TCDD对小鼠腭突间充质细胞增殖和凋亡的影响
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摘要
目的:探讨2,3,7,8-四氯二苯并对二噁英(TCDD)对小鼠腭突间充质细胞增殖和凋亡的影响。方法:采用随机数字表法将SPF级C57BL/6N孕鼠分为TCDD组和对照组,在妊娠第10天(GD10)分别以64μg/kg TCDD和等量玉米油灌胃,取GD13、GD14、GD15胚胎头部,行组织形态学观察。收集GD13正常胎鼠腭组织,分离腭突间充质细胞,用0. 0、0. 1、1. 0、10. 0 nmol/L TCDD处理72 h后,采用MTT法检测细胞增殖并计算增殖抑制率,检测Brdu阳性率,采用荧光定量RT-PCR和Western blot法检测细胞中PCNA、Bax、Caspase-3 mRNA和蛋白表达水平。结果:组织形态学显示TCDD组胎鼠双侧腭突未接触和融合。随着TCDD作用浓度的增加,小鼠腭突间充质细胞增殖抑制率逐渐增加,Brdu阳性率逐渐降低,细胞中Bax、Caspase-3 mRNA和蛋白表达逐渐增加,而PCNA mRNA和蛋白表达逐渐降低(P <0. 05)。结论:TCDD可能通过抑制腭突间充质细胞的增殖并促进其凋亡,导致腭裂的发生。
Aim: To investigate the effects of 2,3,7,8-tetrachlorodibenzo-p-doxin( TCDD) on proliferation and apoptosis of mouse embryonic palatal mesenchymal( EPM) cell. Methods: The SPF-class C567 BL/6 N pregnant mice were allocated into TCDD group and control group randomly. On gestation day 10( GD10),pregnant mice were gavaged by TCDD at64 μg/kg( TCDD group) and equal amount of corn oil( control group). Coronal sections of GD13 to GD15 embryos were taken and stained with HE to observe morphological changes. GD13 normal fetal mouse palatal tissue was collected,and EPM cells were isolated and cultured with different doses of TCDD( 0. 0,0. 1,1. 0,10. 0 nmol/L) for 72 hours,the cell proliferation was detected by MTT assay,and the Brdu positive rate was detected. Real-time RT-PCR and Western blot were used to determine the expression levels of PCNA,Bax,Caspase-3 mRNA and protein. Results: Morphological results showed bilateral shelves did not contact and fusion in TCDD group. With the increase of TCDD dose,the cell proliferation inhibition rate increased gradually,and the Brdu positive rate gradually decreased( P < 0. 05); the expressions of Bax,Caspase-3 mRNA and protein gradually increased,while the expression of PCNA mRNA and protein gradually decreased( P < 0. 05). Conclusion: TCDD may cause cleft palate by inhibiting the proliferation of EPM cells and promoting apoptosis.
Aim: To investigate the effects of 2,3,7,8-tetrachlorodibenzo-p-doxin( TCDD) on proliferation and apoptosis of mouse embryonic palatal mesenchymal( EPM) cell. Methods: The SPF-class C567 BL/6 N pregnant mice were allocated into TCDD group and control group randomly. On gestation day 10( GD10),pregnant mice were gavaged by TCDD at64 μg/kg( TCDD group) and equal amount of corn oil( control group). Coronal sections of GD13 to GD15 embryos were taken and stained with HE to observe morphological changes. GD13 normal fetal mouse palatal tissue was collected,and EPM cells were isolated and cultured with different doses of TCDD( 0. 0,0. 1,1. 0,10. 0 nmol/L) for 72 hours,the cell proliferation was detected by MTT assay,and the Brdu positive rate was detected. Real-time RT-PCR and Western blot were used to determine the expression levels of PCNA,Bax,Caspase-3 mRNA and protein. Results: Morphological results showed bilateral shelves did not contact and fusion in TCDD group. With the increase of TCDD dose,the cell proliferation inhibition rate increased gradually,and the Brdu positive rate gradually decreased( P < 0. 05); the expressions of Bax,Caspase-3 mRNA and protein gradually increased,while the expression of PCNA mRNA and protein gradually decreased( P < 0. 05). Conclusion: TCDD may cause cleft palate by inhibiting the proliferation of EPM cells and promoting apoptosis.
