高胆红素血症新生大鼠脑组织激活素A、Caspase-3表达的研究
摘要
目的高胆红素血症(hyperbilirubinemia)是新生儿期最常见的疾病,严重者可致死或造成中枢神经系统伤残而终生受累。激活素A(activin A,ACT A)是一种糖蛋白激素,最新研究证实,ACT A参与缺氧缺血性脑损伤(hypoxic-ischemic brain damage,HIBD)的发病过程,并且外源性ACT A对新生大鼠HIBD具有神经保护作用。Caspase-3是细胞凋亡过程中的关键酶,通过降解Caspase-3及相应底物可促进细胞凋亡。本研究采用动物实验的方法,研究新生大鼠高胆红素血症不同程度脑损伤后ACT A、Caspase-3表达的变化规律,旨在为进一步探讨高胆红素血症新生儿中枢神经系统损伤的发病机制与有效的防治措施提供实验依据。
方法将100只新生7日龄SD大鼠随机分为正常对照组(C组,n=10)和实验组(T组,n=90),T组又根据腹腔注射胆红素剂量的依次增加分为T1,T2和T3组,每组30只。C组SD大鼠腹腔注射生理盐水1.0ml,T1组SD大鼠腹腔注射胆红素50μg/g,T2组腹腔注射胆红素100μg/g,T3组腹腔注射胆红素200μg/g。各实验组根据造模结束后不同时间点(0h、4h、8h、12h、24h)分为5组,每组6只大鼠。观察各组幼鼠不同时间点的神经行为异常如抽搐、翻滚、俯伏、对外界刺激的逃避反应减弱等;检测各组幼鼠血清胆红素浓度、脑组织胆红素浓度及脑组织含水量;取各组幼鼠脑组织置4%多聚甲醛固定、常规梯度乙醇脱水、二甲苯透明、浸蜡、包埋,冠状切片(厚4μm),常规苏木素-伊红(hematoxylin eosin,HE)染色,光镜下观察组织病理改变,免疫组织化学方法检测脑组织内源性ACTA和Caspase-3的表达。数据以(?)±s表示,应用SPSS13.0软件进行统计学处理。
结果C组及T1组幼鼠于造模结束后各时间点均未见明显的神经行为异常。T2组、T3组幼鼠分别于造模后12h、8h出现翻滚、震颤、俯伏及对外界刺激的逃避反应减弱等表现,T3组幼鼠行为异常表现较T2组明显。造模结束后0h,各组幼鼠血清胆红素浓度、脑组织胆红素浓度及脑组织含水量无显著性差异(P＞0.05)。T1组血清胆红素浓度于造模后8h、12h均高于C组(P＜0.05),T1组脑组织胆红素含量、脑组织含水量及ACT A、Caspase-3表达于造模后各时间点与C组比较均无统计学意义(P＞0.05)。T2、T3组血清胆红素浓度、脑组织胆红素浓度、脑组织含水量及ACT A、Caspase-3表达于造模后各时间点均明显高于C组(P＜0.01)。脑组织胆红素浓度与脑组织含水量及脑组织ACT A、Caspase-3表达均呈正相关(r=0.876,P＜0.01;r=0.886,P＜0.01;r=0.900,P＜0.01)。C组、T1组大脑皮质、海马区神经元排列整齐,结构完整,未见胆红素沉积;T2、T3组神经元数量减少,结构紊乱,并可见不等量的胆红素沉积。ACT A阳性细胞主要出现在大脑皮质及海马区,阳性反应主要位于细胞浆及突起,胞核不着色;Caspase-3阳性细胞以神经元、神经胶质细胞为主,阳性免疫反应位于细胞核,也有一些位于胞浆及突起。
结论
1.高胆红素血症模型鼠均存在不同程度的脑损伤及神经行为异常;其程度与造模后脑组织胆红素含量成正相关。
2.高胆红素血症可诱导模型鼠脑组织Caspase-3表达增加,表明高胆红素血症可加速神经细胞凋亡。
3.高胆红素血症可诱导模型鼠脑组织ACT A表达增加,提示内源性ACT A参与了高胆红素血症所致新生大鼠脑组织损伤的发病过程,可望成为新生儿高胆红素血症及其后所致中枢神经系统损伤病情判定的敏感指标之一。
Objective Hyperbilirubinemia is the most common disease during neonatal period, which leads to neonatal death or induces central nervous system injury.Activin A (ACT A)is a sort of sugar proteinic hormone.The latest research shows that ACT A participates in hypoxic-ischemic brain damage(HIBD),and the exogenous ACT A can protect the brain tissue of neonatal rats with hypoxic-ischemic brain damage. Caspase-3 is the key enzyme in the process of apoptosis.The method of this research adopts animal experiment,it investigates the expression of ACT A and Caspase-3 in different severity of brain injuries of neonatal rats with hyperbilirubinemia.It aims to provide experimental gist to the pathogenesis of central nervous system injury and the valid prevention and cure measure for further study of hyperbilirubinemia.
