不同孕龄胎盘和脐带组织中S100b、NSE、GFAP的表达
摘要
目的
神经元特异性烯醇化酶(NSE)、S100b、胶质纤维酸性蛋白(GFAP)是目前研究较多的缺血缺氧性脑损伤的标记物，NSE特异性定位在神经元内，GFAP、S100b主要集中在星形胶质细胞内。在围产医学，主要通过测定羊水、脐血、新生儿血和新生儿脑脊液中这些指标来预测围产儿脑损伤，国外有研究发现S100b和NSE的表达不仅局限于神经系统，胎盘组织也有相应表达。因此脐血和羊水中S100b和NSE的活性是来自于脑损伤还是来自于胎盘或脐带组织的释放?人们对此提出质疑。S100b、NSE和GFAP究竟在胎盘或脐带组织中呈怎样的表达?不同孕周的变化趋势如何?国内外均未见系统研究报道。我们的研究通过系统评价30名不同孕龄的健康产妇胎盘和脐带组织S100b、NSE和GFAP的免疫活性，探讨哪种指标更有希望成为围产儿缺血缺氧性脑损伤的标记物。
材料和方法
1、试剂
NSE、S100b、GFAP一抗、免疫组化试剂盒均由迈新试剂公司提供。
2、标本来源
组织来自于30名不同孕龄健康产妇的胎盘和脐带(采集标本事先已得到患者和家属的同意)，其中28周以下的来自于10个因为胎儿和母体原因流产的产妇，28-36周的来自于10个早产的产妇，36周以上的来自于10名自然分娩的产妇。多胎妊娠、胎儿脑畸形、21三体综合症及感染的除外。分娩后，立刻取胎盘中心的小叶和胎儿侧脐带组织，并固定在10％多聚甲醛待检。
3、方法
胎盘或脐带组织S100b、NSE和GFAP的检测用免疫组化S-P法，以迈新公司提供的阳性对照片为阳性对照，以PBS缓冲液代替一抗作为阴性对照。
4、结果判定(阳性细胞数)
每张切片随机选6个视野计阳性细胞数，求平均阳性细胞率。
5、统计学处理
所有结果用平均值±标准差((?)±s)表示，利用SPSS13.0统计学处理软件进行正态检验、方差齐性检验，呈正态分布方差齐后，行单因素方差分析，P＜0.05认为在统计学上有显著差异，有显著差异者再进行多重比较。
结果
1、S100b在不同孕龄胎盘的滋养层细胞、血管平滑肌细胞、肌成纤维细胞、巨噬细胞呈现阳性，在胎盘滋养层细胞的表达有随孕龄增长而增加的趋势。在脐带的血管平滑肌细胞、肌纤维母细胞、羊膜上皮细胞、巨噬细胞和单核细胞染色阳性，表达的强弱在不同孕龄之间无明显差别。
2、NSE表达于不同孕龄胎盘的滋养层细胞、血管平滑肌细胞、肌成纤维细胞和巨噬细胞，在脐带的血管平滑肌细胞、肌纤维母细胞、羊膜上皮细胞、巨噬细胞和单核细胞染色阳性，不论是在胎盘还是在脐带组织，NSE表达的强弱在不同孕龄间没有统计学差异。
3、GFAP在不同孕龄胎盘和脐带组织中均未见免疫活性。
结论
不同孕龄胎盘和脐带均有S100b、NSE表达，其中S100b在胎盘滋养细胞的表达随孕龄增加有增强的趋势。S100b、NSE作为围产儿缺血缺氧性脑损伤的标记物可能不具有特异性，因为胎盘和脐带中均有这些物质的释放。GFAP在各期胎盘和脐带中均均未见表达，推测GFAP可能更具有脑特异性，因此我们认为GFAP可能有希望成为围产儿缺血缺氧性脑损伤的指标。
Objective
Presently, S100b and NSE and GFAP are the most common markers of hypoxic-ischemic brain injury .NSE is located in the neurons.S100b and GFAP are present in astrocytes .In perinatology, the significance of neuronal markers such as S100b and NSE in amniotic fluid, cord blood, neonatal blood, and neonatal cerebrospinal fluid has been investigated to predict brain damage in the newborn especially after perinatal asphyxia and preterm labor. However, the expression of S100b and NSE in umbilical cord and placental tissue has been found according to some foreigh studys. Therefore the question arises whether the immunoreactivity of S100b and NSE in (cord) blood and amniotic fluids, indicates exclusively damage in brain tissue or may also result from protein release from placental or umbilical cord tissue. The aim of this study was to investigate the localization and immunoreactivity of S100b and NSE and GFAP systematically in placental and umbilical cord tissue and to make sure if they are suitable markers for perinatal brain damage.
Materials and Methods
1. Agent
The monoclonal antibodies to S100b and NSE and GFAP ,as well as immuno-histochemistry kit are provided by Maixin Biotechnology Company.
2. Materials
Placental and umbilical cord tissues were obtained from 30 healthy pregnant women at different gestational ages. Tissues were collected below 28 weeks' gestation from ten women who underwent elective abortion for fetal or maternal indications, between 28 and 36 weeks' gestation from ten women after preterm delivery, and above 36 weeks' gestation from ten women after spontaneous delivery. Patients with multiple gestation, fetal cerebral malformations, Down syndrome, or infections were excluded from the study. Immediately after delivery, biopsies were taken from the pericentral area of the placenta. Samples from the umbilical cord were taken near the fetus. Tissue was fixed in 10%paraformaldehyde.
3. Methods
The localization and immunoreactivity of S100b and NSE and GFAP are detected by SP immunohistochemistry. Positive comparison film served as a positive control and sections with PBS instead of primary antibody as a negative control.
4. Results assessment
The proportion of positive cells was counted in six randomly selected fields in per section. The average values were used for the total immunohistochemic counts.
