孕期不同阶段铅暴露对大鼠胎盘血栓调节蛋白、核因子-κB表达影响的实验研究
摘要
目的：探讨孕期不同阶段铅暴露对大鼠胎盘血栓调节蛋白(thrombomodulin, TM)、核转录因子NF-κB (nuclear ranscription factor-κB, NF-κB)的影响,推测铅暴露对胎盘的损伤机制。
方法：108只大鼠随机分为A,B,C,D4组,0.025%醋酸铅对实验组大鼠染毒。根据大鼠孕期3周推算,足月剖宫产,孕末期腹腔静脉取血,采用原子吸收光谱仪测定孕期不同阶段低水平铅暴露后的大鼠血铅水平。统计记录各组的平均血铅水平。免疫组织化学方法测定大鼠胎盘内TM、NF-κB的表达。应用实时荧光定量PCR检测大鼠胎盘TMmRNA的表达。
结果：①实验组大鼠孕末期血铅水平均高于对照组(P<0.01)。受孕大鼠血铅水平与胎盘重量呈负相关(r=—0.652、p<0.01)。②NF-κB的OD值实验组(B、C、D组)明显高于对照组(A组),且差别具有统计学意义(P<0.01)。除了B、C两组比较以外,任意两组在NF-κB表达的差异都具有显著性意义。③胎盘组织中TM蛋白表达与TM mRNA的表达趋势基本一致,实验B、C、D组呈依次升高,与A组(对照组)比较差异有显著性(P<0.01),其中B组和C组低于A组低,而D组明显高于A组。
结论：①孕期不同阶段的铅暴露对NF-κB的表达产生不同的影响,其对胞浆细胞器的损害可能干扰母体与胎儿之间营养物质和氧气的交换,这可能是导致异常妊娠的原因之一。②孕期不同阶段铅暴露可影响TM基因mRNA和蛋白的表达；TM表达异常引起的消耗性血管内凝血和胎盘血管化生可能是孕期铅暴露致胎盘损伤的重要机制之一。
Objective To investigate the toxicity of lead exposure on the placenta at different dosages and the relationship with placental expression of NF-κB and thrombomodulin.
Methods 108 wistar rats were randomly divided into four groups (A,B,C,D)for consumption of water with or without 0.025% lead acetate during various gestational periods; Blood lead content was determined by atomic absorption spectrophotometry. Placental NF-κB and thrombomodulin expression was evaluated by immunohistochemistry. TM mRNA was evaluated by fluorescent quantitative real time PCR.
Results Maternal blood lead levels in the rats was significantly higher compared to controls (P<0.01). Maternal blood lead levels in the rats negatively correlated with placental weight(r=0.652, p<0.01). On the other hand, placental expression of NF-κB in lead-exposed rats was significantly higher than that in controls(P<0.01). The placental expression of TM mRNA in lead-exposed rats was corresponding with the expression of TM protein. The tendency was increased continuous in group B, group C and group D. The TM expression in group B and group C was lower than in group A, whereas the expression in group D was higher than in group A (P<0.01).
Conclusion Lead exposure at various gestational periods produce varied effects, with NF-κB activation following lead exposure.Injury to cytoplasmic organelles may interfere with the nutrition and oxygen exchange between mother and fetus, which may be contribute to abnormal pregnancy outcomes. On the other hand, Lead exposure on rat placental may affect the expression of thrombomodulin. This effect could be associated with impairment in the placenta and may represent one mechanism by which prenatal lead exposure, caseuse impairment by consumptive intravascular coagulation and placental vascularization.
