组及空白对照组比较,wnt5a组可检测出I_(Na)。结论 wnt5a可诱导心肌祖细胞CP15-5a向心肌细胞分化。
Objective To investigate the effect of Wnt5a on inducing differentiation of Cp15-5a cell to myocardial cell. Methods Recombinant adenovirus wnt5a(Ad-wnt5a) and Ad-GFP was amplified with human embryo kidney 293 cells(HEK293 cells), and then transfected into CP15-5a cells and 3 experiment groups were set up: wnt5a group, GFP group and blank control group. Flow cytometry was used to detect the transfection efficiency of Ad-wnt5a and Ad-GFP. One week after transfection, the expressions of genes GATA binding protein 4(GATA4) and myocardial enhancement factor 2 C(MEF2C) were analyzed by realtime quantitative PCR(qRT-PCR). Two weeks after transfection, the expressions of cardiac-specific connexin 43(Cx43) and cardiac troponin T(cTnT) in Ad-wnt5a-induced CP15-5a cells were detected by Western blotting and immunofluorescence techniques. The sodium current expression(I_(Na)) was detected by whole cell patch clamp techniques. Results The transfection efficiency of Ad-wnt5a and Ad-GFP was 42.8% and 44.3%, respectively. One week after transduction, the expressions of GATA4 and MEF2C were significantly higher in wnt5a group(1.717±0.220 and 1.847±0.190) than in GFP group(1.003±0.087 and 0.456±0.042, P<0.05) and blank control group(0.961±0.063 and 0.500±0.095, P<0.05), while no significant difference existed between GFP group and blank control group. Two weeks after transduction, the expressions of CX43 and cTnT were significantly higher in wnt5a group(1.597±0.267 and 0.727±0.100) than in GFP group(0.723±0.047 and 0.217±0.021, P<0.05) and blank control group(0.783±0.1333 and 0.253±0.102, P<0.01), while no significant difference existed between GFP group and blank control group. I_(Na) was detected in the wnt5a group compared with GFP group and blank control group. Conclusion wnt5a may induce differentiation of cp15-5a cell into myocardial cell.
Objective To investigate the effect of Wnt5a on inducing differentiation of Cp15-5a cell to myocardial cell. Methods Recombinant adenovirus wnt5a(Ad-wnt5a) and Ad-GFP was amplified with human embryo kidney 293 cells(HEK293 cells), and then transfected into CP15-5a cells and 3 experiment groups were set up: wnt5a group, GFP group and blank control group. Flow cytometry was used to detect the transfection efficiency of Ad-wnt5a and Ad-GFP. One week after transfection, the expressions of genes GATA binding protein 4(GATA4) and myocardial enhancement factor 2 C(MEF2C) were analyzed by realtime quantitative PCR(qRT-PCR). Two weeks after transfection, the expressions of cardiac-specific connexin 43(Cx43) and cardiac troponin T(cTnT) in Ad-wnt5a-induced CP15-5a cells were detected by Western blotting and immunofluorescence techniques. The sodium current expression(I_(Na)) was detected by whole cell patch clamp techniques. Results The transfection efficiency of Ad-wnt5a and Ad-GFP was 42.8% and 44.3%, respectively. One week after transduction, the expressions of GATA4 and MEF2C were significantly higher in wnt5a group(1.717±0.220 and 1.847±0.190) than in GFP group(1.003±0.087 and 0.456±0.042, P<0.05) and blank control group(0.961±0.063 and 0.500±0.095, P<0.05), while no significant difference existed between GFP group and blank control group. Two weeks after transduction, the expressions of CX43 and cTnT were significantly higher in wnt5a group(1.597±0.267 and 0.727±0.100) than in GFP group(0.723±0.047 and 0.217±0.021, P<0.05) and blank control group(0.783±0.1333 and 0.253±0.102, P<0.01), while no significant difference existed between GFP group and blank control group. I_(Na) was detected in the wnt5a group compared with GFP group and blank control group. Conclusion wnt5a may induce differentiation of cp15-5a cell into myocardial cell.
引文
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[23]Motegi Y,Katayama K,Sakurai F,et al.An effective geneknockdown using multiple shRNA-expressing adenovirus vectors[J].J Control Release,2011,153(2):149-153.
[2]Kantor PF,Lougheed J,Dancea A,et al.Presentation,diagnosis,and medical management of heart failure in children:Canadian Cardiovascular Society guidelines[J].Pediatria Polska,2013,29(12):1535.
[3]Katz M,Freimark D,Raichlin E,et al.Risk of early,intermediate,and late rejection following heart transplantation:trends over the past 25 years and relation to changes in medical management tertiary center experience:the sheba heart transplantation registry[J].Clin Transplant,2017,31(10).doi:10.1111/ctr.13063.