5. Statistical treatment
All the results are expressed by means value±standard error (x|-±s) . Comparison of positive cell at different developmental stages was made by one-way analysis of variance followed by Turkey test. Statistical significance was set at P < 0.05.
Results
1. In the placenta, S100b was localized in trophoblast cells, myofibroblasts, smooth muscle cells of the vascular wall, and macrophages. The intensity of immunostaining of S100b increased with advancing gestation. In the umbilical cord , S100b was present in endothelial cells of umbilical vessels, the smooth muscle cells of the vascular wall, myofibroblasts, amnion epithelium, macrophages, and monocytes. No significant difference was found at different gestational ages
2. In the placenta, NSE immunoreactivity was found in trophoblast cells, myofibroblasts, smooth muscle cells of the vascular wall, and macrophages. In the umbilical cord , NSE was localized in endothelial cells of umbilical vessels, the smooth muscle cells of the vascular wall, myofibroblasts, amnion epithelium, macrophages, and monocytes. There was no apparent change in the amount of immunoreactive NSE present in the placenta and umbilical cord at different gestational ages
3. Glial fibrillary acidic protein showed no immunoreactivity in the placenta and umbilical cord of all 30 cases.
Conclusion
This study demonstrated that fetoplacental tissues contain S100b and NSE, suggesting that these tissues may, at least in part, be responsible for the high level found in the fetal circulation. Although the significance of placental S100b and NSE is unknown, this origin should be taken into account when this protein is used as a marker of brain injury in the fetus or infant at birth. However, GFAP showed no immunoreactivity in the umbilical cord and the placenta .So GFAP has better brain specificity . Brain-restricted proteins such as glial fibrillary acidic protein seems more promising as a marker of brain injury.
神经元特异性烯醇化酶(NSE)、S100b、胶质纤维酸性蛋白(GFAP)是目前研究较多的缺血缺氧性脑损伤的标记物，NSE特异性定位在神经元内，GFAP、S100b主要集中在星形胶质细胞内。在围产医学，主要通过测定羊水、脐血、新生儿血和新生儿脑脊液中这些指标来预测围产儿脑损伤，国外有研究发现S100b和NSE的表达不仅局限于神经系统，胎盘组织也有相应表达。因此脐血和羊水中S100b和NSE的活性是来自于脑损伤还是来自于胎盘或脐带组织的释放?人们对此提出质疑。S100b、NSE和GFAP究竟在胎盘或脐带组织中呈怎样的表达?不同孕周的变化趋势如何?国内外均未见系统研究报道。我们的研究通过系统评价30名不同孕龄的健康产妇胎盘和脐带组织S100b、NSE和GFAP的免疫活性，探讨哪种指标更有希望成为围产儿缺血缺氧性脑损伤的标记物。
材料和方法
1、试剂
NSE、S100b、GFAP一抗、免疫组化试剂盒均由迈新试剂公司提供。
2、标本来源
组织来自于30名不同孕龄健康产妇的胎盘和脐带(采集标本事先已得到患者和家属的同意)，其中28周以下的来自于10个因为胎儿和母体原因流产的产妇，28-36周的来自于10个早产的产妇，36周以上的来自于10名自然分娩的产妇。多胎妊娠、胎儿脑畸形、21三体综合症及感染的除外。分娩后，立刻取胎盘中心的小叶和胎儿侧脐带组织，并固定在10％多聚甲醛待检。
3、方法
胎盘或脐带组织S100b、NSE和GFAP的检测用免疫组化S-P法，以迈新公司提供的阳性对照片为阳性对照，以PBS缓冲液代替一抗作为阴性对照。
4、结果判定(阳性细胞数)
每张切片随机选6个视野计阳性细胞数，求平均阳性细胞率。
5、统计学处理
所有结果用平均值±标准差((?)±s)表示，利用SPSS13.0统计学处理软件进行正态检验、方差齐性检验，呈正态分布方差齐后，行单因素方差分析，P＜0.05认为在统计学上有显著差异，有显著差异者再进行多重比较。
结果
1、S100b在不同孕龄胎盘的滋养层细胞、血管平滑肌细胞、肌成纤维细胞、巨噬细胞呈现阳性，在胎盘滋养层细胞的表达有随孕龄增长而增加的趋势。在脐带的血管平滑肌细胞、肌纤维母细胞、羊膜上皮细胞、巨噬细胞和单核细胞染色阳性，表达的强弱在不同孕龄之间无明显差别。
2、NSE表达于不同孕龄胎盘的滋养层细胞、血管平滑肌细胞、肌成纤维细胞和巨噬细胞，在脐带的血管平滑肌细胞、肌纤维母细胞、羊膜上皮细胞、巨噬细胞和单核细胞染色阳性，不论是在胎盘还是在脐带组织，NSE表达的强弱在不同孕龄间没有统计学差异。
3、GFAP在不同孕龄胎盘和脐带组织中均未见免疫活性。
结论
不同孕龄胎盘和脐带均有S100b、NSE表达，其中S100b在胎盘滋养细胞的表达随孕龄增加有增强的趋势。S100b、NSE作为围产儿缺血缺氧性脑损伤的标记物可能不具有特异性，因为胎盘和脐带中均有这些物质的释放。GFAP在各期胎盘和脐带中均均未见表达，推测GFAP可能更具有脑特异性，因此我们认为GFAP可能有希望成为围产儿缺血缺氧性脑损伤的指标。
Objective
Presently, S100b and NSE and GFAP are the most common markers of hypoxic-ischemic brain injury .NSE is located in the neurons.S100b and GFAP are present in astrocytes .In perinatology, the significance of neuronal markers such as S100b and NSE in amniotic fluid, cord blood, neonatal blood, and neonatal cerebrospinal fluid has been investigated to predict brain damage in the newborn especially after perinatal asphyxia and preterm labor. However, the expression of S100b and NSE in umbilical cord and placental tissue has been found according to some foreigh studys. Therefore the question arises whether the immunoreactivity of S100b and NSE in (cord) blood and amniotic fluids, indicates exclusively damage in brain tissue or may also result from protein release from placental or umbilical cord tissue. The aim of this study was to investigate the localization and immunoreactivity of S100b and NSE and GFAP systematically in placental and umbilical cord tissue and to make sure if they are suitable markers for perinatal brain damage.
