大鼠局灶性脑缺血再灌注早期DNA修复蛋白Ku70的表达及意义
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摘要
脑缺血再灌注早期，损伤区域会迅速产生大量的氧自由基，通过直接或间接的作用引起神经元DNA氧化损伤，包括DNA碱基损伤、单链断裂和双链断裂。这种氧化性DNA损伤可以通过自身DNA修复系统进行修复，但是，如果DNA损伤持久或严重，超出自身修复能力，则诱导神经元死亡。研究表明脑缺血再灌注早期DNA修复酶的表达减少可能与随后发生的细胞死亡有关。神经细胞的死亡是脑缺血后神经功能受损的基础，对DNA损伤修复的研究有可能深入揭示缺血神经元的死亡机制，为缺血性脑血管病的治疗提供新的策略。缺血预处理和中药丹参对于缺血性脑损伤都有显著的神经保护作用，关于二者的脑保护机制已有多方面的探索，但仍未完全明确，是否可能通过调节内源性DNA修复活性而发挥保护作用还有待进一步研究。
本研究采用大脑中动脉线拴法建立大鼠局灶性脑缺血再灌注模型，利用HE染色方法、免疫组织化学方法和原位末端标记法(TUNEL法)，首先观察大脑中动脉阻塞(MCAO)2h再灌注0.5h、2h、6h、12h、24h、48h后缺血区额顶叶皮质和尾壳核DNA修复蛋白Ku70的表达及神经元凋亡情况，分析二者的变
第四军医大学硕士学位论文
化在时间上的关系;然后分别观察缺血预处理和中药丹参对脑缺
血再灌注后Ku7O表达的影响，初步探讨脑缺血再灌注早期
DNA修复酶Ku7O的表达变化规律及其意义，并从DNA损伤修
复的角度探讨缺血预处理和中药丹参对抗脑缺血再灌注损伤的分
子机制。
实验结果如下:
(l)大鼠大脑中动脉阻塞再灌注(MCAO服)后损伤区域主要
位于额叶皮质、顶叶皮质和尾壳核。
(2)正常对照组、假手术组及缺血再灌注对侧额顶叶皮质、尾
壳核可见Ku70广泛表达，定位于细胞核:MCAOZh、再灌注
0.5h，缺血侧尾壳核Ku7O阳性细胞数开始减少(P<0.05)，再
灌注Zh，额顶叶皮质Ku70阳性细胞数开始减少(尸<0.05)，
于再灌注6h，上述区域Ku70表达水平显著降低(P<0.01)，
并持续至再灌注48h;实施缺血预处理或给予腹腔注射丹参后，
明显抑制了Ku7O表达水平的降低，各时间点Ku7O阳性细胞数
均较相应对照组明显升高(尸<0.01)。
(3)正常对照组、假手术组及缺血再灌注对侧未见TUNEL阳性
细胞:‘MCAOZh、再灌注0.sh一2h，缺血区偶见散在的TUNEL阳
性细胞:再灌注6h，TUNEL阳性细胞数开始增多(P<0.01)，
随着再灌注时间的延长，阳性细胞数不断增高，再灌注24h和
48h，TUNEL阳性细胞数达到高峰(尸<0.01):实施缺血预处
理或给予丹参腹腔注射，明显抑制了脑缺血再灌注后细胞凋亡的
发生，各时间点TUNEL阳性细胞数均较相应对照组显著减少
(P<0 .01)。
(4) Ku蛋白在损伤区域内全面减少发生在缺血性神经元凋亡
高峰出现之前，二者呈负相关关系。
第四军医大学硕士学位论文
综上，本研究的初步结论如下:
(1)脑缺血再灌注早期，损伤区域Ku蛋白表达下降，可能
是导致DNA双链断裂无法得到修复、细胞随之死亡的机制之
(2)缺血预处理可能通过上调DNA修复蛋白Ku的表达或括
性而增强DNA修复能力，从而发挥其内源性神经保护作用。
(3)丹参可能通过抑制缺血再灌注后DNA修复蛋白Ku的减
少，保护机体自身DNA修复功能免遭破坏而发挥其神经保护作
用。
A large number of free radicals are produced in a burst-like manner at the early stage of cerebral ischemia/reperfusion and mainly cause oxidative DNA damage. Neuronal DNA damage caused by oxidative stress consistes of the lesion of base, DNA single strand breaks (SSB) and DNA double strand breaks (DSB). Oxidative DNA damage after ischemia / reperfusion can be repaired by DNA repair system, but continuous or severe DNA damage against DNA repair could induce neuronal death, such as necrosis or apoptosis. It has been reported that early decrease of DNA repair enzyme probably has correlation with subsequent DNA-damaged cell death after cerebral ischemia / reperfusion. As neuronal death was the basis of brain injury, researches on DNA damage and repair would probably reveal the mechanism of ischemic neuronal death, and will bring a new strategy for treating ischemic cerebrovascular diseases. It has been proved that both ischemic preconditioning (IPC) and radix salviae miltiorrhizae (RSM) have significant
neuroprotection against ischemic injury in the brain. A number of studies have been performed to clarify the protective effect and mechanism, however, whether IPC and RSM have any effects on the DNA damage and repair in the neuropathological process following cerebral ischemia/reperfusion still remains to be investigated.