方法：108只大鼠随机分为A,B,C,D4组,0.025%醋酸铅对实验组大鼠染毒。根据大鼠孕期3周推算,足月剖宫产,孕末期腹腔静脉取血,采用原子吸收光谱仪测定孕期不同阶段低水平铅暴露后的大鼠血铅水平。统计记录各组的平均血铅水平。免疫组织化学方法测定大鼠胎盘内TM、NF-κB的表达。应用实时荧光定量PCR检测大鼠胎盘TMmRNA的表达。
结果：①实验组大鼠孕末期血铅水平均高于对照组(P<0.01)。受孕大鼠血铅水平与胎盘重量呈负相关(r=—0.652、p<0.01)。②NF-κB的OD值实验组(B、C、D组)明显高于对照组(A组),且差别具有统计学意义(P<0.01)。除了B、C两组比较以外,任意两组在NF-κB表达的差异都具有显著性意义。③胎盘组织中TM蛋白表达与TM mRNA的表达趋势基本一致,实验B、C、D组呈依次升高,与A组(对照组)比较差异有显著性(P<0.01),其中B组和C组低于A组低,而D组明显高于A组。
结论：①孕期不同阶段的铅暴露对NF-κB的表达产生不同的影响,其对胞浆细胞器的损害可能干扰母体与胎儿之间营养物质和氧气的交换,这可能是导致异常妊娠的原因之一。②孕期不同阶段铅暴露可影响TM基因mRNA和蛋白的表达；TM表达异常引起的消耗性血管内凝血和胎盘血管化生可能是孕期铅暴露致胎盘损伤的重要机制之一。
Objective To investigate the toxicity of lead exposure on the placenta at different dosages and the relationship with placental expression of NF-κB and thrombomodulin.
Methods 108 wistar rats were randomly divided into four groups (A,B,C,D)for consumption of water with or without 0.025% lead acetate during various gestational periods; Blood lead content was determined by atomic absorption spectrophotometry. Placental NF-κB and thrombomodulin expression was evaluated by immunohistochemistry. TM mRNA was evaluated by fluorescent quantitative real time PCR.
Results Maternal blood lead levels in the rats was significantly higher compared to controls (P<0.01). Maternal blood lead levels in the rats negatively correlated with placental weight(r=0.652, p<0.01). On the other hand, placental expression of NF-κB in lead-exposed rats was significantly higher than that in controls(P<0.01). The placental expression of TM mRNA in lead-exposed rats was corresponding with the expression of TM protein. The tendency was increased continuous in group B, group C and group D. The TM expression in group B and group C was lower than in group A, whereas the expression in group D was higher than in group A (P<0.01).
Conclusion Lead exposure at various gestational periods produce varied effects, with NF-κB activation following lead exposure.Injury to cytoplasmic organelles may interfere with the nutrition and oxygen exchange between mother and fetus, which may be contribute to abnormal pregnancy outcomes. On the other hand, Lead exposure on rat placental may affect the expression of thrombomodulin. This effect could be associated with impairment in the placenta and may represent one mechanism by which prenatal lead exposure, caseuse impairment by consumptive intravascular coagulation and placental vascularization.
引文
[1]张慧英.血栓调节蛋白的研究及其意义[J].生理科学进展,1993,23(4)：323-326
[2]马海燕,李红,等.铅的胎盘毒性[J].《中国临床医生》,2006,34(9)：21-23
[3]Evans TJ, James2Kracke MR, Kleiboeker SB, et al. Lead enters Rcho21trophoblastic cellsbycalcium transportmechanismsand com-plexes with cytosolic calcium-binding proteins[J]. Toxicol Appl Pharmacol,2003,186(2):772-89
[4]MantonW I,Angle C R,Stanrek KL,et al.Release of lead from bone in pregnancy and lactation[J].Envion Res,2003,92(2):139-151.
[5]Lafond J, HamelA, TakserL,et al. Low environmental contamination by lead in pregnantwomen: effecton calcium transfer in human placental syncytiotrophoblasts[J]. J Toxicol Environ Health A.2004,67(14):1069-1079
[6]李红,马海燕,赵延祜.围产期铅毒性[J].中华围产医学杂志,2004,9：315-317
[7]马海燕,李红,王教辰,等.孕期不同阶段铅暴露对大鼠胎盘和仔鼠的影响[J].中华预防医学志,2006,2：101-104.