Methods Totally 100 seven-day-old SD rats were randomly assigned to the normal control group(n=10)and the experiment group(n=90).The rats of experiment group were medially subdivided into 3 groups:T1,T2,and T3 groups(30 for each) according to the increased bilirubin injected intraperitoneally.The specimens were collected at each time point(0,4,8,12,24 hours)after the bilirubin injection.Observe the behavior disorder of every group at each time point.The bilirubin contents in the brain and serum were measured and the degree of the brain edema was assessed by wet/dry weight ratios of the brain tissue.Brain tissues were fixed in 4% paraformaldehyde at certain time point for a certain period,then anhydrated in gradient ethanol,luciditied in xylene,embed in paraffin.Observe the pathology changes of brain tissue with H&E staining and the expression of ACT A and Caspase-3 by immunohistochemistry staining under light microscope.All data were expressed as mean±standard variance((?)±s).All statistical analyses were completed by the package of SPSS13.0.
Results The serum bilirubin content of T1 group increased obviously at 8h,12h than those in C group(P＜0.05).There was no significant difference in the brain bilirubin content,the brain edema and the expression of activin A and Caspase-3 between T1 group and C group(P＞0.05).The bilirubin contents in the brain and serum,the brain edema and the expression of activin A and Caspase-3 of T2,T3 group increased respectively at each time points after the bilirubin injection(P＜0.01,vs C). There was positive relation between the brain bilirubin content and the brain edema, the expression of activin A and Caspase-3(r=0.876,P＜0.01;r=0.886,P＜0.01; r=0.900,P＜0.01).The neurons of C group had complete structure and lined up in order.The neurons of T2,T3 group shrank obviously,and normal structure and arrangement disappeared.In T2,T3 group,the expression of activin A began at 8h, reached a peak at 12h and was still in high level until 24h after the bilirubin injection, while the expression of Caspase-3 began at 4h and peaked at 24h after the bilirubin injection.The positive cells of activin A and Caspase-3 mostly located in cerebral cortex and hippocampus.Positive reaction of activin A localized mainly in cytoplasm,axon and dendron,nonstaining in nucleus.Positive reaction of Caspase-3 localized mainly in nucleus,and also in cytoplasm,axon and dendron.
Conclusions
1.There are different severity of brain injury and behavior disorder in neonatal rats with hyperbilirubinemia.And there is positive relation between the severity and the brain bilirubin content.
2.The expression of Caspase-3 increases dynamically in neonatal rats with hyperbilirubinemia.It indicates the bilirubin in brain tissue induces apoptosis.
3.The expression of ACT A increases in neonatal rats with hyperbilirubinemia.It indicates that ACT A may play an important role in pathogenic procedure of brain injury in neonatal rats caused by hyperbilirubinemia.ACT A may be used as a sensitive index in hyperbilirubinemia and brain injury.
方法将100只新生7日龄SD大鼠随机分为正常对照组(C组,n=10)和实验组(T组,n=90),T组又根据腹腔注射胆红素剂量的依次增加分为T1,T2和T3组,每组30只。C组SD大鼠腹腔注射生理盐水1.0ml,T1组SD大鼠腹腔注射胆红素50μg/g,T2组腹腔注射胆红素100μg/g,T3组腹腔注射胆红素200μg/g。各实验组根据造模结束后不同时间点(0h、4h、8h、12h、24h)分为5组,每组6只大鼠。观察各组幼鼠不同时间点的神经行为异常如抽搐、翻滚、俯伏、对外界刺激的逃避反应减弱等;检测各组幼鼠血清胆红素浓度、脑组织胆红素浓度及脑组织含水量;取各组幼鼠脑组织置4%多聚甲醛固定、常规梯度乙醇脱水、二甲苯透明、浸蜡、包埋,冠状切片(厚4μm),常规苏木素-伊红(hematoxylin eosin,HE)染色,光镜下观察组织病理改变,免疫组织化学方法检测脑组织内源性ACTA和Caspase-3的表达。数据以(?)±s表示,应用SPSS13.0软件进行统计学处理。
结果C组及T1组幼鼠于造模结束后各时间点均未见明显的神经行为异常。T2组、T3组幼鼠分别于造模后12h、8h出现翻滚、震颤、俯伏及对外界刺激的逃避反应减弱等表现,T3组幼鼠行为异常表现较T2组明显。造模结束后0h,各组幼鼠血清胆红素浓度、脑组织胆红素浓度及脑组织含水量无显著性差异(P＞0.05)。T1组血清胆红素浓度于造模后8h、12h均高于C组(P＜0.05),T1组脑组织胆红素含量、脑组织含水量及ACT A、Caspase-3表达于造模后各时间点与C组比较均无统计学意义(P＞0.05)。T2、T3组血清胆红素浓度、脑组织胆红素浓度、脑组织含水量及ACT A、Caspase-3表达于造模后各时间点均明显高于C组(P＜0.01)。脑组织胆红素浓度与脑组织含水量及脑组织ACT A、Caspase-3表达均呈正相关(r=0.876,P＜0.01;r=0.886,P＜0.01;r=0.900,P＜0.01)。C组、T1组大脑皮质、海马区神经元排列整齐,结构完整,未见胆红素沉积;T2、T3组神经元数量减少,结构紊乱,并可见不等量的胆红素沉积。ACT A阳性细胞主要出现在大脑皮质及海马区,阳性反应主要位于细胞浆及突起,胞核不着色;Caspase-3阳性细胞以神经元、神经胶质细胞为主,阳性免疫反应位于细胞核,也有一些位于胞浆及突起。
结论
1.高胆红素血症模型鼠均存在不同程度的脑损伤及神经行为异常;其程度与造模后脑组织胆红素含量成正相关。
2.高胆红素血症可诱导模型鼠脑组织Caspase-3表达增加,表明高胆红素血症可加速神经细胞凋亡。