In this study, we made the model of rat middle cerebral artery occlusion/ reperfusion and used H&E stain method, immuno-histochemical technology and TUNEL detection, in order to explore the relationship between the expression of Ku70 protein-which is critical to the repair of DNA DSB and neuronal death after focal cerebral ischemia/reperfusion, and the possible molecular mechanisms by which IPC and RSM protect brain. Firstly, we observed the expression of Ku70 protein and neuronal apoptosis in the ischemic cortex and caudatum after MCAO 2h reperfusion 0.5h, 2h, 6h, 12h, 24h, 48h, and evaluated the temporal relationship between Ku70 protein and neuronal apoptosis. Then we investigated the influence of IPC and RSM on the expression of Ku70 protein after focal cerebral ischemia/reperfusion.
Our results showed:
(1) HE stain indicated the model of rat MCAO/R was credible, and the damaged regions included cortex and caudate putamen.
(2) Immunohistochemical method showed the nuclear expression of Ku70 protein in the nomal control group, sham operation group and controlateral nonischemic hemisphere. The expression of Ku70 started to decrease at 0.5h of reperfusion in the caudate putamen and at 2h in the cortex (P<0.05), The number of Ku70 positive cells was decreased remarkably in the entire damaged area as early as 6
hours after reperfusion and remained reduced until 48 hours. IPC (MCAO 20min) or RSM (15g/kg i.p.) significantly suppressed the early reduction of Ku70 expression in ischemic cortex and caudate putamen, compared with controls, the number of Ku70 positive cells was significantly higher at different reperfusion time (P<0.01).
(3) There were no TUNEL positive cells in the nomal control group, sham operation group and controlateral nonischemic hemisphere. TUNEL positive cells begin to rise from 6h after reperfusion, reached peak at 24h and 48h (P<0.01). IPC (MCAO 20min) or RSM (15g/kg i.p.) reduced significantly TUNEL positive cells in ischemic cortex and caudate putamen at different reperfusion time after MCAO 2h (P<0.01).
(4) The number of Ku 70 positive cells was decreased remarkably in the entire damaged area as early as 6 hours after ischemia/reperfusion and neuronal apoptosis occurred after the reduction of Ku expression.
Our results demonstrate:
(1) Expression of DNA repair protein Ku70 decreased remarkably in the damaged regions at early stage of cerebral ischemia/reperfusion, which results in DNA DSB unrepaired or incompletely repaired, It may involve the mechanism of subsequent neuronal apoptosis.