Materials and Methods
1. Agent
The monoclonal antibodies to S100b and NSE and GFAP ,as well as immuno-histochemistry kit are provided by Maixin Biotechnology Company.
2. Materials
Placental and umbilical cord tissues were obtained from 30 healthy pregnant women at different gestational ages. Tissues were collected below 28 weeks' gestation from ten women who underwent elective abortion for fetal or maternal indications, between 28 and 36 weeks' gestation from ten women after preterm delivery, and above 36 weeks' gestation from ten women after spontaneous delivery. Patients with multiple gestation, fetal cerebral malformations, Down syndrome, or infections were excluded from the study. Immediately after delivery, biopsies were taken from the pericentral area of the placenta. Samples from the umbilical cord were taken near the fetus. Tissue was fixed in 10%paraformaldehyde.
3. Methods
The localization and immunoreactivity of S100b and NSE and GFAP are detected by SP immunohistochemistry. Positive comparison film served as a positive control and sections with PBS instead of primary antibody as a negative control.
4. Results assessment
The proportion of positive cells was counted in six randomly selected fields in per section. The average values were used for the total immunohistochemic counts.
5. Statistical treatment
All the results are expressed by means value±standard error (x|-±s) . Comparison of positive cell at different developmental stages was made by one-way analysis of variance followed by Turkey test. Statistical significance was set at P < 0.05.
Results
1. In the placenta, S100b was localized in trophoblast cells, myofibroblasts, smooth muscle cells of the vascular wall, and macrophages. The intensity of immunostaining of S100b increased with advancing gestation. In the umbilical cord , S100b was present in endothelial cells of umbilical vessels, the smooth muscle cells of the vascular wall, myofibroblasts, amnion epithelium, macrophages, and monocytes. No significant difference was found at different gestational ages
2. In the placenta, NSE immunoreactivity was found in trophoblast cells, myofibroblasts, smooth muscle cells of the vascular wall, and macrophages. In the umbilical cord , NSE was localized in endothelial cells of umbilical vessels, the smooth muscle cells of the vascular wall, myofibroblasts, amnion epithelium, macrophages, and monocytes. There was no apparent change in the amount of immunoreactive NSE present in the placenta and umbilical cord at different gestational ages
3. Glial fibrillary acidic protein showed no immunoreactivity in the placenta and umbilical cord of all 30 cases.
Conclusion
This study demonstrated that fetoplacental tissues contain S100b and NSE, suggesting that these tissues may, at least in part, be responsible for the high level found in the fetal circulation. Although the significance of placental S100b and NSE is unknown, this origin should be taken into account when this protein is used as a marker of brain injury in the fetus or infant at birth. However, GFAP showed no immunoreactivity in the umbilical cord and the placenta .So GFAP has better brain specificity . Brain-restricted proteins such as glial fibrillary acidic protein seems more promising as a marker of brain injury.
引文
1 Wijnberger LD, Nikkels PG, Van Donden AJ, et al. Expression in the placenta of neuronal markers for perinatal brain damage. Pediatr Res, 2002, 51(4):492-496.
2 Marinoni E,Dilorio R,Gazzolo D,et al. Ontogenetic localization and distribution of S-100beta protein in human placental tissues. Obstet Gynecol.,2002 Jun,99(6): 1093-1099.
3 Michetti F, Massaro A, Russo G, et al. The S-100 antigen in cerebrospinal fluid as a possible index of cell injury in the nervous system. J Neurol Sci, 1980, 44(2-3):259-263.
4 Gazzolo D, Marinoni E, Di Iorio R, et al. Measurement of urinary S 100B protein concentrations for the early identification of brain damage in asphyxiated full-term infants. Arch Pediatr Adolesc Med, 2003 Dec,157(12): 1163-1168.
5 Gazzolo D, Marinoni E, Di Iorio R, et al. Urinary S100B protein measurements: A tool for the early identification of hypoxic-ischemic encephalopathy in asphyxiated full-term infants. Crit Care Med, 2004 Jan, 32(1):131-136.
6 Perlman JM, Tack ED. Renal injury in asphyxiated newborn infant: relation to neurologic outcome. J Pediatr, 1988, 113(5): 875-879.
7 Nagdyman N, Komen W, Ko HK, et al. Early biochemical indicators of hypoxic-ischemic encephslopathy after birth asphyxia. Pediatric Research, 2001 Apr, 49(1):502-506.
8 Nagdyman N, Grimmer I, Scholz T, et al. Predictive value of brain-specific proteins in serun for neurodevelopmental outcome after brain birth asphyxia. Pediatr Res, 2003 Aug, 54(2);270-275.
9 Gozzolo D,Vinesi P, Marinoni E, et al. S100B Protein Concentrations in Cord Blood: Correlations with Gestational Age in Term and Preterm Deliveries. Clin Chem, 2000 Jul, 46(7):998-1000.
10 王庆红，宋健辉，王霞，等．血清S100B蛋白在新生儿窒息后脑损伤中的临床意义．中国当代儿科杂志，2005，7(4)：318-320．
11 Thorngren-Jerneck K, Alling C, Herbst A, et al. S100 Protein in Serum as a Prognostic Marker for Cerebral Injury in Term Newborn Infants with Hypoxic Ischemic Encephalopathy. Pediatr. Res, March 1, 2004,55(3): 406-412.
12 刘春艳，董文斌，陈跃，等．血清神经元特异性烯醇化酶与新生儿缺氧缺血性脑病的关系研究．中国优生与遗传杂志，2005，13(2)：85-86．
13 黄燕萍，李小权，王安生，等．神经元特异性烯醇化酶在早期评估新生儿缺氧缺血性脑病预后中的价值．新生儿科杂志，2005，20(1)：23-26．
14 Verdu Perez A, Falero MP, Arroyos A, et al.Blood neuronal specific enolase in newborns with perinatal asphyxia. Rev Neurol, 2001 Apr, 32(8):714-717.
