转录因子Hand2在妊娠期糖尿病胎鼠心脏发育过程中的表达
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摘要
目的:了解妊娠糖尿病(gestational diabetes mellitus,GDM)母鼠胎鼠心脏发育过程中碱性螺旋-环-螺旋蛋白(basic helix-loop-helix,bHLH)转录因子Hand2表达的变化规律,探讨其在GDM胎鼠心脏发育异常中的作用机制。方法:114只成年雌性SD大鼠,空白对照组(n=24)、GDM组(n=30)、阴性对照组(n=30)及GDM+胰岛素干预组(n=30);空白对照组不予任何处理;GDM组腹腔注射2%链脲佐菌素(streptozotocin,STZ;每只40 mg/kg);阴性对照组腹腔注射等量的柠檬酸-柠檬酸钠缓冲液(STZ溶剂);GDM+胰岛素干预组在GDM建模成功后皮下注射中效胰岛素控制空腹血糖在正常范围。给药72 h后每天测血糖及体质量,各组分别于孕12 d(embryotic day 12,E12)、E15和E19剖取胎鼠心脏组织HE染色观察心脏组织病理变化,免疫组化检测Hand2蛋白的表达,实时荧光定量PCR检测Hand2 mRNA的表达,Western blotting检测Hand2蛋白表达的变化。结果:各组Hand2蛋白的表达呈动态变化:E12可见表达,E15时表达增加,E19时Hand2蛋白表达最高,E12和E15 GDM组Hand2 mRNA及蛋白在胎鼠心肌细胞中的表达呈现下降趋势,差异有统计学意义(P<0.05)。结论:妊娠糖尿病母鼠其胎鼠心脏发育异常的发生率明显升高;Hand2 mRNA及蛋白在妊娠糖尿病胎鼠E12和E15心脏表达水平明显下降,提示与GDM胎鼠心脏发育异常相关。
AIM: To study the effect of Hand2( one of basic helix-loop-helix proteins' transcription factors)expression on the development of the cardiac tissues in the fetal rats from gestational diabetes mellitus( GDM) parents,and to investigate the potential pathogenesis of GDM-induced congenital cardiac defects in rats. METHODS: The adult Sprague-Dawley female rats were randomly divided into blank control group( n = 24),GDM group( n = 30),negative control group( n = 30) and insulin intervened group( n = 30). The GDM model was established by intraperitoneal injection of 2%streptozotocin( STZ,40 mg /kg body weight) to the pregnant rats on the successive day. The rats in insulin intervened group were injected with intermediate-acting insulin in order to keep the fasting blood glucose in the normal range. The rats in negative control group were injected with citric acid-sodium citrate buffer solution in the same position. Blood glucose and body weight were examined every day 72 h after STZ injection. On E12,E15 and E19,the rats were anesthetized and the embryonic cardiac tissues were collected after caesarean section. The histopathological changes of the cardiac tissues were observed under microscope with HE staining. The expression of Hand2 was analyzed by the method of immunohistochemistry. The expression of Hand2 in the cardiac tissues at mRNA and protein levels was determined by real-time fluorescence quantitative PCR and Western blotting. RESULTS: During the development of embryonic heart,the protein expression of Hand2 in the cardiac tissues was showed dynamic changes. Observed on E12,obviously increased on E15,and at the highest level on E19. Compared with the other 3 groups,the protein and mRNA expression of Hand2 in GDM group was decreased at the time points of E12 and E15. CONCLUSION: The morbidity of fetal cardiac malformation is significantly increased in GDM group,suggesting that Hand2 may be involved in the development of cardiac malformation in GDM.
AIM: To study the effect of Hand2( one of basic helix-loop-helix proteins' transcription factors)expression on the development of the cardiac tissues in the fetal rats from gestational diabetes mellitus( GDM) parents,and to investigate the potential pathogenesis of GDM-induced congenital cardiac defects in rats. METHODS: The adult Sprague-Dawley female rats were randomly divided into blank control group( n = 24),GDM group( n = 30),negative control group( n = 30) and insulin intervened group( n = 30). The GDM model was established by intraperitoneal injection of 2%streptozotocin( STZ,40 mg /kg body weight) to the pregnant rats on the successive day. The rats in insulin intervened group were injected with intermediate-acting insulin in order to keep the fasting blood glucose in the normal range. The rats in negative control group were injected with citric acid-sodium citrate buffer solution in the same position. Blood glucose and body weight were examined every day 72 h after STZ injection. On E12,E15 and E19,the rats were anesthetized and the embryonic cardiac tissues were collected after caesarean section. The histopathological changes of the cardiac tissues were observed under microscope with HE staining. The expression of Hand2 was analyzed by the method of immunohistochemistry. The expression of Hand2 in the cardiac tissues at mRNA and protein levels was determined by real-time fluorescence quantitative PCR and Western blotting. RESULTS: During the development of embryonic heart,the protein expression of Hand2 in the cardiac tissues was showed dynamic changes. Observed on E12,obviously increased on E15,and at the highest level on E19. Compared with the other 3 groups,the protein and mRNA expression of Hand2 in GDM group was decreased at the time points of E12 and E15. CONCLUSION: The morbidity of fetal cardiac malformation is significantly increased in GDM group,suggesting that Hand2 may be involved in the development of cardiac malformation in GDM.
