高血压脑出血早期血肿周围脑组织细胞凋亡与凝血酶表达相关性的研究
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摘要
目的探讨高血压脑出血(HICH)早期血肿周围脑组织细胞凋亡与凝血酶表达的关系。
方法取36例高血压脑出血患者血肿周围脑组织作为实验组,按发病至手术的时间分为5组：<6h(7例)、6～12 h(11例)、12～24 h(8例)、24～48h(6例)、48～72 h(4例)。另将侧脑室肿瘤患者经皮层入路取得的正常脑组织作为对照组。采用脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)检测神经细胞凋亡率,免疫组织化学方法检测凝血酶表达。
结果对照组脑组织基本无TUNEL阳性细胞(0.32±0.037)；<6 h组有轻微表达(7.19±2.53,P<0.05),发病6h后逐渐增高(15.11±3.69,P<0.01),12～24h增高显著(28.26±7.83,P<0.01)并于24～48 h达高峰(53.79±6.35,P<0.01),48～72h组有所回落,但仍维持较高水平(38.23±3.29,P<0.01)。凝血酶表达变化与此一致。相关分析显示：细胞凋亡与凝血酶表达呈显著正相关(r=0.7451,P<0.05或P<0.01)。
结论HICH患者血肿周围脑组织中存在细胞凋亡,凝血酶表达是细胞凋亡过程中的一个关键事件
Objective To investigate the relationship between cell apoptosis and expression of thrombin in the perihematoma tissue in early hypertensive intracerebral hemorrhage (HICH).
Methods 36 samples were obtained from the perihematoma region of HICH patients and divided into 5 groups according to the time from onset to operation:6hours (n=7),6～12hours (n=11),12～24hours (n=8),24～48hours (n=6),48～72hours (n=4). The normal control group were collected from the patients with lateral ventricle tumor who were operation by transcortical approach.The terminal deoxynucleotidyl-transferase mediated dUTP-biotin nick end labeling (TUNEL) method was used to detect apoptosis and the expression of thrombin was observed by immunohistochemistry.
Results the TUNEL positive cells was almost no expression in the control group (0.32±0.037) and it have a slight expression in<6 h group(7.19±2.53,P< 0.05).It increased gradually 6hours after the onset of HICH (15.11±3.69, P< 0.01)and reached the maximum at 24～48 hours (53.79±6.35, P< 0.01).48-72h group has dropped slightly, but still maintain a high level (38.23±3.29, P<0.01). The expression of the thrombin was consistent with the apoptosis. The correlation analysis showed that the TUNEL positive cells was positively correlated to the expression of the thrombin (P<0.05 or P<0.01).
Conclusion Apoptosis cells exist in perihematomal region among hypertensive intracerebral hemorrhage.The expression of thrombin is a critical step in apoptotic cell death after hypertensive intracerebral hemorrhage.
方法取36例高血压脑出血患者血肿周围脑组织作为实验组,按发病至手术的时间分为5组：<6h(7例)、6～12 h(11例)、12～24 h(8例)、24～48h(6例)、48～72 h(4例)。另将侧脑室肿瘤患者经皮层入路取得的正常脑组织作为对照组。采用脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)检测神经细胞凋亡率,免疫组织化学方法检测凝血酶表达。
结果对照组脑组织基本无TUNEL阳性细胞(0.32±0.037)；<6 h组有轻微表达(7.19±2.53,P<0.05),发病6h后逐渐增高(15.11±3.69,P<0.01),12～24h增高显著(28.26±7.83,P<0.01)并于24～48 h达高峰(53.79±6.35,P<0.01),48～72h组有所回落,但仍维持较高水平(38.23±3.29,P<0.01)。凝血酶表达变化与此一致。相关分析显示：细胞凋亡与凝血酶表达呈显著正相关(r=0.7451,P<0.05或P<0.01)。
结论HICH患者血肿周围脑组织中存在细胞凋亡,凝血酶表达是细胞凋亡过程中的一个关键事件
Objective To investigate the relationship between cell apoptosis and expression of thrombin in the perihematoma tissue in early hypertensive intracerebral hemorrhage (HICH).
Methods 36 samples were obtained from the perihematoma region of HICH patients and divided into 5 groups according to the time from onset to operation:6hours (n=7),6～12hours (n=11),12～24hours (n=8),24～48hours (n=6),48～72hours (n=4). The normal control group were collected from the patients with lateral ventricle tumor who were operation by transcortical approach.The terminal deoxynucleotidyl-transferase mediated dUTP-biotin nick end labeling (TUNEL) method was used to detect apoptosis and the expression of thrombin was observed by immunohistochemistry.