15 Tekgul H, Yalaz M, Kutukculer N, et al.Value of Biochemical Markers for Outcome in Term Infants With Asphyxia. Obstetrical & Gynecological Survey, 2005, 60(5):293-295.
16 Elimian A, Figueroa R, Patel K, et al.Reference values of amniotic fluid neuron-specific enolase [J]. Maternal-fetal Med, 2001,10(3): 155-158.
17 Zinsmeyer J, Marangos PJ, Issel EP, et al. Neuron specific enolase in amniotic fluid—a possible indicator for fetal distress and brain implication. J Perinat Med, 1987,15(3): 199-202.
18 Elimian A, Figueroa R, Verma U, et al. Amniotic fluid neuron-specific enolase: a role in predicting neonatal neurologic injury? Obstet Gynecol,1998,92(1): 546-550.
19 Van Eldik LJ, Wainwright MS. The Janus face of glial-derived S100B: Beneficial and detrimental functions in the brain. Rest Neurol Neurosci, 2003,21 (3-4):97-108.
20 Foerch C,Curdt I,Yan B, et al .Serum glial fibrillary acidic protein as a biomarker for intracerebral haemorrhage in patients with acute stroke.J Neurol Neurosurg Psychiatry, 2006, 77(2):181-184.
21 Vos PE,Lamers K,Hendriks Met al.Glial and neuronal protein in serum predict outcome after severe traumatic brain injury.Neurology, 2004,62(8): 1303-1310.
22 Blennow M, Hagberg H, Rosengren L. Glial fibrillary acidic protein in the cerebrospinal fluid: a possible indicator of prognosis in full-term asphyxiated newborn infants?. Pediatr Res, 1995,37(3):260-264.
23 Blennow M, Rosengren L, Johsson S, et al. Glial fibrillary acidic protein is increased in the cerebrospinal fluid of preterm infants with abnormal neurological findings. Acta Paediatr, 1996,85(4):485-489.
1 Vannucci RC, Perlman JM. Intervention for perinatal hypoxic-ischemic encephalopathy. Pediatrics, 1997, 100(6): 1004-1014.
2 du Plessis AJ, Johnston MV. Hypoxic-ischemic brain injury in the newborn: cellular mechanisms and potential strategies for neuroprotection. Clin Perinatol, 1997,24(3):627-654.
3 Volpe JJ. Intracranial hemorrhage: germinal matrix-intraventricular hemorrhage of premature infant. Neurology of the newborn, 1995,403-463.
4 Volpe JJ. Hypoxic-ischemic encephalopathy: clinical aspects.In: Volpe JJ eds. Neurology of the newborn..Philadelphia: W.B. Saunders, 1995:314-370.
5 Michetti F, Massaro A, Russo G, et al..The S-100 antigen in cerebrospinal fluid as a possible index of cell injury in the nervous system. J Neurol Sci, 1980, 44(2-3):259-263.
6 Nagdyman N, Komen W, Ko HK, et al. Early biochemical indicators of hypoxic-ischemic encephslopathy after birth asphyxia. Pediatric Research,2001 Apr,49(1):502-506.
7 Gozzolo D,Vinesi P, Marinoni E, et al. S100B Protein Concentrations in Cord Blood: Correlations with Gestational Age in Term and Preterm Deliveries. Clin Chem, 2000 Jul, 46(7):998-1000.
8 王庆红，宋健辉，王霞，等．血清S100B蛋白在新生儿窒息后脑损伤中的临床意义．中国当代儿科杂志，2005，7(4)：318-320．
9 Thorngren-Jerneck K, Alling C,Herbst A, et al.S100 Protein in Serum as a Prognostic Marker for Cerebral Injury in Term Newborn Infants with Hypoxic Ischemic Encephalopathy. Pediatr. Res, March 1, 2004,55(3): 406-412.
10 Gazzolo D, Marinoni E, Di Iorio R, et al. Measurement of urinary S100B protein concentrations for the early identification of brain damage in asphyxiated full-term infants Arch Pediatr Adolesc Med, 2003 Dec, 157(12): 1163-8.
11 Gazzolo D, Marinoni E, Di Iorio R, et al. Urinary S100B protein measurements: A tool for the early identification of hypoxic-ischemic encephalopathy in asphyxiated full-term infants. Crit Care Med, 2004 Jan, 32(1):131-6.
12 Perlman JM, Tack ED. Renal injury in asphyxiated newborn infant: relation to neurologic outcome. J Pediatr, 1988,113(5): 875-879.
13 刘春艳，董文斌，陈跃，等．血清神经元特异性烯醇化酶与新生儿缺氧缺血性脑病的关系研究．中国优生与遗传杂志，2005，13(2)：85-86．
14 黄燕萍，李小权，王安生，等．神经元特异性烯醇化酶在早期评估新生儿缺氧缺血性脑病预后中的价值．新生儿科杂志，2005，20(1)：23-26．
15 Verdu Perez A, Falero MP, Arroyos A, et al. Blood neuronal specific enolase in newborns with perinatal asphyxia. Rev Neurol,2001 Apr 16-30,32(8):714-7.
16 Tekgul H, Yalaz M, Kutukculer N, et al. Value of Biochemical Markers for Outcome inTerm infants With Asphyxia. Obstetrical & GynecologicalSurvey, 2005,60(5):293-295.
17 Van Eldik LJ, Wainwright MS. The Janus face of glial-derived S100B: Beneficial and detrimental functions in the brain. Rest Neurol Neurosci, 2003, 21(3-4):97-108.
18 Wijnberger LD, Nikkels PG, van Dongen AJ, et al. Expression in the placenta of neuronal markers for perinatal brain damage. Pediatr Res,2002 Apr,51(4):492-6.