引文
[1]杨慧霞.妊娠合并糖尿病[M].第2版.北京:人民卫生出版社,2008.25-30.
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[4]Tsuchihashi T,Maeda J,Shin CH,et al.Hand2 function in second heart field progenitors is essential for cardiogenesis[J].Dev Biol,2011,351(1):62-69.
[5]Holler KL,Hendershot TJ,Troy SE,et al.Targeted deletion of Hand2 in cardiac neural crest-derived cells influences cardiac gene expression and outflow tract development[J].Dev Biol,2010,341(1):291-304.
[6]孙凤杰,任建兵,黄晓,等.GATA-4在妊娠期糖尿病胎鼠心脏中的表达[J].中国病理生理杂志,2013,29(3):449-454.
[7]柳国胜,罗瑶,赵立华,等.链脲霉素实验性大鼠妊娠期糖尿病对子鼠心肌细胞超微结构的影响[J].暨南大学学报(医学版),2007,28(4):374-378.
[8]毛东伟,赵一平,李守柔,等.高血糖致胎鼠神经管畸形的发生机理及牛磺酸拮抗作用的研究[J].中华妇产科杂志,2004,39(3):169-172.
[9]丰有吉,沈铿,马丁,等.妇产科学[M].第2版.北京:人民卫生出版社,2010.140-143.
[10]Cripe SM,O'Brien W,Gelaye B,et al.Perinatal outcomes of southeast Asians with pregnancies complicated by gestational diabetes mellitus or preeclampsia[J].J Immigr Minor Health,2012,14(5):747-753.
[11]Wren C,Birrell G,Hawthorne G.Cardiovascular malformations in infants of diabetic mothers[J].Heart,2003,89(10):1217-1220.
[12]Kumar SD,Dheen ST,Tay SS.Maternal diabetes induces congenital heart defects in mice by altering the expression of genes involved in cardiovascular development[J].Cardiovasc Diabetol,2007,6:34.
[13]Lisowski LA,Verheijen PM,Copel JA,et al.Congenital heart disease in pregnancies complicated by maternal diabetes mellitus.An international clinical collaboration,literature review,andmeta-analysis[J].Herz,2010,35(1):19-26.
[14]Olson EN.Gene regulatory networks in the evolution and development of the heart[J].Science,2006,313(5795):1922-1927.
[15]Firulli AB,McFadden DG,Lin Q,et al.Heart and extraembryonic mesodermal defects in mouse embryos lacking the bHLH transcription factor Hand1[J].Nat Genet,1998,18(3):266-270.
[16]Firulli AB,Firulli BA,Wang J,et al.Gene replacement strategies to test the functional redundancy of basic helixloop-helix transcription factor[J].Pediatr Cardiol,2010,31(3):438-448.
[17]Liang J,Gui Y,Wang W,et al.Elevated glucose induces congenital heart defects by altering the expression of tbx5,tbx20,and has2 in developing zebrafish embryos[J].Birth Defects Res A Clin Mol Teratol,2010,88(6):480-486.
[18]孙淑娜,桂永浩,宋后燕,等.hand2基因对斑马鱼胚胎发育影响的实验研究[J].复旦学报:医学版,2008,35(3):523-527.
[19]Morikawa Y,Cserjesi P.Cardiac neural crest expression of Hand2 regulates outflow and second heart field development[J].Circ Res,2008,103(12):1422-1429.
[20]McFadden DG,McAnally J,Richardson JA,et al.Misexpression of dHAND induces ectopic digits in the developing limb bud in the absence of direct DNA binding[J].Development,2002,129(13):3077-3088.
[21]Liu N,Barbosa AC,Chapman SL,et al.DNA bindingdependent and-independent functions of the Hand2 transcription factor during mouse embryogenesis[J].Development,2009,136(6):933-942.
[22]Zhao Y,Samal E,Srivastava D.Serum response factor ergulates a muscle-specific microRNA that targets Hand2during cardio genesis[J].Nature,2005,436(7048):181-182.