3.高胆红素血症可诱导模型鼠脑组织ACT A表达增加,提示内源性ACT A参与了高胆红素血症所致新生大鼠脑组织损伤的发病过程,可望成为新生儿高胆红素血症及其后所致中枢神经系统损伤病情判定的敏感指标之一。
Objective Hyperbilirubinemia is the most common disease during neonatal period, which leads to neonatal death or induces central nervous system injury.Activin A (ACT A)is a sort of sugar proteinic hormone.The latest research shows that ACT A participates in hypoxic-ischemic brain damage(HIBD),and the exogenous ACT A can protect the brain tissue of neonatal rats with hypoxic-ischemic brain damage. Caspase-3 is the key enzyme in the process of apoptosis.The method of this research adopts animal experiment,it investigates the expression of ACT A and Caspase-3 in different severity of brain injuries of neonatal rats with hyperbilirubinemia.It aims to provide experimental gist to the pathogenesis of central nervous system injury and the valid prevention and cure measure for further study of hyperbilirubinemia.
Methods Totally 100 seven-day-old SD rats were randomly assigned to the normal control group(n=10)and the experiment group(n=90).The rats of experiment group were medially subdivided into 3 groups:T1,T2,and T3 groups(30 for each) according to the increased bilirubin injected intraperitoneally.The specimens were collected at each time point(0,4,8,12,24 hours)after the bilirubin injection.Observe the behavior disorder of every group at each time point.The bilirubin contents in the brain and serum were measured and the degree of the brain edema was assessed by wet/dry weight ratios of the brain tissue.Brain tissues were fixed in 4% paraformaldehyde at certain time point for a certain period,then anhydrated in gradient ethanol,luciditied in xylene,embed in paraffin.Observe the pathology changes of brain tissue with H&E staining and the expression of ACT A and Caspase-3 by immunohistochemistry staining under light microscope.All data were expressed as mean±standard variance((?)±s).All statistical analyses were completed by the package of SPSS13.0.
Results The serum bilirubin content of T1 group increased obviously at 8h,12h than those in C group(P＜0.05).There was no significant difference in the brain bilirubin content,the brain edema and the expression of activin A and Caspase-3 between T1 group and C group(P＞0.05).The bilirubin contents in the brain and serum,the brain edema and the expression of activin A and Caspase-3 of T2,T3 group increased respectively at each time points after the bilirubin injection(P＜0.01,vs C). There was positive relation between the brain bilirubin content and the brain edema, the expression of activin A and Caspase-3(r=0.876,P＜0.01;r=0.886,P＜0.01; r=0.900,P＜0.01).The neurons of C group had complete structure and lined up in order.The neurons of T2,T3 group shrank obviously,and normal structure and arrangement disappeared.In T2,T3 group,the expression of activin A began at 8h, reached a peak at 12h and was still in high level until 24h after the bilirubin injection, while the expression of Caspase-3 began at 4h and peaked at 24h after the bilirubin injection.The positive cells of activin A and Caspase-3 mostly located in cerebral cortex and hippocampus.Positive reaction of activin A localized mainly in cytoplasm,axon and dendron,nonstaining in nucleus.Positive reaction of Caspase-3 localized mainly in nucleus,and also in cytoplasm,axon and dendron.
Conclusions
1.There are different severity of brain injury and behavior disorder in neonatal rats with hyperbilirubinemia.And there is positive relation between the severity and the brain bilirubin content.
2.The expression of Caspase-3 increases dynamically in neonatal rats with hyperbilirubinemia.It indicates the bilirubin in brain tissue induces apoptosis.
3.The expression of ACT A increases in neonatal rats with hyperbilirubinemia.It indicates that ACT A may play an important role in pathogenic procedure of brain injury in neonatal rats caused by hyperbilirubinemia.ACT A may be used as a sensitive index in hyperbilirubinemia and brain injury.
引文
[1]Date M,Matsuzaki K,Matsushita M,et al.Differential regulation of activin A for hepatocyte growth and fibronectin synthesis in rat liver injury[J].J Hepatol,2000,32(2):251-260.