19 邓锦娥，潘秋兰．血清NSE、CK-MB、尿p2-MG测定对新生儿窒息后治疗的意义．中国热带医学，2005，5(6)：1291-1292
20 Huang CC, Wang ST, Chang YC,et al. Measurement of the urinary lactate: creatinine ratio for the early identification of newborn infants at risk for hypoxic-ischemic encephalopathy. N Engl J Med, 1999, 341(5): 328-335.
21 Turker G, Babaoglu K, Gokalp AS, et al. Cord blood cardiac troponin Ⅰ as an early predictor of short-term outcome in perinatal hypoxia. Biol Neonate, 2004,86(2): 131-7.
22 Turker G, Babaoglu K, Duman C, et al. The effect of blood gas and Apgar score on cord blood cardiac troponin I. J Matem Fetal Neonatal Med, 2004 Nov, 16(5):315-9.
23 “九五”攻关项目HIE治疗协作组．新生儿缺氧缺血性脑病治疗方案(试行稿)[J]．中国实用儿科杂志，2000，15(6)：381-382．
24 Sola A, Berrios M, Sheldon RA, et al. Fructose-1,6-bisphosphate after hypoxic ischemic injury is protective to the neonatal rat brain. Brain Res, 1996,741(1-2):294-299.
25 Rogido M, Husson I, Bonnier C, et al. Fructose-1, 6 bisphosphate prevents excitotoxic neuronal cell death in the neonatal mouse brain.Brain Res Dev,2003,140(2) :287-297
26 Tutak E,Satar M,Zorludemir S,et al.Neuroprotective effects of indomethacin and amino guanidine in the newborn rats with hypoxic-ischemic cerebral injury.Neurochem Res,2005 Aug,30(8) :937-42．
27 Palmer C,Towfighi J,Roberts RL,et al.Allopurinol administered after inducing hypoxiaischemia reduces brain injury in 7-day-old rats.Pediatr Res,1993,33(4) :405-411．
28 Tutunculer F,Eskiocak S,Basaran UN,et al.The protective role of melatonin in experimental hypoxic brain damage.Pediatr Int,2005 Aug;47(4) :434-9．
29 Singh D,Kumar P,Majumdar S,et al.Effect of phenobarbital on free radicals in neonates with hypoxic ischemic encephalopathy:a randomized controlled trial.J Perinat Med,2004,32 (3) :278-81．
30 Sze KH,Sim TC,Wong E,et al.Effect of nimodipine on memoryafter cerebral infarction.Acta Neurol Scand,1998,97(6) :386-392．
31 Johnston MV.Excitotoxicity in perinatal brain injury.Brain Pathol,2005 Jul,15(3) :234-40．
32 Garnier Y,Middelanis J,Jensen A,et al.Neuroprotective effects of magnesium on metabolic disturbances in fetal hippocampal slices after oxygen-glucose deprivation:mediation by nitric oxide system.J Soc Gynecol Investig,2002,9(2) :86-92．
33 Tsuji M,Higuchi Y,Shiraishi K,et al.Protective effect of aminoguanidine on hypoxic-ischemic brain damage and temporal profile of brain nitric oxide in neonatal rat.Pediatr Res,2000 Jan,47(1) :79-83．
34 Feng Y,Piletz JE,Leblanc MH.Agmatine suppresses nitric oxide production and attenuates hypoxic-ischemic brain injury in neonatal rats.Pediatr Res,2002 Oct,52(4) :606-11．
35 Gluckman PD,Wyatt JS,Azzopardi RB,et al.Selective head cooling with mild systemic hypothermia to improve neurodevelopmental outcome following neonatal encephalopathy.Pediatr Res,2004,55(3) :582A
36 Shankaran S,Laptook AR,Ehrenkranz RA,et al.Safety of whole body hypothermia for hypoxic-ischemic encephalopathy (HIE).Pediatr Res,2004,55(2) :582A.
37 Edwards AD,Azzopardi DV.Therapeutic hypothermia following perinatal asphyxia.Arch Dis Child Fetal Neonatal Ed,2006 Mar,91(2) :F127-31．
38 Hellstrom-Westas L.Hypothermia after perinatal asphyxia reduces the risk of brain damage.But it's too early to recommend the method for routine treatment.Lakartidningen,2005 Oct 17-23,102(42) :3030-1．
39 Li Q,Stephenson D.Postischemic administration of basic fibroblast growth factor improves sensorimotor function and reduces infarct size following permanent focalcerebral ischemia in the rat.Exp Neurol,2002 Oct,177(2) :531-7．
40 Holtzman DM,Sheldon RA,Jaffe W,et al..Nerve growth factor protects the neonatal brain against hypoxic-ischemic injury.Ann Neurol,1996,39(1) :114-122．
41 ChengY,Gidday JM,Yan Q,et al.Marked age-dependent neuroprotection by brain-derived neurotrophic factor against neonatal hypoxic-ischemic brain injury.Ann Neurol,1997,41(4) :521-529．
42 Johnston BM,Mallard EC,Williams CE,et al.Insulin-like growth factor-1 is a potent neuronal rescue agent followinghypoxic-ischemic injury in fetal lambs.J Clin Invest,1996,97(1) :300-308．
43 Brywe KG,Mallard C,Gustavsson M,et al.IGF-I neuroprotection in the immature brain after hypoxia-ischemia,involvement of Akt and GSK3beta?Eur J Neurosci,2005 Mar,21(6) :1489-502．
44 Fisher JW.Erythropoietin:physiology and pharmacology update.Exp Biol Med,2003,228(1) :1-14．
45 Juul SE,Anderson DK,Li Y,et al.Erythropoietin and erythropoietin receptor in the developing human central nervous system.Pediatr Res,1998,43(1) :40-49．
46 Digicaylioglu M,Lipton SA.Erythropoietin-mediated neuroprotection involves cross-talk between Jak2 and NF-kappaB signalling cascades.Nature,2001,412(6848) :641-647
47 Solaroglu I,Solaroglu A,Kaptanoglu E,et al.Erythropoietin prevents ischemia-reperfusion from inducing oxidative damage in fetal rat brain.Childs Nerv Syst,2003,19(1) :19-22．
48 Kumral A,Baskin H,Gokmen N,et al.Selective inhibition of nitric oxide in hypoxic-ischemic brain model in newborn rats:is it an explanation for the protective role of erythropoietin?Biol Neonate,2004,85(1) :51-54．
49 Shingo T,Sorokan ST,Shimazaki T,et al.Erythropoietin regulates the in vitro and in vivo production of neuronal progenitors by mammalian forebrain neural stem cells.J Neurosci,2001,21(24) :9733-9743．
50 Masuda S,Nagao N,Takahata K,et al.Functional erythropoietin receptor of the cells with neural characteristics.Comparison with receptor properties of erythroid cells.J Biol Chem,1993,268(15) :11208-11216．
51 Gorio A,Gokmen N,Erbayraktar S,et al.Recombinant human erythropoietin counteracts secondary injury and markedly enhances neurological recovery from experimental spinal cord trauma.Proc Natl Acad Sci USA,2002,99(14) :9450-9455．
52 Sola A,Wen TC,Hamrick SE,et al.Potential for protection and repair following injury to the developing brain:a role for erythropoietin?Pediatr Res,2005 May;57(5 Pt 2) :110R-117R.