[8]FalconM, Vinas P, Luna A. Placental lead and outcome of pregnancy[J]. Toxicology,2003,185(1 2):59-66
[9]李红,马海燕,王云英,等.不同孕期大鼠铅暴露胎盘一氧化氮／一氧化氮合酶系统与其超微结构改变的相关性[J].实用儿科临床杂志,2006,21(18)：1230-1231
[10]ShaamashAH, Elsonosy ED, ZakhariMM, et al. Placental nitricoxide synthase (NOS) activity and nitric oxide (NO) production in normal pregnancy, pre2eclampsia and eclampsia[J]. Int JGynaecolObstet,2001,72(2):127-133
[11]Villeda Hernandez J, MendezArmentaM, BarrosoMoguel R, etal.Morp2hometric analysis of brain lesions in rat fetu2ses prenatally exposed to low level lead acetate:correlation with lipid peroxidation[J]. HistolHistopathol,2006,21(6):609-17
[12]马海燕,李红,王教辰,徐风森.妊娠期低水平铅暴露时胎盘组织中金属硫蛋白的表达[J].中华妇产科杂志,2006,41(10)：676-679
[13]ShimadaA, Yamamoto E, Morita T, et al. Ultrastructural demon stration ofmercury granules in the placenta ofmetallothionein null pregnantmice after exposure tomercury vapor[J]. Toxicol Pat2hol.2004,32(5):519-526
[14]McAleerMF, TuanRS. Metallothionein protectsag ainst severe oxidative stress induced apoptosisof human trophoblastic cells[J]. I VitrMol Tox2icol.2001,14(3):229-231
[15]DuboisB, Arnold B, Opdenakker G. Gelatinase B deficiency impairs reproduction[J]. J Clin Invest, 2000,106:627-628
[16]李向红,马海燕,李红,张采欣,徐风森等.不同阶段铅暴露对大鼠妊娠结局及胎盘MMP-9表达 的影响[J].中国优生与遗传杂志,2008,16(10)：41-43
[17]郑汝强.血栓调节蛋白生物学特性及相关疾病研究进展[J].中华新医学,2005,6(1)：71-72
[18]Aso Y,lunkai T,Takemura Y,et al.Mechanisms of elevation of seurm and urinary conentration of soluble thrombodulin in diabetic patients:possible as a marker for vascular endothelial injury.Metabolism,1998,47:362-365
[19]叶方.创伤性休克大鼠肾脏血栓调节蛋白表达的变化和意义[J].南京医科大学学报(自然科学版),2005,25(2)：113-115
[20]Fujimi S,Ogurah,Tanaka H,et al.Increased production of leukocyte microparticles with enhanced expression of adhesion molecules from activated polymorphonuclear leukocyte in severely injuried patients[J].Trauma,2003,54(1):114-119
[21]Kokame K, Zheng X, Sadler JE. J Biol Chem,1998,273(20):12-35
[22]Bouma BN, MosnierLO, MeijersJC, et al. Thromb Haemost,1999,82(6):1703-1708.
[23]Ma ruyama I, et al J Cell Blot 1985,101:383-385
[24]Ishii H. et al. Blood 1986,57:362-368
[25]孙金波,单晓清.血栓调节蛋白与冠心病[J].国外医学内科学分册,2002,29(4)：149-152
[26]朱海燕,沈洪,等.动脉微栓塞组织血栓调节蛋白变化的实验研究[J].中国全科医学,2006,9(8)：637-639
[27]CornuzJ,Person SD,Creager MA,et al. Importance of findings on the initial evaluationfor cancer in patients with symptomatic idiopathic deep venous thrombosisJ. Ann Int Med,1996,125:785-787
[28]杨忠思,谭齐贤.血栓调节蛋白与肿瘤研究进展[J].青岛大学医学院学报,2003,39(1)：106-108
[29]Curg PM,Butcher DN,Fisher C, et al.Mod2Pathol,2000,13(2):107
[30]马水清,白春梅,边旭明.血栓调节蛋白在妊高症患者血浆中的检测[J].中华围产医学杂志,2000,3(1)：43～44
[31]沈丽丽,杨群英,陈淡璋,等.新生儿窒息可溶性血栓调节蛋白及其临床意义[J].实用儿科临床杂志,1999,14(6)：328-331
[32]Sen R, Baltimore D. Multiple nuclear factors interact with the immunoglobulin enhancer sequences [J]. Cell 1986; 46 (5):705-716.