[2]Mohan A,Asselin J,Sargent IL,et al.Effect of cytokines and growth factors on the secretion of inhibin A,activin A and follistatin by term placental villous trophoblasts in culture[J].Eur J Endocrinol,2001,145(4):505-511.
[3]朱辉,刘国艺,李庆雷,等.激活素A对离体培养大鼠卵泡发育的影响[J].基础医学与临床,2001,21(6):560-562.
[4]Peng C,Ohno T,Loo YK,et al.Human ovary and placenta express messenger RNA for multiple activin receptors[J].Life Sci,1999,64(12):983-994.
[5]Vanttinen T,Kuulasmaa T,Liu J,et al.Expression of activin/inhibin receptor and binding protein genes and regulation of activin/inhibin peptide secretion in human adrenocortical cells[J].J Clin Endocrinol Metab,2002,87(9):4257-4263.
[6]Phillips D J,Jones KL,Scheerlinck JY,et al.Evidence for activin A and follistatin involvement in the systemic inflammatory response[J].Mol Cell Endocrinol,2001,180(1-2):155-162.
[7]Mitsuhiro MR,Eguchi S,Yamashita H.Regulation mechanisms of retinal pigment epithelial cell migration by the TGF—beta superfamily[J].Acta Ophthalmol Scand,2003,81(6):630-638.
[8]Bilezikjian LM,Corrigan AZ,Blount AL,et al.Regulation and actions of Smad 7in the modulation of activin,inhibin,and transforming growth factor-beta signaling in anterior pituitary cells[J].Endocrinology,2001,142(3):1065-1072.
[9]Funaba M,Ikeda T,Ogawa K,et al.Role of activin A in murine mast cells:modulation of cell growth,differentiation,and migration[J].J Leukoc Biol,2003,73(6):793-801.
[10]Sugiyama M,Ichida T,Sato T,et al.Expression of activin A is increased in cirrhotic and fibrotic rat livers[J].Gastroenterology,1998,114(3):550-558.
[11]王晓琴,安丽,王平,等.缺氧缺血性脑损伤与激活素A表达变化的相关性研究[J].山东大学学报(医学版),2007,45(6):565-568.
[12]安丽,朱薇薇,王萍,等.激活素对新生大鼠缺氧缺血性脑损伤的防护作用及其机制[J].中华儿科杂志,2005,43(6):465-466.
[13]张慧娟,刘伯宁,Sanjiv M,等.Inhibin/Activin信号对卵巢癌生长调控的初步研究[J].现代妇产科进展,2003,12(1):12-15.
[14]金汉珍,黄德珉,官希吉,等.实用新生儿学[M].第三版.北京:人民卫生出版社,2002,297-304.
[15]王恩多.ATP合成酶和Na~+-K~+-ATP酶[J].科学,1998,50(1):60-62.
[16]杨光英,孔祥英,吴晓翠.胆红素脑病与神经细胞凋亡[J].医学综述,2004,10(3):166-167.
[17]Ehlers MD,Tingley WG,Huganir RL.Regulated subcellular distribution of the NR_1 subunit of the NMDA receptor[J].Science,1995,269(5231):1734-1737.
[18]Ishii T,Moriy Oshi K,Sukihara H,et al.Molecular characterization of the N-methyl-D-aspartate receptor subunits[J].Biol them,1993,268(4):2836-2843.
[19]Tingley WG,Roche KW,Thompson AK,et al.Regulation of NR phosphorylation by alternative splicing of the C-terminal domain[J].Nature,1993,364(6432):70-73.
[20]Omkumar RV,Kiely MJ,Rosenstein AJ.Identification of a phosphorylation site for calcium/calmodulin-dependent protein kinase in the NR2B subunit of the N-methyl-D-aspartate receptor[J].Biol Chem,1996,271(49):31670-31678.
[21]Bliss TV,Collingridge GL.A synaptic model of memory:long-term potentiation in the hippocampus[J].Nature,1993,361(6407):31-39.
[22]Vikram R Rao,Steven Finkbeiner.NMDA and AMPA receptors:old channels,new tricks[J].Trends Neurosci,2007,30(6):284-291.
[23]Hu B,Sun SG,Tong ET.NMDA and AMPA receptors mediate intracellular calcium increase in rat cortical astrocytes[J].Acta Pharmacol sin,2004,25(6):714-720.
[24]Arvanian VL,Mendell LM.Removal of NMDA receptor Mg(2+)block extends the action of NT-3 on synaptic transmission in neonatal rat motoneurons[J].J Neurophysiol,2001,86(1):123-129.
[25]Inglefield JR,Schwartz-bloom RD.Activation of excitatory amino acid receptors in the rat hippocampal slice increase intracellular cl~- and cell volume[J].J Neurochem,1998,71(4):1396-1404.