[23]Zhao Y,Ransom JF,Li A,et al.Dysregulation of cardiogenesis,cardiac conduction,and cell cycle in mice lacking miRNA-1-2[J].Cell,2007,129(2):303-317.
[2]Carolan M,Davey MA,Biro MA,et al.Maternal age,ethnicity and gestational diabetes mellitus[J].Midwifery,2012,28(6):778-783.
[3]Correa A,Gilboa SM,Besser LM,et al.Diabetes mellitus and birth defects[J].Am J Obstet Gynecol,2008,199(3):231-239.
[4]Tsuchihashi T,Maeda J,Shin CH,et al.Hand2 function in second heart field progenitors is essential for cardiogenesis[J].Dev Biol,2011,351(1):62-69.
[5]Holler KL,Hendershot TJ,Troy SE,et al.Targeted deletion of Hand2 in cardiac neural crest-derived cells influences cardiac gene expression and outflow tract development[J].Dev Biol,2010,341(1):291-304.
[6]孙凤杰,任建兵,黄晓,等.GATA-4在妊娠期糖尿病胎鼠心脏中的表达[J].中国病理生理杂志,2013,29(3):449-454.
[7]柳国胜,罗瑶,赵立华,等.链脲霉素实验性大鼠妊娠期糖尿病对子鼠心肌细胞超微结构的影响[J].暨南大学学报(医学版),2007,28(4):374-378.
[8]毛东伟,赵一平,李守柔,等.高血糖致胎鼠神经管畸形的发生机理及牛磺酸拮抗作用的研究[J].中华妇产科杂志,2004,39(3):169-172.
[9]丰有吉,沈铿,马丁,等.妇产科学[M].第2版.北京:人民卫生出版社,2010.140-143.
[10]Cripe SM,O'Brien W,Gelaye B,et al.Perinatal outcomes of southeast Asians with pregnancies complicated by gestational diabetes mellitus or preeclampsia[J].J Immigr Minor Health,2012,14(5):747-753.
[11]Wren C,Birrell G,Hawthorne G.Cardiovascular malformations in infants of diabetic mothers[J].Heart,2003,89(10):1217-1220.
[12]Kumar SD,Dheen ST,Tay SS.Maternal diabetes induces congenital heart defects in mice by altering the expression of genes involved in cardiovascular development[J].Cardiovasc Diabetol,2007,6:34.
[13]Lisowski LA,Verheijen PM,Copel JA,et al.Congenital heart disease in pregnancies complicated by maternal diabetes mellitus.An international clinical collaboration,literature review,andmeta-analysis[J].Herz,2010,35(1):19-26.
[14]Olson EN.Gene regulatory networks in the evolution and development of the heart[J].Science,2006,313(5795):1922-1927.
[15]Firulli AB,McFadden DG,Lin Q,et al.Heart and extraembryonic mesodermal defects in mouse embryos lacking the bHLH transcription factor Hand1[J].Nat Genet,1998,18(3):266-270.
[16]Firulli AB,Firulli BA,Wang J,et al.Gene replacement strategies to test the functional redundancy of basic helixloop-helix transcription factor[J].Pediatr Cardiol,2010,31(3):438-448.
[17]Liang J,Gui Y,Wang W,et al.Elevated glucose induces congenital heart defects by altering the expression of tbx5,tbx20,and has2 in developing zebrafish embryos[J].Birth Defects Res A Clin Mol Teratol,2010,88(6):480-486.
[18]孙淑娜,桂永浩,宋后燕,等.hand2基因对斑马鱼胚胎发育影响的实验研究[J].复旦学报:医学版,2008,35(3):523-527.
[19]Morikawa Y,Cserjesi P.Cardiac neural crest expression of Hand2 regulates outflow and second heart field development[J].Circ Res,2008,103(12):1422-1429.
[20]McFadden DG,McAnally J,Richardson JA,et al.Misexpression of dHAND induces ectopic digits in the developing limb bud in the absence of direct DNA binding[J].Development,2002,129(13):3077-3088.
[21]Liu N,Barbosa AC,Chapman SL,et al.DNA bindingdependent and-independent functions of the Hand2 transcription factor during mouse embryogenesis[J].Development,2009,136(6):933-942.
[22]Zhao Y,Samal E,Srivastava D.Serum response factor ergulates a muscle-specific microRNA that targets Hand2during cardio genesis[J].Nature,2005,436(7048):181-182.
[23]Zhao Y,Ransom JF,Li A,et al.Dysregulation of cardiogenesis,cardiac conduction,and cell cycle in mice lacking miRNA-1-2[J].Cell,2007,129(2):303-317.