引文
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[14]HU X,GAO JH,LIAO YJ,et al. 2,3,7,8-tetrachlorodibenzo-p-dioxin delays palatal shelf elevation and suppresses Wnt5a and lymphoid enhancing-binding factor 1 signaling in developing palate[J]. Cleft Palate Craniofaci J,2015,52(1):54
[15]王浩宇,安峰,吴靖芳,等. BCL-2与P53在腭发育及腭裂形成中的作用[J].毒理学杂志,2009,23(4):267
[16]SEIFERT A,RAU S,KUELLERTZ G,et al. TCDD induces cell migration via NFATc1/ATX-signaling in MCF-7 cells[J]. Toxicol Lett,2009,184(1):26
[17]CHEN SC,LIAO TL,WEI YH,et al. Endocrine disruptor,dioxin(TCDD)-induced mitochondrial dysfunction and apoptosis in human trophoblast-like JAR cells[J]. Mol Hum Reprod,2010,16(5):361
[18]KHAN Z,BISEN PS. Oncoapoptotic signaling and deregulated target genes in cancers:special reference to oral cancer[J]. Biochim Biophys Acta,2013,1836(1):123
[19]SETIA S,SANYAL SN. Downregulation of NF-kappa B and PCNA in the regulatory pathways of apoptosis by cyclooxygenase-2 inhibitors in experimental lung cancer[J]. Mol Cell Biochem,2012,369(1/2):75
[20]BISWAS S,SHI Q,MATISE L,et al. A role for proapoptotic Bax and Bak in T-cell differentiation and transformation[J]. Blood,2010,116(24):5237
[21]HASTAK K,GUPTA S,AHMAD N,et al. Role of p53 and NF-kappaB in epigallocatechin-3-gallate-induced apoptosis of LNCa P cells[J]. Oncogene,2003,22(31):4851
[22]XU WL,MI YQ,HE P,et al. Gamma-tocotrienol inhibits proliferation and induces apoptosis via the mitochondrial pathway in human cervical cancer He La cells[J]. Molecules,2017,22(8):1299
[2] LEITE IC,PAUMGARTTEN FJ,KOIFMAN S. Chemical exposure during pregnancy and oral clefts in newborns[J].Cad Saude Publica,2002,18(1):17
[3] YOSHIZAWA K,HEATHERLY A,MALARKEY DE,et al.A critical comparison of murine pathology and epidemiological data of TCDD,PCB126,and Pe CDF[J]. Toxicol Pathol,2007,35(7):865
[4] ABBOTT BD. The etiology of cleft palate:a 50-year search for mechanistic and molecular understanding[J]. Birth Defects Res B Dev Reprod Toxicol,2010,89(4):266
[5] YAMADA T,MISHIMA K,FUJIWARA K,et al. Cleft lip and palate in mice treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin:a morphological in vivo study[J]. Congenit Anom(Kyoto),2006,46(1):21
[6] BUSH JO,JIANG RL. Palatogenesis:morphogenetic and molecular mechanisms of secondary palate development[J]. Development,2012,139(2):231
[7] TAKAGI TN,MATSUI KA,YAMASHITA K,et al. Pathogenesis of cleft palate in mouse embryos exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD)[J]. Teratog Carcinog Mutagen,2000,20(2):73
[8] JHEON AH,SCHNEIDER RA. The cells that fill the bill:neural crest and the evolution of craniofacial development[J]. J Dent Res,2009,88(1):12
[9] BALDRIDGE MG,MARKS GT,RAWLINS RG,et al. Very low-dose(femtomolar)2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD)disrupts steroidogenic enzyme mRNAs and steroid secretion by human luteinizing granulosa cells[J]. Reprod Toxicol,2015,52(1):57
[10]LI Y,WANG K,JIANG YZ,et al. 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD)inhibits human ovarian cancer cell proliferation[J]. Cell Oncol(Dordr),2014,37(6):429
[11]ZHANG LA,MA J,TAKEUCHI M,et al. Suppression of experimental autoimmune uveoretinitis by inducing differentiation of regulatory T cells via activation of aryl hydrocarbon receptor[J]. Invest Ophthalmol Vis Sci,2010,51(4):2109
[12]张丁文,袁心刚,傅跃先,等. TCDD诱导C57BL/6J胎鼠腭裂过程中组蛋白H4乙酰化的表达[J].中华整形外科杂志,2018,34(4):305
[13]何晓梦,刘翠苹,蒲亚兰,等.以形态与组织学为基础筛选诱导胎鼠腭裂的四氯二苯二噁英最适剂量[J].卫生研究,2013,42(2):277
[14]HU X,GAO JH,LIAO YJ,et al. 2,3,7,8-tetrachlorodibenzo-p-dioxin delays palatal shelf elevation and suppresses Wnt5a and lymphoid enhancing-binding factor 1 signaling in developing palate[J]. Cleft Palate Craniofaci J,2015,52(1):54
[15]王浩宇,安峰,吴靖芳,等. BCL-2与P53在腭发育及腭裂形成中的作用[J].毒理学杂志,2009,23(4):267
[16]SEIFERT A,RAU S,KUELLERTZ G,et al. TCDD induces cell migration via NFATc1/ATX-signaling in MCF-7 cells[J]. Toxicol Lett,2009,184(1):26
[17]CHEN SC,LIAO TL,WEI YH,et al. Endocrine disruptor,dioxin(TCDD)-induced mitochondrial dysfunction and apoptosis in human trophoblast-like JAR cells[J]. Mol Hum Reprod,2010,16(5):361
[18]KHAN Z,BISEN PS. Oncoapoptotic signaling and deregulated target genes in cancers:special reference to oral cancer[J]. Biochim Biophys Acta,2013,1836(1):123
[19]SETIA S,SANYAL SN. Downregulation of NF-kappa B and PCNA in the regulatory pathways of apoptosis by cyclooxygenase-2 inhibitors in experimental lung cancer[J]. Mol Cell Biochem,2012,369(1/2):75
[20]BISWAS S,SHI Q,MATISE L,et al. A role for proapoptotic Bax and Bak in T-cell differentiation and transformation[J]. Blood,2010,116(24):5237
[21]HASTAK K,GUPTA S,AHMAD N,et al. Role of p53 and NF-kappaB in epigallocatechin-3-gallate-induced apoptosis of LNCa P cells[J]. Oncogene,2003,22(31):4851
[22]XU WL,MI YQ,HE P,et al. Gamma-tocotrienol inhibits proliferation and induces apoptosis via the mitochondrial pathway in human cervical cancer He La cells[J]. Molecules,2017,22(8):1299