[26]Sung KW,Kirby M,Mcdonald MP,et al.Abnormal GABA_A receptor-mediated currents in dorsal root ganglion neurons isolated from Na-K-2Cl cotransporter null mice[J].J Neurosci,2000,20(20):7531-7538.
[27]Grojean S,Koziel V,Vert P,et al.Bilirubin induces apoptosis via activation of NMDA receptor in developing Rat brain neurons[J].Exp Neurol,2000,166(2):334-341.
[28]贲晓明,秦玉明,陈舜年,等.[Ca~(2+)]i介导胆红素神经毒性与NMDA受体拮抗剂GAPA治疗作用[J].新生儿科杂志,2000,15(4):165-166.
[29]Wennberg RP.The Blood-Brain Barrier and Bilirubin Encephalopathy[J].Cell Mol Neurobiol,2000,20(1):97-109.
[30]Mayor F Jr,Dfez-Guerra J,ualdivieso F,et al.Effect of bilirubin on the membrane potential of rat brain synaptosomes[J].J NeuroChem,1986,47(2):363-369.
[31]陈舜年,贲晓明,李佩红,等.胆红素脑病模型制作与鉴定[J].新生儿科杂志,1997,12(4):166-168.
[32]贲晓明,陈舜年,秦玉明,等.胆红素脑病模型鼠EAA神经递质的检测[J].新生儿科杂志,2000,15(1):14-16.
[33]贲晓明,陈舜年,秦玉明,等.NMDA受体拮抗剂治疗胆红素脑病的实验研究[J].中国生育健康杂志,2000,11(1):33-35.
[34]Roger C,Koziel V,vert P,et al.Effects of bilirubin infusion on local cerebral glucose utilization in the immature rat[J].Brain Res Dev Brain Res,1993,76(1):115-130.
[35]Ostrow JD,Celic L,MukerJee P.Molecular and micellar associations in the PH-dependent stable and metastable dissolution of unconjugated bilirubin by bile salts[J].J Lipid Res,1988,29(3):335-348.
[36]邵洁,俞善昌,陈舜年,等.胆红素对突触膜Na~+-K~+-ATP酶影响的实验研究[J].中华儿科杂志,1997,35(10):524-526.
[37] Deliconstantions G. Structure activity relationship of cholesterol and steroid hormones with respect to their effects on the Ca2+-stimulated ATPase and lipid fluidity of synaptosomal plasma membranes from dog and rabbit brain[J]. Comp Biochem Physiol B,1988,89(3):585-594.
[38] Blagosklonny MV, Chuman Y, Bergan RC, et al. Mitogen-activated protein kinase pathway is dispensable for microtubule active drug-induced Raf-1/Bcl-2[J]. Leukermia,1999,13(7):1028-1036.
[39] Dimitrov T, Krajcsi P, Hermiston TW, et al. Adenovirus E3-10.4K/14.5K protein complex inhibits tumor necrosis factor-induced translocation of cytosolic phospholipase A2 to membranes[J]. Virol,1997,71 (4):2830-2837.
[40] Krajewska, Stanislaw krajewski, Maryla et al. Release of Caspase-9 from mitochondria during neuronal apoptosis and cerebral ischemia [J]. PNAS,1999,96(10):5752-5757.
[41] Liu X, Kin CN, Yang J, et al. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c[J]. Cell,1996,86(1):147-157.
[42] Wolvetang EJ, Johnson KL, Krauer K, et al. Mitochondrial respiratory chain inhibitors induces apoptosis[J]. FEBS Lett, 1994,399(1-2):40-44.
[43] Rodrigues CM, Sola S, Silva R. Bilirubin and amyloid-beta peptide induced cytochrome C release through mitochondrial membrane permeabilization[J]. Mol Med,2000,6(11):936-946.
[44] Wang J, Lenardo MJ. Roles of Caspases in apoptosis, development, and cytokine maturation revealed by homozygous gene deficiencies [J]. J Cell Sci,2000,113(5):753-757.
[45] Nunez G, Benedict MA, Hu Y, et al. Caspases : The proteases of the apoptotic pathway[J]. Oncogene,1998,17(25):3237-3245.
[46] Schulze-osthoff k, Ferrari D Los M, et al. Apoptosis signaling by death receptors[J]. Eur J biochem, 1998,254(3):439-459.
[47] JS Kim, L He, JJ Lemasters. Mitochondrial permeability transition: a common pathway to necrosis and apoptosis [J]. Biochem Biophys Res Commun,2003,304(3):463-470.
[48]Takeyama A,Miki S,Hirakawa A,et al.Role of the Mitochondrial Permeability Transition and Cytochrome c Release in Hydrogen Peroxide-Induced Apoptosis[J].Exp Cell Res,2002,274(1):16-24.
[49]I Lavrik,A Golks,PH Krammer.Death receptor signaling[J].J Cell Sci,2005,118(2):265-267.
[50]Xu L,Budihardjo I,Hua Z,et al.Bid,a bcl-2 interacting protein,mediates Cytochrome c release from mitochondria in response to activation of cell surface death receptors[J].Cell,1998,94(4):481-490.