53 Ingebrigtsen T,Romner B.Biochemical serum markers of traumatic brain injury.J Trauma,2002,52(4) :798-808．
2 Marinoni E,Dilorio R,Gazzolo D,et al. Ontogenetic localization and distribution of S-100beta protein in human placental tissues. Obstet Gynecol.,2002 Jun,99(6): 1093-1099.
3 Michetti F, Massaro A, Russo G, et al. The S-100 antigen in cerebrospinal fluid as a possible index of cell injury in the nervous system. J Neurol Sci, 1980, 44(2-3):259-263.
4 Gazzolo D, Marinoni E, Di Iorio R, et al. Measurement of urinary S 100B protein concentrations for the early identification of brain damage in asphyxiated full-term infants. Arch Pediatr Adolesc Med, 2003 Dec,157(12): 1163-1168.
5 Gazzolo D, Marinoni E, Di Iorio R, et al. Urinary S100B protein measurements: A tool for the early identification of hypoxic-ischemic encephalopathy in asphyxiated full-term infants. Crit Care Med, 2004 Jan, 32(1):131-136.
6 Perlman JM, Tack ED. Renal injury in asphyxiated newborn infant: relation to neurologic outcome. J Pediatr, 1988, 113(5): 875-879.
7 Nagdyman N, Komen W, Ko HK, et al. Early biochemical indicators of hypoxic-ischemic encephslopathy after birth asphyxia. Pediatric Research, 2001 Apr, 49(1):502-506.
8 Nagdyman N, Grimmer I, Scholz T, et al. Predictive value of brain-specific proteins in serun for neurodevelopmental outcome after brain birth asphyxia. Pediatr Res, 2003 Aug, 54(2);270-275.
9 Gozzolo D,Vinesi P, Marinoni E, et al. S100B Protein Concentrations in Cord Blood: Correlations with Gestational Age in Term and Preterm Deliveries. Clin Chem, 2000 Jul, 46(7):998-1000.
10 王庆红，宋健辉，王霞，等．血清S100B蛋白在新生儿窒息后脑损伤中的临床意义．中国当代儿科杂志，2005，7(4)：318-320．
11 Thorngren-Jerneck K, Alling C, Herbst A, et al. S100 Protein in Serum as a Prognostic Marker for Cerebral Injury in Term Newborn Infants with Hypoxic Ischemic Encephalopathy. Pediatr. Res, March 1, 2004,55(3): 406-412.
12 刘春艳，董文斌，陈跃，等．血清神经元特异性烯醇化酶与新生儿缺氧缺血性脑病的关系研究．中国优生与遗传杂志，2005，13(2)：85-86．
13 黄燕萍，李小权，王安生，等．神经元特异性烯醇化酶在早期评估新生儿缺氧缺血性脑病预后中的价值．新生儿科杂志，2005，20(1)：23-26．
14 Verdu Perez A, Falero MP, Arroyos A, et al.Blood neuronal specific enolase in newborns with perinatal asphyxia. Rev Neurol, 2001 Apr, 32(8):714-717.
15 Tekgul H, Yalaz M, Kutukculer N, et al.Value of Biochemical Markers for Outcome in Term Infants With Asphyxia. Obstetrical & Gynecological Survey, 2005, 60(5):293-295.
16 Elimian A, Figueroa R, Patel K, et al.Reference values of amniotic fluid neuron-specific enolase [J]. Maternal-fetal Med, 2001,10(3): 155-158.
17 Zinsmeyer J, Marangos PJ, Issel EP, et al. Neuron specific enolase in amniotic fluid—a possible indicator for fetal distress and brain implication. J Perinat Med, 1987,15(3): 199-202.
18 Elimian A, Figueroa R, Verma U, et al. Amniotic fluid neuron-specific enolase: a role in predicting neonatal neurologic injury? Obstet Gynecol,1998,92(1): 546-550.
19 Van Eldik LJ, Wainwright MS. The Janus face of glial-derived S100B: Beneficial and detrimental functions in the brain. Rest Neurol Neurosci, 2003,21 (3-4):97-108.
20 Foerch C,Curdt I,Yan B, et al .Serum glial fibrillary acidic protein as a biomarker for intracerebral haemorrhage in patients with acute stroke.J Neurol Neurosurg Psychiatry, 2006, 77(2):181-184.
21 Vos PE,Lamers K,Hendriks Met al.Glial and neuronal protein in serum predict outcome after severe traumatic brain injury.Neurology, 2004,62(8): 1303-1310.