Results the TUNEL positive cells was almost no expression in the control group (0.32±0.037) and it have a slight expression in<6 h group(7.19±2.53,P< 0.05).It increased gradually 6hours after the onset of HICH (15.11±3.69, P< 0.01)and reached the maximum at 24～48 hours (53.79±6.35, P< 0.01).48-72h group has dropped slightly, but still maintain a high level (38.23±3.29, P<0.01). The expression of the thrombin was consistent with the apoptosis. The correlation analysis showed that the TUNEL positive cells was positively correlated to the expression of the thrombin (P<0.05 or P<0.01).
Conclusion Apoptosis cells exist in perihematomal region among hypertensive intracerebral hemorrhage.The expression of thrombin is a critical step in apoptotic cell death after hypertensive intracerebral hemorrhage.
引文
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[1].Lee, J.C., G.S. Cho, B.O. Choi, et al., Intracerebral hemorrhage-induced brain injury is aggravated in senescence-accelerated prone mice. Stroke,2006.37(1):216-222.
[2]. Zain, J., Y.Q. Huang, X. Feng, et al., Concentration-dependent dual effect of thrombin on impaired growth/apoptosis or mitogenesis in tumor cells. Blood,2000.95(10):3133-3138.
[3]. Citron, B.A., I.V. Smirnova, P.M. Arnold, et al., Upregulation of neurotoxic serine proteases, prothrombin, and protease-activated receptor 1 early after spinal cord injury. J Neurotrauma, 2000.17(12):1191-1203.
[4]. Ishii, H., H.H. Salem, C.E. Bell, et al., Thrombomodulin, an endothelial anticoagulant protein, is absent from the human brain. Blood,1986.67(2):362-365.
[5]. Ishida, Y., A. Nagai, S. Kobayashi, et al., Upregulation of protease-activated receptor-1 in astrocytes in Parkinson disease:astrocyte-mediated neuroprotection through increased levels of glutathioneperoxidase. J Neuropathol Exp Neurol,2006.65(1):66-77.
[6].Henrich-Noack, P., M. Riek-Burchardt, K. Baldauf, et al., Focal ischemia induces expression of protease-activated receptorl (PAR1) and PAR3 on microglia and enhances PAR4 labeling in the penumbra. Brain Res,2006.1070(1):232-241.
[7]. Zheng, G.Q., X.T. Wang, X.M. Wang, et al., Long-time course of protease-activated receptor-1 expression after intracerebral hemorrhage in rats. Neurosci Lett,2009.459(2):62-65.
[8].Nagy, Z., K. Kolev, E. Csonka, et al., Contraction of human brain endothelial cells induced by thrombogenic and fibrinolytic factors. An in vitro cell culture model. Stroke,1995. 26(2):265-270.
[9].Kenya Kawakita, M.,*(?), M. Nobuyuki Kawai,*, M. Yasuhiro Kuroda, (?), et al., Expression of matrix metalloproteinse-9 in thrombin-induced brain edema formation in rats. Stroke and Cerebrovascular Diseases,2006.15(3):88-95.
[10]. 代大伟,王德生,and 蔡军,.鼠脑内注射凝血酶后水通道蛋白4的表达变化.中国临床康复,2006.10(30)86-88.
[11]. Tang, Y, D. Cai, and Y. Chen, Thrombin inhibits aquaporin 4 expression through protein kinase C-dependent pathway in cultured astrocytes. J Mol Neurosci,2007.31(1):83-93.
[12]. Donovan, F.M., C.J. Pike, C.W. Cotman, et al, Thrombin induces apoptosis in cultured neurons and astrocytes via a pathway requiring tyrosine kinase and RhoA activities. J Neurosci,1997.17(14):5316-5326.
[13]. Rao, H.V., L. Thirumangalakudi, P. Desmond, et al., Cyclin Dl, cdk4, and Bim are involved in thrombin-induced apoptosis in cultured cortical neurons. J Neurochem,2007.101(2):498-505.
[14]. Rao, H.V., L. Thirumangalakudi, and P. Grammas, Cyclin C and cyclin dependent kinases 1,2 and 3 in thrombin-induced neuronal cell cycle progression and apoptosis. Neurosci Lett,2009. 450(3):347-350.