[51]Keane RWA,Srunivasan LM,Foster ME et al.Activation of CPP32 during apoptosis of neurons and astrocytes[J].JNeurosciRes,1997,48(2):168-180.
[52]Kroemer G.Mitochondrial control of apoptosis:an introduction[J].Biochem Biophys Res Commun,2003,304(3):433-435.
[53]Kirsch DG,Doseff A,Chau BN,et al.Caspase-3-dependent cleavage of Bcl-2Promotes Release of Cytochrome C[J].J Biol Chem,1999,274(30):21155-21161.
[54]Matapurkar A,Lazebnik Y.Requirement of cytochrome c for apoptosis in human cells[J].Cell Death Differ,2006,13(12):2062-2067.
[55]Kasie Cole,J Regino Perez-Polo.Neuronal trauma model:in search on Thanatos[J].Int J Devl Neurosci,2004,22(7):485-496.
[56]Seth Love.Apoptosis and brain ischemia[J].Progress in Neuro-Psychopharmacology & Biological Psychiatry,2003,27(2):267-282.
[57]Green DR.Apoptotic Pathway:Ten Minutes to Dead[J].Cell,2005,121(5):671-674.
[58]袁长青,丁振华.Caspase的活化及其在细胞凋亡中的作用[J].生理科学进展,2002,33(3):220-224.
[59]Susin SA Lorenzo HK,Zamzami N,et al.Mitochcondrial release of Caspase-2and Caspase-9 during the apoptosis process[J].J Exp Med,1999,189(18):381.
[60]Perrone S,Bracci R.New biomarkers of fetal-neonatal hypoxic stress[J].Acta Paediatrica,2002,91(438):135-138.
[61]Florio P,Perrone S.Activin A Plasma Levels at Birth:An Index of Fetal Hypoxia in Preterm Newbom[J].Pediatric Research,2003,54(5):696-700.
[62]Zhang G,Ohsawa Y,Kametaka S,et al.Regulation of FLRG expression in rat primary astroglial cells and injured brain tissue by transforming growth factor-beta 1(TGF-beta 1)[J].J Neurosci Res,2003,72(1):33-45.
[63]Perrone S,Bracci R,Buonocore G.New biomarkers of fetal-neonatal hypoxic stress[J].Acta Pediatr suppl,2002,91(438):135-138.
[64]安丽,陈丽,赵平.激活素研究进展[J].医学综述,2005,11(8):682-684.
[2]Mohan A,Asselin J,Sargent IL,et al.Effect of cytokines and growth factors on the secretion of inhibin A,activin A and follistatin by term placental villous trophoblasts in culture[J].Eur J Endocrinol,2001,145(4):505-511.
[3]朱辉,刘国艺,李庆雷,等.激活素A对离体培养大鼠卵泡发育的影响[J].基础医学与临床,2001,21(6):560-562.
[4]Peng C,Ohno T,Loo YK,et al.Human ovary and placenta express messenger RNA for multiple activin receptors[J].Life Sci,1999,64(12):983-994.
[5]Vanttinen T,Kuulasmaa T,Liu J,et al.Expression of activin/inhibin receptor and binding protein genes and regulation of activin/inhibin peptide secretion in human adrenocortical cells[J].J Clin Endocrinol Metab,2002,87(9):4257-4263.
[6]Phillips D J,Jones KL,Scheerlinck JY,et al.Evidence for activin A and follistatin involvement in the systemic inflammatory response[J].Mol Cell Endocrinol,2001,180(1-2):155-162.
[7]Mitsuhiro MR,Eguchi S,Yamashita H.Regulation mechanisms of retinal pigment epithelial cell migration by the TGF—beta superfamily[J].Acta Ophthalmol Scand,2003,81(6):630-638.
[8]Bilezikjian LM,Corrigan AZ,Blount AL,et al.Regulation and actions of Smad 7in the modulation of activin,inhibin,and transforming growth factor-beta signaling in anterior pituitary cells[J].Endocrinology,2001,142(3):1065-1072.
[9]Funaba M,Ikeda T,Ogawa K,et al.Role of activin A in murine mast cells:modulation of cell growth,differentiation,and migration[J].J Leukoc Biol,2003,73(6):793-801.
[10]Sugiyama M,Ichida T,Sato T,et al.Expression of activin A is increased in cirrhotic and fibrotic rat livers[J].Gastroenterology,1998,114(3):550-558.
[11]王晓琴,安丽,王平,等.缺氧缺血性脑损伤与激活素A表达变化的相关性研究[J].山东大学学报(医学版),2007,45(6):565-568.
[12]安丽,朱薇薇,王萍,等.激活素对新生大鼠缺氧缺血性脑损伤的防护作用及其机制[J].中华儿科杂志,2005,43(6):465-466.