[33]汪福州,景亮.对核因子NF-κB在免疫反应和细胞凋亡中作用的认识[J].中华麻醉,2007,9(4)：34-37
[34]Henkel T, Zabel U, van Zee K, et al. Intramolecular masking of the nuclear location signal and dimerization domain in the p recursor for the p50 NF2kappa B subunit. Cell,1992,68 (6): 1121-1133
[35]Xia Y, Pauza ME, FengL, et al. Rel B regulation of chemokine expression modulates local infiammation. AmJ Pathol,1997,151:375-387.
[36]Djavaheri-Mergny M, Amelotti M, Mathieu J, et al. NF-kappaB activation represses tumor necrosis factor-alpha-induced autophagy. J Biol Chem,2006,281 (41):30373～30382
[37]Ivanov VN,Ronai Z.p38 Protects human melanoma cells from UV-induced apoptosis through down-regulation of NF-kappaB zctivitu and Fas expression.Oncogene 2000;19:3003-3012
[38]Campbell KJ,Rocha S,Perkin ND.Active repression of antipoptotic gene expression by Rel(p65) NF-kappaB.Mol Cell 2004;13:853-865.
[39]Chen X,Kandasamy K,Srivastava RK.Differential roles of Rel(p65) and c-Rel subunits of nuclear factor kappaB in tumor uecrosis factor-related bapoptosis-inducing ligand singaling.Cancer Res 2003;63:1059-1066.
[40]Aya K, AlhawagriM, Hagen2Stap leton A, et al. NF-(kappa)B inducing kinase controls lymphocyte and osteoclast activities in inflammatory arthritis. J Clin Invest,2005.115 (7): 1848～1854.
[41]Shin SR, Sanchez-Velar N, Sherr DH, et al.7,12-dimethyl-benz(a) anthracene treatment of a c-rel mouse mammary tumor cell line induces ep ithelial to mesenchymal transition via activation of nuclear factor-kappaB [J]. Cancer Res,2006,66 (5):2570-2575.
[42]Kucharczak J, SmimonsMJ, Fan Y, et al. To be, or not to be:NF-kappaB is the answer-Role of Rel/NF-kappaB in the regulation of apop tosis [J]. Oncogene,2003,22 (56):8961-8982.
[43]Arsura M, Cavin LG. Nuclear factor2kappaB and liver carcino-genesis[J]. Cancer Lett,2005, 229 (2):157-169.
[44]Shishodia S, Aggarwal BB. Nuclear factor2kappaB:a friend or a foe in cancer? [J]. Biochem Pharmacol,2004,68 (6):1071-1080.
[45]史艳晖卢圣栋.转录因子NF-κB的研究现状及其应用前景[J].中国生物工程杂志,2007,27(4)：110-104
[46]蒋荣珍,陈汉平.核因子KB在妊高征患者胎盘组织的定位及定量研究,中国优生与遗传杂志[J],2004,12(3)：46-48
[47]王春香,王庆华,钟青.核因子在前置胎盘患者胎盘组织中的表达[J].中国现代医生,2008,46(18)：91-92
[2]马海燕,李红,等.铅的胎盘毒性[J].《中国临床医生》,2006,34(9)：21-23
[3]Evans TJ, James2Kracke MR, Kleiboeker SB, et al. Lead enters Rcho21trophoblastic cellsbycalcium transportmechanismsand com-plexes with cytosolic calcium-binding proteins[J]. Toxicol Appl Pharmacol,2003,186(2):772-89
[4]MantonW I,Angle C R,Stanrek KL,et al.Release of lead from bone in pregnancy and lactation[J].Envion Res,2003,92(2):139-151.