[15].Thirumangalakudi, L., H.V. Rao, and P. Grammas, Involvement of PGE2 and PGDH but not COX-2 in thrombin-induced cortical neuron apoptosis. Neurosci Lett,2009.452(2):172-175.
[16]. Gong, C., J.T. Hoff, and R.F. Keep, Acute inflammatory reaction following experimental intracerebral hemorrhage in rat. Brain Res,2000.871(1):57-65.
[17]. Chen, D. and A. Dorling, Critical roles for thrombin in acute and chronic inflammation. J Thromb Haemost,2009.7 (1):122-126.
[18]. Leung, L.L., T. Nishimura, and T. Myles, Regulation of tissue inflammation by thrombin-activatable carboxypeptidase B (or TAFI). Adv Exp Med Biol,2008.63 (2):61-69.
[19].Nishimura, T., T. Myles, A.M. Piliponsky, et al., Thrombin-activatable procarboxypeptidase B regulates activated complement C5a in vivo. Blood,2007.109(5):1992-1997.
[20]. Haver, V.M. and D.H. Namm, Generation of a vasoactive substance in human plasma during coagulation. Evidence of thrombin-induced contraction of rabbit aorta and dog coronary artery. Blood Vessels,1983.20(2):92-98.
[21]. Kai, Y., Y. Maeda, T. Sasaki, et al., Basic and translational research on proteinase-activated receptors:the role of thrombin receptor in cerebral vasospasm in subarachnoid hemorrhage. J Pharmacol Sci,2008.108(4):426-432.
[22].Lee, K.R., N. Kawai, S. Kim, et al., Mechanisms of edema formation after intracerebral hemorrhage:effects of thrombin on cerebral blood flow, blood-brain barrier permeability, and cell survival in a rat model. J Neurosurg,1997.86(2):272-278.
[23]. Lee, K.R., I. Drury, E. Vitarbo, et al., Seizures induced by intracerebral injection of thrombin: a model of intracerebral hemorrhage. J Neurosurg,1997.87(1):73-78.
[24]. Zhang, Y., J.C. Feng, J. Wu, et al., Protective effects of hirudin on acute experimental intracerebral hemorrhage. Zhongguo Zhong Yao Za Zhi,2006.31(1):69-72.
[25].Matsuoka, H. and R. Hamada, Role of thrombin in CNS damage associated with intracerebral haemorrhage:opportunity for pharmacological intervention? CNS Drugs,2002. 16(8):509-516.
[26].Nagatsuna, T., S. Nomura, E. Suehiro, et al., Systemic administration of argatroban reduces secondary brain damage in a rat model of intracerebral hemorrhage:histopathological assessment. Cerebrovasc Dis,2005.19(3):192-200.
[2].Kazui, S., K. Minematsu, H. Yamamoto, et al., Predisposing factors to enlargement of spontaneous intracerebral hematoma. Stroke,1997.28(12):2370-2375.
[3].Kazui, S., H. Naritomi, H. Yamamoto, et al., Enlargement of spontaneous intracerebral hemorrhage. Incidence and time course. Stroke,1996.27(10):1783-1787.
[4].Brott, T., J. Broderick, R. Kothari, et al., Early hemorrhage growth in patients with intracerebral hemorrhage. Stroke,1997.28(1):1-5.
[5].Mayer, S.A., A. Lignelli, M.E. Fink, et al., Perilesional blood flow and edema formation in acute intracerebral hemorrhage:a SPECT study. Stroke,1998.29(9):1791-1798.
[6].Mendelow, A.D., Mechanisms of ischemic brain damage with intracerebral hemorrhage. Stroke,1993.24(12 Suppl):I115-117; discussion I118-119.
[7].Xi, G., R.F. Keep, and J.T. Hoff, Mechanisms of brain injury after intracerebral haemorrhage. Lancet Neurol,2006.5(1):53-63.
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[1].Lee, J.C., G.S. Cho, B.O. Choi, et al., Intracerebral hemorrhage-induced brain injury is aggravated in senescence-accelerated prone mice. Stroke,2006.37(1):216-222.
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[5]. Ishida, Y., A. Nagai, S. Kobayashi, et al., Upregulation of protease-activated receptor-1 in astrocytes in Parkinson disease:astrocyte-mediated neuroprotection through increased levels of glutathioneperoxidase. J Neuropathol Exp Neurol,2006.65(1):66-77.
[6].Henrich-Noack, P., M. Riek-Burchardt, K. Baldauf, et al., Focal ischemia induces expression of protease-activated receptorl (PAR1) and PAR3 on microglia and enhances PAR4 labeling in the penumbra. Brain Res,2006.1070(1):232-241.