[13]张慧娟,刘伯宁,Sanjiv M,等.Inhibin/Activin信号对卵巢癌生长调控的初步研究[J].现代妇产科进展,2003,12(1):12-15.
[14]金汉珍,黄德珉,官希吉,等.实用新生儿学[M].第三版.北京:人民卫生出版社,2002,297-304.
[15]王恩多.ATP合成酶和Na~+-K~+-ATP酶[J].科学,1998,50(1):60-62.
[16]杨光英,孔祥英,吴晓翠.胆红素脑病与神经细胞凋亡[J].医学综述,2004,10(3):166-167.
[17]Ehlers MD,Tingley WG,Huganir RL.Regulated subcellular distribution of the NR_1 subunit of the NMDA receptor[J].Science,1995,269(5231):1734-1737.
[18]Ishii T,Moriy Oshi K,Sukihara H,et al.Molecular characterization of the N-methyl-D-aspartate receptor subunits[J].Biol them,1993,268(4):2836-2843.
[19]Tingley WG,Roche KW,Thompson AK,et al.Regulation of NR phosphorylation by alternative splicing of the C-terminal domain[J].Nature,1993,364(6432):70-73.
[20]Omkumar RV,Kiely MJ,Rosenstein AJ.Identification of a phosphorylation site for calcium/calmodulin-dependent protein kinase in the NR2B subunit of the N-methyl-D-aspartate receptor[J].Biol Chem,1996,271(49):31670-31678.
[21]Bliss TV,Collingridge GL.A synaptic model of memory:long-term potentiation in the hippocampus[J].Nature,1993,361(6407):31-39.
[22]Vikram R Rao,Steven Finkbeiner.NMDA and AMPA receptors:old channels,new tricks[J].Trends Neurosci,2007,30(6):284-291.
[23]Hu B,Sun SG,Tong ET.NMDA and AMPA receptors mediate intracellular calcium increase in rat cortical astrocytes[J].Acta Pharmacol sin,2004,25(6):714-720.
[24]Arvanian VL,Mendell LM.Removal of NMDA receptor Mg(2+)block extends the action of NT-3 on synaptic transmission in neonatal rat motoneurons[J].J Neurophysiol,2001,86(1):123-129.
[25]Inglefield JR,Schwartz-bloom RD.Activation of excitatory amino acid receptors in the rat hippocampal slice increase intracellular cl~- and cell volume[J].J Neurochem,1998,71(4):1396-1404.
[26]Sung KW,Kirby M,Mcdonald MP,et al.Abnormal GABA_A receptor-mediated currents in dorsal root ganglion neurons isolated from Na-K-2Cl cotransporter null mice[J].J Neurosci,2000,20(20):7531-7538.
[27]Grojean S,Koziel V,Vert P,et al.Bilirubin induces apoptosis via activation of NMDA receptor in developing Rat brain neurons[J].Exp Neurol,2000,166(2):334-341.
[28]贲晓明,秦玉明,陈舜年,等.[Ca~(2+)]i介导胆红素神经毒性与NMDA受体拮抗剂GAPA治疗作用[J].新生儿科杂志,2000,15(4):165-166.
[29]Wennberg RP.The Blood-Brain Barrier and Bilirubin Encephalopathy[J].Cell Mol Neurobiol,2000,20(1):97-109.
[30]Mayor F Jr,Dfez-Guerra J,ualdivieso F,et al.Effect of bilirubin on the membrane potential of rat brain synaptosomes[J].J NeuroChem,1986,47(2):363-369.
[31]陈舜年,贲晓明,李佩红,等.胆红素脑病模型制作与鉴定[J].新生儿科杂志,1997,12(4):166-168.
[32]贲晓明,陈舜年,秦玉明,等.胆红素脑病模型鼠EAA神经递质的检测[J].新生儿科杂志,2000,15(1):14-16.
[33]贲晓明,陈舜年,秦玉明,等.NMDA受体拮抗剂治疗胆红素脑病的实验研究[J].中国生育健康杂志,2000,11(1):33-35.
[34]Roger C,Koziel V,vert P,et al.Effects of bilirubin infusion on local cerebral glucose utilization in the immature rat[J].Brain Res Dev Brain Res,1993,76(1):115-130.
[35]Ostrow JD,Celic L,MukerJee P.Molecular and micellar associations in the PH-dependent stable and metastable dissolution of unconjugated bilirubin by bile salts[J].J Lipid Res,1988,29(3):335-348.
[36]邵洁,俞善昌,陈舜年,等.胆红素对突触膜Na~+-K~+-ATP酶影响的实验研究[J].中华儿科杂志,1997,35(10):524-526.
[37] Deliconstantions G. Structure activity relationship of cholesterol and steroid hormones with respect to their effects on the Ca2+-stimulated ATPase and lipid fluidity of synaptosomal plasma membranes from dog and rabbit brain[J]. Comp Biochem Physiol B,1988,89(3):585-594.