22 Blennow M, Hagberg H, Rosengren L. Glial fibrillary acidic protein in the cerebrospinal fluid: a possible indicator of prognosis in full-term asphyxiated newborn infants?. Pediatr Res, 1995,37(3):260-264.
23 Blennow M, Rosengren L, Johsson S, et al. Glial fibrillary acidic protein is increased in the cerebrospinal fluid of preterm infants with abnormal neurological findings. Acta Paediatr, 1996,85(4):485-489.
1 Vannucci RC, Perlman JM. Intervention for perinatal hypoxic-ischemic encephalopathy. Pediatrics, 1997, 100(6): 1004-1014.
2 du Plessis AJ, Johnston MV. Hypoxic-ischemic brain injury in the newborn: cellular mechanisms and potential strategies for neuroprotection. Clin Perinatol, 1997,24(3):627-654.
3 Volpe JJ. Intracranial hemorrhage: germinal matrix-intraventricular hemorrhage of premature infant. Neurology of the newborn, 1995,403-463.
4 Volpe JJ. Hypoxic-ischemic encephalopathy: clinical aspects.In: Volpe JJ eds. Neurology of the newborn..Philadelphia: W.B. Saunders, 1995:314-370.
5 Michetti F, Massaro A, Russo G, et al..The S-100 antigen in cerebrospinal fluid as a possible index of cell injury in the nervous system. J Neurol Sci, 1980, 44(2-3):259-263.
6 Nagdyman N, Komen W, Ko HK, et al. Early biochemical indicators of hypoxic-ischemic encephslopathy after birth asphyxia. Pediatric Research,2001 Apr,49(1):502-506.
7 Gozzolo D,Vinesi P, Marinoni E, et al. S100B Protein Concentrations in Cord Blood: Correlations with Gestational Age in Term and Preterm Deliveries. Clin Chem, 2000 Jul, 46(7):998-1000.
8 王庆红，宋健辉，王霞，等．血清S100B蛋白在新生儿窒息后脑损伤中的临床意义．中国当代儿科杂志，2005，7(4)：318-320．
9 Thorngren-Jerneck K, Alling C,Herbst A, et al.S100 Protein in Serum as a Prognostic Marker for Cerebral Injury in Term Newborn Infants with Hypoxic Ischemic Encephalopathy. Pediatr. Res, March 1, 2004,55(3): 406-412.
10 Gazzolo D, Marinoni E, Di Iorio R, et al. Measurement of urinary S100B protein concentrations for the early identification of brain damage in asphyxiated full-term infants Arch Pediatr Adolesc Med, 2003 Dec, 157(12): 1163-8.
11 Gazzolo D, Marinoni E, Di Iorio R, et al. Urinary S100B protein measurements: A tool for the early identification of hypoxic-ischemic encephalopathy in asphyxiated full-term infants. Crit Care Med, 2004 Jan, 32(1):131-6.
12 Perlman JM, Tack ED. Renal injury in asphyxiated newborn infant: relation to neurologic outcome. J Pediatr, 1988,113(5): 875-879.
13 刘春艳，董文斌，陈跃，等．血清神经元特异性烯醇化酶与新生儿缺氧缺血性脑病的关系研究．中国优生与遗传杂志，2005，13(2)：85-86．
14 黄燕萍，李小权，王安生，等．神经元特异性烯醇化酶在早期评估新生儿缺氧缺血性脑病预后中的价值．新生儿科杂志，2005，20(1)：23-26．
15 Verdu Perez A, Falero MP, Arroyos A, et al. Blood neuronal specific enolase in newborns with perinatal asphyxia. Rev Neurol,2001 Apr 16-30,32(8):714-7.
16 Tekgul H, Yalaz M, Kutukculer N, et al. Value of Biochemical Markers for Outcome inTerm infants With Asphyxia. Obstetrical & GynecologicalSurvey, 2005,60(5):293-295.
17 Van Eldik LJ, Wainwright MS. The Janus face of glial-derived S100B: Beneficial and detrimental functions in the brain. Rest Neurol Neurosci, 2003, 21(3-4):97-108.
18 Wijnberger LD, Nikkels PG, van Dongen AJ, et al. Expression in the placenta of neuronal markers for perinatal brain damage. Pediatr Res,2002 Apr,51(4):492-6.
19 邓锦娥，潘秋兰．血清NSE、CK-MB、尿p2-MG测定对新生儿窒息后治疗的意义．中国热带医学，2005，5(6)：1291-1292
20 Huang CC, Wang ST, Chang YC,et al. Measurement of the urinary lactate: creatinine ratio for the early identification of newborn infants at risk for hypoxic-ischemic encephalopathy. N Engl J Med, 1999, 341(5): 328-335.
21 Turker G, Babaoglu K, Gokalp AS, et al. Cord blood cardiac troponin Ⅰ as an early predictor of short-term outcome in perinatal hypoxia. Biol Neonate, 2004,86(2): 131-7.
22 Turker G, Babaoglu K, Duman C, et al. The effect of blood gas and Apgar score on cord blood cardiac troponin I. J Matem Fetal Neonatal Med, 2004 Nov, 16(5):315-9.
23 “九五”攻关项目HIE治疗协作组．新生儿缺氧缺血性脑病治疗方案(试行稿)[J]．中国实用儿科杂志，2000，15(6)：381-382．
24 Sola A, Berrios M, Sheldon RA, et al. Fructose-1,6-bisphosphate after hypoxic ischemic injury is protective to the neonatal rat brain. Brain Res, 1996,741(1-2):294-299.