[5]Lafond J, HamelA, TakserL,et al. Low environmental contamination by lead in pregnantwomen: effecton calcium transfer in human placental syncytiotrophoblasts[J]. J Toxicol Environ Health A.2004,67(14):1069-1079
[6]李红,马海燕,赵延祜.围产期铅毒性[J].中华围产医学杂志,2004,9：315-317
[7]马海燕,李红,王教辰,等.孕期不同阶段铅暴露对大鼠胎盘和仔鼠的影响[J].中华预防医学志,2006,2：101-104.
[8]FalconM, Vinas P, Luna A. Placental lead and outcome of pregnancy[J]. Toxicology,2003,185(1 2):59-66
[9]李红,马海燕,王云英,等.不同孕期大鼠铅暴露胎盘一氧化氮／一氧化氮合酶系统与其超微结构改变的相关性[J].实用儿科临床杂志,2006,21(18)：1230-1231
[10]ShaamashAH, Elsonosy ED, ZakhariMM, et al. Placental nitricoxide synthase (NOS) activity and nitric oxide (NO) production in normal pregnancy, pre2eclampsia and eclampsia[J]. Int JGynaecolObstet,2001,72(2):127-133
[11]Villeda Hernandez J, MendezArmentaM, BarrosoMoguel R, etal.Morp2hometric analysis of brain lesions in rat fetu2ses prenatally exposed to low level lead acetate:correlation with lipid peroxidation[J]. HistolHistopathol,2006,21(6):609-17
[12]马海燕,李红,王教辰,徐风森.妊娠期低水平铅暴露时胎盘组织中金属硫蛋白的表达[J].中华妇产科杂志,2006,41(10)：676-679
[13]ShimadaA, Yamamoto E, Morita T, et al. Ultrastructural demon stration ofmercury granules in the placenta ofmetallothionein null pregnantmice after exposure tomercury vapor[J]. Toxicol Pat2hol.2004,32(5):519-526
[14]McAleerMF, TuanRS. Metallothionein protectsag ainst severe oxidative stress induced apoptosisof human trophoblastic cells[J]. I VitrMol Tox2icol.2001,14(3):229-231
[15]DuboisB, Arnold B, Opdenakker G. Gelatinase B deficiency impairs reproduction[J]. J Clin Invest, 2000,106:627-628
[16]李向红,马海燕,李红,张采欣,徐风森等.不同阶段铅暴露对大鼠妊娠结局及胎盘MMP-9表达 的影响[J].中国优生与遗传杂志,2008,16(10)：41-43
[17]郑汝强.血栓调节蛋白生物学特性及相关疾病研究进展[J].中华新医学,2005,6(1)：71-72
[18]Aso Y,lunkai T,Takemura Y,et al.Mechanisms of elevation of seurm and urinary conentration of soluble thrombodulin in diabetic patients:possible as a marker for vascular endothelial injury.Metabolism,1998,47:362-365
[19]叶方.创伤性休克大鼠肾脏血栓调节蛋白表达的变化和意义[J].南京医科大学学报(自然科学版),2005,25(2)：113-115
[20]Fujimi S,Ogurah,Tanaka H,et al.Increased production of leukocyte microparticles with enhanced expression of adhesion molecules from activated polymorphonuclear leukocyte in severely injuried patients[J].Trauma,2003,54(1):114-119
[21]Kokame K, Zheng X, Sadler JE. J Biol Chem,1998,273(20):12-35
[22]Bouma BN, MosnierLO, MeijersJC, et al. Thromb Haemost,1999,82(6):1703-1708.