[7]. Zheng, G.Q., X.T. Wang, X.M. Wang, et al., Long-time course of protease-activated receptor-1 expression after intracerebral hemorrhage in rats. Neurosci Lett,2009.459(2):62-65.
[8].Nagy, Z., K. Kolev, E. Csonka, et al., Contraction of human brain endothelial cells induced by thrombogenic and fibrinolytic factors. An in vitro cell culture model. Stroke,1995. 26(2):265-270.
[9].Kenya Kawakita, M.,*(?), M. Nobuyuki Kawai,*, M. Yasuhiro Kuroda, (?), et al., Expression of matrix metalloproteinse-9 in thrombin-induced brain edema formation in rats. Stroke and Cerebrovascular Diseases,2006.15(3):88-95.
[10]. 代大伟,王德生,and 蔡军,.鼠脑内注射凝血酶后水通道蛋白4的表达变化.中国临床康复,2006.10(30)86-88.
[11]. Tang, Y, D. Cai, and Y. Chen, Thrombin inhibits aquaporin 4 expression through protein kinase C-dependent pathway in cultured astrocytes. J Mol Neurosci,2007.31(1):83-93.
[12]. Donovan, F.M., C.J. Pike, C.W. Cotman, et al, Thrombin induces apoptosis in cultured neurons and astrocytes via a pathway requiring tyrosine kinase and RhoA activities. J Neurosci,1997.17(14):5316-5326.
[13]. Rao, H.V., L. Thirumangalakudi, P. Desmond, et al., Cyclin Dl, cdk4, and Bim are involved in thrombin-induced apoptosis in cultured cortical neurons. J Neurochem,2007.101(2):498-505.
[14]. Rao, H.V., L. Thirumangalakudi, and P. Grammas, Cyclin C and cyclin dependent kinases 1,2 and 3 in thrombin-induced neuronal cell cycle progression and apoptosis. Neurosci Lett,2009. 450(3):347-350.
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[16]. Gong, C., J.T. Hoff, and R.F. Keep, Acute inflammatory reaction following experimental intracerebral hemorrhage in rat. Brain Res,2000.871(1):57-65.
[17]. Chen, D. and A. Dorling, Critical roles for thrombin in acute and chronic inflammation. J Thromb Haemost,2009.7 (1):122-126.
[18]. Leung, L.L., T. Nishimura, and T. Myles, Regulation of tissue inflammation by thrombin-activatable carboxypeptidase B (or TAFI). Adv Exp Med Biol,2008.63 (2):61-69.
[19].Nishimura, T., T. Myles, A.M. Piliponsky, et al., Thrombin-activatable procarboxypeptidase B regulates activated complement C5a in vivo. Blood,2007.109(5):1992-1997.
[20]. Haver, V.M. and D.H. Namm, Generation of a vasoactive substance in human plasma during coagulation. Evidence of thrombin-induced contraction of rabbit aorta and dog coronary artery. Blood Vessels,1983.20(2):92-98.
[21]. Kai, Y., Y. Maeda, T. Sasaki, et al., Basic and translational research on proteinase-activated receptors:the role of thrombin receptor in cerebral vasospasm in subarachnoid hemorrhage. J Pharmacol Sci,2008.108(4):426-432.
[22].Lee, K.R., N. Kawai, S. Kim, et al., Mechanisms of edema formation after intracerebral hemorrhage:effects of thrombin on cerebral blood flow, blood-brain barrier permeability, and cell survival in a rat model. J Neurosurg,1997.86(2):272-278.
[23]. Lee, K.R., I. Drury, E. Vitarbo, et al., Seizures induced by intracerebral injection of thrombin: a model of intracerebral hemorrhage. J Neurosurg,1997.87(1):73-78.
[24]. Zhang, Y., J.C. Feng, J. Wu, et al., Protective effects of hirudin on acute experimental intracerebral hemorrhage. Zhongguo Zhong Yao Za Zhi,2006.31(1):69-72.
[25].Matsuoka, H. and R. Hamada, Role of thrombin in CNS damage associated with intracerebral haemorrhage:opportunity for pharmacological intervention? CNS Drugs,2002. 16(8):509-516.
[26].Nagatsuna, T., S. Nomura, E. Suehiro, et al., Systemic administration of argatroban reduces secondary brain damage in a rat model of intracerebral hemorrhage:histopathological assessment. Cerebrovasc Dis,2005.19(3):192-200.