[4]Bernstein D.Introduction to the series:chal lenges and opportunities in pediatric heart failure and transplantation[J].Circulation,2014,129(1):112-114.
[5]Zhang HT,Du Y,Cao FF,et al.National consensus on low cardiac output syndrome in China[J].Med J Chin PLA,2017,42(11):933-944.[张海涛,杜雨,曹芳芳,等.低心排血量综合征中国专家共识[J].解放军医学杂志,2017,42(11):933-944.]
[6]Koyanagi M,Bushoven P,Iwasaki M,et al.Notch signaling contributes to the expression of cardiac markers in human circulating progenitor cells[J].Circ Res,2007,101(11):1139-1145.
[7]Koyanagi M,Iwasaki M,Haendeler J,et al.Wnt5a increases cardiac gene expressions of cultured human circulating progenitor cells via a PKC delta activation[J].PLoS One,2009,4(6):e5765.
[8]Fang SH,Liu SX,Chen Y.β-catenin is involved in BMP9-induced differentiation of C3H10T1/2 cells into cardiomyocytelike cells[J].Chin J Cel Mol Im,2016,32(6):750-754.[房松华,刘书霞,陈沅.β联蛋白参与骨形成蛋白9诱导的C3H10T1/2细胞向心肌细胞样细胞分化[J].细胞与分子免疫学杂志,2016,32(6):750-754.]
[9]Pfister O,Della Verde G,Kuster GM,et al.Regenerative therapy for cardiovascular disease[J].Transl Res,2014,163(4):307-320.
[10]Deng NB,Zeng YQ,Han TL,et al.hHO-1 combined with GATA-4 transduction promotes myocardial transdifferentiation and antiapoptosis of rat mesenchymal stem cells[J].Med J Chin PLA,2017,42(4):314-319.[邓宁波,曾元清,韩腾龙,等.hHO-1联合GATA-4修饰大鼠骨髓间充质干细胞抗凋亡及向心肌分化的实验研究[J].解放军医学杂志,2017,42(4):314-319.]
[11]Chamuleau SA,Vrijsen KR,Rokosh DG,et al.Cell therapy for ischaemic heart disease:focus on the role of resident cardiac stem cells[J].Neth Heart J,2009,17(5):199-207.
[12]Srivastava D.Making or breaking the heart:from lineage determination to morphogenesis[J].Cell,2006,126(6):1037-1048.
[13]Cesselli D,Aleksova A,Mazzega E,et al.Cardiac stem cell aging and heart failure[J].Pharmacol Res,2018,127:26-32.
[14]Li M,Chen Y,Bi Y,et al.Establishment and characterization of the reversibly immortalized mouse fetal heart progenitors[J].Int J Med Sci,2013,10(8):1035-1046.
[15]Malek Mohammadi M,Kattih B,Grund A,et al.The transcription factor GATA4 promotes myocardial regeneration in neonatal mice[J].EMBO Mol Med,2017,9(2):265-279.
[16]Gordon JW.Regulation of cardiac myocyte cell death and differentiation by myocardin[J].Mol Cell Biochem,2018,437(1-2):119-131.
[17]England J,Pang KL,Parnall M,et al.Cardiac troponin T is necessary for normal development in the embryonic chick heart[J].J Anat,2016,229(3):436-449.
[18]Chopra SS,Stroud DM,Watanabe H,et al.Voltage-gated sodium channels are required for heart development in zebrafish[J].Circ Res,2010,106(8):1342-1350.
[19]Qin L,Yin YT,Zheng FJ,et al.WNT5A promotes stemness characteristics in nasopharyngeal carcinoma cells leading to metastasis and tumorigenesis[J].Oncotarget,2015,6(12):10239-10252.
[20]Kikuchi A,Yamamoto H,Sato A,et al.Wnt5a:its signalling,functions and implication in diseases[J].Acta Physiol(Oxf),2012,204(1):17-33.
[21]Bhatt PM,Malgor R.Wnt5a:a player in the pathogenesis of atherosclerosis and other inflammatory disorders[J].Atherosclerosis,2014,237(1):155-162.
[22]Palpant NJ,Yasuda S,Macdougald O,et al.Non-canonical Wnt signaling enhances differentiation of Sca1+/c-kit+adiposederived murine stromal vascular cells into spontaneously beating cardiac myocytes[J].J Mol Cell Cardioly,2007,43(3):362-370.
[23]Motegi Y,Katayama K,Sakurai F,et al.An effective geneknockdown using multiple shRNA-expressing adenovirus vectors[J].J Control Release,2011,153(2):149-153.