[23]Ma ruyama I, et al J Cell Blot 1985,101:383-385
[24]Ishii H. et al. Blood 1986,57:362-368
[25]孙金波,单晓清.血栓调节蛋白与冠心病[J].国外医学内科学分册,2002,29(4)：149-152
[26]朱海燕,沈洪,等.动脉微栓塞组织血栓调节蛋白变化的实验研究[J].中国全科医学,2006,9(8)：637-639
[27]CornuzJ,Person SD,Creager MA,et al. Importance of findings on the initial evaluationfor cancer in patients with symptomatic idiopathic deep venous thrombosisJ. Ann Int Med,1996,125:785-787
[28]杨忠思,谭齐贤.血栓调节蛋白与肿瘤研究进展[J].青岛大学医学院学报,2003,39(1)：106-108
[29]Curg PM,Butcher DN,Fisher C, et al.Mod2Pathol,2000,13(2):107
[30]马水清,白春梅,边旭明.血栓调节蛋白在妊高症患者血浆中的检测[J].中华围产医学杂志,2000,3(1)：43～44
[31]沈丽丽,杨群英,陈淡璋,等.新生儿窒息可溶性血栓调节蛋白及其临床意义[J].实用儿科临床杂志,1999,14(6)：328-331
[32]Sen R, Baltimore D. Multiple nuclear factors interact with the immunoglobulin enhancer sequences [J]. Cell 1986; 46 (5):705-716.
[33]汪福州,景亮.对核因子NF-κB在免疫反应和细胞凋亡中作用的认识[J].中华麻醉,2007,9(4)：34-37
[34]Henkel T, Zabel U, van Zee K, et al. Intramolecular masking of the nuclear location signal and dimerization domain in the p recursor for the p50 NF2kappa B subunit. Cell,1992,68 (6): 1121-1133
[35]Xia Y, Pauza ME, FengL, et al. Rel B regulation of chemokine expression modulates local infiammation. AmJ Pathol,1997,151:375-387.
[36]Djavaheri-Mergny M, Amelotti M, Mathieu J, et al. NF-kappaB activation represses tumor necrosis factor-alpha-induced autophagy. J Biol Chem,2006,281 (41):30373～30382
[37]Ivanov VN,Ronai Z.p38 Protects human melanoma cells from UV-induced apoptosis through down-regulation of NF-kappaB zctivitu and Fas expression.Oncogene 2000;19:3003-3012
[38]Campbell KJ,Rocha S,Perkin ND.Active repression of antipoptotic gene expression by Rel(p65) NF-kappaB.Mol Cell 2004;13:853-865.
[39]Chen X,Kandasamy K,Srivastava RK.Differential roles of Rel(p65) and c-Rel subunits of nuclear factor kappaB in tumor uecrosis factor-related bapoptosis-inducing ligand singaling.Cancer Res 2003;63:1059-1066.
[40]Aya K, AlhawagriM, Hagen2Stap leton A, et al. NF-(kappa)B inducing kinase controls lymphocyte and osteoclast activities in inflammatory arthritis. J Clin Invest,2005.115 (7): 1848～1854.
[41]Shin SR, Sanchez-Velar N, Sherr DH, et al.7,12-dimethyl-benz(a) anthracene treatment of a c-rel mouse mammary tumor cell line induces ep ithelial to mesenchymal transition via activation of nuclear factor-kappaB [J]. Cancer Res,2006,66 (5):2570-2575.
[42]Kucharczak J, SmimonsMJ, Fan Y, et al. To be, or not to be:NF-kappaB is the answer-Role of Rel/NF-kappaB in the regulation of apop tosis [J]. Oncogene,2003,22 (56):8961-8982.
[43]Arsura M, Cavin LG. Nuclear factor2kappaB and liver carcino-genesis[J]. Cancer Lett,2005, 229 (2):157-169.
[44]Shishodia S, Aggarwal BB. Nuclear factor2kappaB:a friend or a foe in cancer? [J]. Biochem Pharmacol,2004,68 (6):1071-1080.
[45]史艳晖卢圣栋.转录因子NF-κB的研究现状及其应用前景[J].中国生物工程杂志,2007,27(4)：110-104
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