[38] Blagosklonny MV, Chuman Y, Bergan RC, et al. Mitogen-activated protein kinase pathway is dispensable for microtubule active drug-induced Raf-1/Bcl-2[J]. Leukermia,1999,13(7):1028-1036.
[39] Dimitrov T, Krajcsi P, Hermiston TW, et al. Adenovirus E3-10.4K/14.5K protein complex inhibits tumor necrosis factor-induced translocation of cytosolic phospholipase A2 to membranes[J]. Virol,1997,71 (4):2830-2837.
[40] Krajewska, Stanislaw krajewski, Maryla et al. Release of Caspase-9 from mitochondria during neuronal apoptosis and cerebral ischemia [J]. PNAS,1999,96(10):5752-5757.
[41] Liu X, Kin CN, Yang J, et al. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c[J]. Cell,1996,86(1):147-157.
[42] Wolvetang EJ, Johnson KL, Krauer K, et al. Mitochondrial respiratory chain inhibitors induces apoptosis[J]. FEBS Lett, 1994,399(1-2):40-44.
[43] Rodrigues CM, Sola S, Silva R. Bilirubin and amyloid-beta peptide induced cytochrome C release through mitochondrial membrane permeabilization[J]. Mol Med,2000,6(11):936-946.
[44] Wang J, Lenardo MJ. Roles of Caspases in apoptosis, development, and cytokine maturation revealed by homozygous gene deficiencies [J]. J Cell Sci,2000,113(5):753-757.
[45] Nunez G, Benedict MA, Hu Y, et al. Caspases : The proteases of the apoptotic pathway[J]. Oncogene,1998,17(25):3237-3245.
[46] Schulze-osthoff k, Ferrari D Los M, et al. Apoptosis signaling by death receptors[J]. Eur J biochem, 1998,254(3):439-459.
[47] JS Kim, L He, JJ Lemasters. Mitochondrial permeability transition: a common pathway to necrosis and apoptosis [J]. Biochem Biophys Res Commun,2003,304(3):463-470.
[48]Takeyama A,Miki S,Hirakawa A,et al.Role of the Mitochondrial Permeability Transition and Cytochrome c Release in Hydrogen Peroxide-Induced Apoptosis[J].Exp Cell Res,2002,274(1):16-24.
[49]I Lavrik,A Golks,PH Krammer.Death receptor signaling[J].J Cell Sci,2005,118(2):265-267.
[50]Xu L,Budihardjo I,Hua Z,et al.Bid,a bcl-2 interacting protein,mediates Cytochrome c release from mitochondria in response to activation of cell surface death receptors[J].Cell,1998,94(4):481-490.
[51]Keane RWA,Srunivasan LM,Foster ME et al.Activation of CPP32 during apoptosis of neurons and astrocytes[J].JNeurosciRes,1997,48(2):168-180.
[52]Kroemer G.Mitochondrial control of apoptosis:an introduction[J].Biochem Biophys Res Commun,2003,304(3):433-435.
[53]Kirsch DG,Doseff A,Chau BN,et al.Caspase-3-dependent cleavage of Bcl-2Promotes Release of Cytochrome C[J].J Biol Chem,1999,274(30):21155-21161.
[54]Matapurkar A,Lazebnik Y.Requirement of cytochrome c for apoptosis in human cells[J].Cell Death Differ,2006,13(12):2062-2067.
[55]Kasie Cole,J Regino Perez-Polo.Neuronal trauma model:in search on Thanatos[J].Int J Devl Neurosci,2004,22(7):485-496.
[56]Seth Love.Apoptosis and brain ischemia[J].Progress in Neuro-Psychopharmacology & Biological Psychiatry,2003,27(2):267-282.
[57]Green DR.Apoptotic Pathway:Ten Minutes to Dead[J].Cell,2005,121(5):671-674.
[58]袁长青,丁振华.Caspase的活化及其在细胞凋亡中的作用[J].生理科学进展,2002,33(3):220-224.
[59]Susin SA Lorenzo HK,Zamzami N,et al.Mitochcondrial release of Caspase-2and Caspase-9 during the apoptosis process[J].J Exp Med,1999,189(18):381.
[60]Perrone S,Bracci R.New biomarkers of fetal-neonatal hypoxic stress[J].Acta Paediatrica,2002,91(438):135-138.
[61]Florio P,Perrone S.Activin A Plasma Levels at Birth:An Index of Fetal Hypoxia in Preterm Newbom[J].Pediatric Research,2003,54(5):696-700.
[62]Zhang G,Ohsawa Y,Kametaka S,et al.Regulation of FLRG expression in rat primary astroglial cells and injured brain tissue by transforming growth factor-beta 1(TGF-beta 1)[J].J Neurosci Res,2003,72(1):33-45.
[63]Perrone S,Bracci R,Buonocore G.New biomarkers of fetal-neonatal hypoxic stress[J].Acta Pediatr suppl,2002,91(438):135-138.
[64]安丽,陈丽,赵平.激活素研究进展[J].医学综述,2005,11(8):682-684.