25 Rogido M, Husson I, Bonnier C, et al. Fructose-1, 6 bisphosphate prevents excitotoxic neuronal cell death in the neonatal mouse brain.Brain Res Dev,2003,140(2) :287-297
26 Tutak E,Satar M,Zorludemir S,et al.Neuroprotective effects of indomethacin and amino guanidine in the newborn rats with hypoxic-ischemic cerebral injury.Neurochem Res,2005 Aug,30(8) :937-42．
27 Palmer C,Towfighi J,Roberts RL,et al.Allopurinol administered after inducing hypoxiaischemia reduces brain injury in 7-day-old rats.Pediatr Res,1993,33(4) :405-411．
28 Tutunculer F,Eskiocak S,Basaran UN,et al.The protective role of melatonin in experimental hypoxic brain damage.Pediatr Int,2005 Aug;47(4) :434-9．
29 Singh D,Kumar P,Majumdar S,et al.Effect of phenobarbital on free radicals in neonates with hypoxic ischemic encephalopathy:a randomized controlled trial.J Perinat Med,2004,32 (3) :278-81．
30 Sze KH,Sim TC,Wong E,et al.Effect of nimodipine on memoryafter cerebral infarction.Acta Neurol Scand,1998,97(6) :386-392．
31 Johnston MV.Excitotoxicity in perinatal brain injury.Brain Pathol,2005 Jul,15(3) :234-40．
32 Garnier Y,Middelanis J,Jensen A,et al.Neuroprotective effects of magnesium on metabolic disturbances in fetal hippocampal slices after oxygen-glucose deprivation:mediation by nitric oxide system.J Soc Gynecol Investig,2002,9(2) :86-92．
33 Tsuji M,Higuchi Y,Shiraishi K,et al.Protective effect of aminoguanidine on hypoxic-ischemic brain damage and temporal profile of brain nitric oxide in neonatal rat.Pediatr Res,2000 Jan,47(1) :79-83．
34 Feng Y,Piletz JE,Leblanc MH.Agmatine suppresses nitric oxide production and attenuates hypoxic-ischemic brain injury in neonatal rats.Pediatr Res,2002 Oct,52(4) :606-11．
35 Gluckman PD,Wyatt JS,Azzopardi RB,et al.Selective head cooling with mild systemic hypothermia to improve neurodevelopmental outcome following neonatal encephalopathy.Pediatr Res,2004,55(3) :582A
36 Shankaran S,Laptook AR,Ehrenkranz RA,et al.Safety of whole body hypothermia for hypoxic-ischemic encephalopathy (HIE).Pediatr Res,2004,55(2) :582A.
37 Edwards AD,Azzopardi DV.Therapeutic hypothermia following perinatal asphyxia.Arch Dis Child Fetal Neonatal Ed,2006 Mar,91(2) :F127-31．
38 Hellstrom-Westas L.Hypothermia after perinatal asphyxia reduces the risk of brain damage.But it's too early to recommend the method for routine treatment.Lakartidningen,2005 Oct 17-23,102(42) :3030-1．
39 Li Q,Stephenson D.Postischemic administration of basic fibroblast growth factor improves sensorimotor function and reduces infarct size following permanent focalcerebral ischemia in the rat.Exp Neurol,2002 Oct,177(2) :531-7．
40 Holtzman DM,Sheldon RA,Jaffe W,et al..Nerve growth factor protects the neonatal brain against hypoxic-ischemic injury.Ann Neurol,1996,39(1) :114-122．
41 ChengY,Gidday JM,Yan Q,et al.Marked age-dependent neuroprotection by brain-derived neurotrophic factor against neonatal hypoxic-ischemic brain injury.Ann Neurol,1997,41(4) :521-529．
42 Johnston BM,Mallard EC,Williams CE,et al.Insulin-like growth factor-1 is a potent neuronal rescue agent followinghypoxic-ischemic injury in fetal lambs.J Clin Invest,1996,97(1) :300-308．
43 Brywe KG,Mallard C,Gustavsson M,et al.IGF-I neuroprotection in the immature brain after hypoxia-ischemia,involvement of Akt and GSK3beta?Eur J Neurosci,2005 Mar,21(6) :1489-502．
44 Fisher JW.Erythropoietin:physiology and pharmacology update.Exp Biol Med,2003,228(1) :1-14．
45 Juul SE,Anderson DK,Li Y,et al.Erythropoietin and erythropoietin receptor in the developing human central nervous system.Pediatr Res,1998,43(1) :40-49．
46 Digicaylioglu M,Lipton SA.Erythropoietin-mediated neuroprotection involves cross-talk between Jak2 and NF-kappaB signalling cascades.Nature,2001,412(6848) :641-647
47 Solaroglu I,Solaroglu A,Kaptanoglu E,et al.Erythropoietin prevents ischemia-reperfusion from inducing oxidative damage in fetal rat brain.Childs Nerv Syst,2003,19(1) :19-22．
48 Kumral A,Baskin H,Gokmen N,et al.Selective inhibition of nitric oxide in hypoxic-ischemic brain model in newborn rats:is it an explanation for the protective role of erythropoietin?Biol Neonate,2004,85(1) :51-54．
49 Shingo T,Sorokan ST,Shimazaki T,et al.Erythropoietin regulates the in vitro and in vivo production of neuronal progenitors by mammalian forebrain neural stem cells.J Neurosci,2001,21(24) :9733-9743．
50 Masuda S,Nagao N,Takahata K,et al.Functional erythropoietin receptor of the cells with neural characteristics.Comparison with receptor properties of erythroid cells.J Biol Chem,1993,268(15) :11208-11216．
51 Gorio A,Gokmen N,Erbayraktar S,et al.Recombinant human erythropoietin counteracts secondary injury and markedly enhances neurological recovery from experimental spinal cord trauma.Proc Natl Acad Sci USA,2002,99(14) :9450-9455．
52 Sola A,Wen TC,Hamrick SE,et al.Potential for protection and repair following injury to the developing brain:a role for erythropoietin?Pediatr Res,2005 May;57(5 Pt 2) :110R-117R.
53 Ingebrigtsen T,Romner B.Biochemical serum markers of traumatic brain injury.J Trauma,2002,52(4) :798-808．