(2) The neuroprotective mechanism of IPC is associated with upregulating expression or activity of DNA repair
本研究采用大脑中动脉线拴法建立大鼠局灶性脑缺血再灌注模型，利用HE染色方法、免疫组织化学方法和原位末端标记法(TUNEL法)，首先观察大脑中动脉阻塞(MCAO)2h再灌注0.5h、2h、6h、12h、24h、48h后缺血区额顶叶皮质和尾壳核DNA修复蛋白Ku70的表达及神经元凋亡情况，分析二者的变
第四军医大学硕士学位论文
化在时间上的关系;然后分别观察缺血预处理和中药丹参对脑缺
血再灌注后Ku7O表达的影响，初步探讨脑缺血再灌注早期
DNA修复酶Ku7O的表达变化规律及其意义，并从DNA损伤修
复的角度探讨缺血预处理和中药丹参对抗脑缺血再灌注损伤的分
子机制。
实验结果如下:
(l)大鼠大脑中动脉阻塞再灌注(MCAO服)后损伤区域主要
位于额叶皮质、顶叶皮质和尾壳核。
(2)正常对照组、假手术组及缺血再灌注对侧额顶叶皮质、尾
壳核可见Ku70广泛表达，定位于细胞核:MCAOZh、再灌注
0.5h，缺血侧尾壳核Ku7O阳性细胞数开始减少(P<0.05)，再
灌注Zh，额顶叶皮质Ku70阳性细胞数开始减少(尸<0.05)，
于再灌注6h，上述区域Ku70表达水平显著降低(P<0.01)，
并持续至再灌注48h;实施缺血预处理或给予腹腔注射丹参后，
明显抑制了Ku7O表达水平的降低，各时间点Ku7O阳性细胞数
均较相应对照组明显升高(尸<0.01)。
(3)正常对照组、假手术组及缺血再灌注对侧未见TUNEL阳性
细胞:‘MCAOZh、再灌注0.sh一2h，缺血区偶见散在的TUNEL阳
性细胞:再灌注6h，TUNEL阳性细胞数开始增多(P<0.01)，
随着再灌注时间的延长，阳性细胞数不断增高，再灌注24h和
48h，TUNEL阳性细胞数达到高峰(尸<0.01):实施缺血预处
理或给予丹参腹腔注射，明显抑制了脑缺血再灌注后细胞凋亡的
发生，各时间点TUNEL阳性细胞数均较相应对照组显著减少
(P<0 .01)。
(4) Ku蛋白在损伤区域内全面减少发生在缺血性神经元凋亡
高峰出现之前，二者呈负相关关系。
第四军医大学硕士学位论文
综上，本研究的初步结论如下:
(1)脑缺血再灌注早期，损伤区域Ku蛋白表达下降，可能
是导致DNA双链断裂无法得到修复、细胞随之死亡的机制之
(2)缺血预处理可能通过上调DNA修复蛋白Ku的表达或括
性而增强DNA修复能力，从而发挥其内源性神经保护作用。
(3)丹参可能通过抑制缺血再灌注后DNA修复蛋白Ku的减
少，保护机体自身DNA修复功能免遭破坏而发挥其神经保护作
用。
A large number of free radicals are produced in a burst-like manner at the early stage of cerebral ischemia/reperfusion and mainly cause oxidative DNA damage. Neuronal DNA damage caused by oxidative stress consistes of the lesion of base, DNA single strand breaks (SSB) and DNA double strand breaks (DSB). Oxidative DNA damage after ischemia / reperfusion can be repaired by DNA repair system, but continuous or severe DNA damage against DNA repair could induce neuronal death, such as necrosis or apoptosis. It has been reported that early decrease of DNA repair enzyme probably has correlation with subsequent DNA-damaged cell death after cerebral ischemia / reperfusion. As neuronal death was the basis of brain injury, researches on DNA damage and repair would probably reveal the mechanism of ischemic neuronal death, and will bring a new strategy for treating ischemic cerebrovascular diseases. It has been proved that both ischemic preconditioning (IPC) and radix salviae miltiorrhizae (RSM) have significant
neuroprotection against ischemic injury in the brain. A number of studies have been performed to clarify the protective effect and mechanism, however, whether IPC and RSM have any effects on the DNA damage and repair in the neuropathological process following cerebral ischemia/reperfusion still remains to be investigated.
In this study, we made the model of rat middle cerebral artery occlusion/ reperfusion and used H&E stain method, immuno-histochemical technology and TUNEL detection, in order to explore the relationship between the expression of Ku70 protein-which is critical to the repair of DNA DSB and neuronal death after focal cerebral ischemia/reperfusion, and the possible molecular mechanisms by which IPC and RSM protect brain. Firstly, we observed the expression of Ku70 protein and neuronal apoptosis in the ischemic cortex and caudatum after MCAO 2h reperfusion 0.5h, 2h, 6h, 12h, 24h, 48h, and evaluated the temporal relationship between Ku70 protein and neuronal apoptosis. Then we investigated the influence of IPC and RSM on the expression of Ku70 protein after focal cerebral ischemia/reperfusion.
Our results showed:
(1) HE stain indicated the model of rat MCAO/R was credible, and the damaged regions included cortex and caudate putamen.
(2) Immunohistochemical method showed the nuclear expression of Ku70 protein in the nomal control group, sham operation group and controlateral nonischemic hemisphere. The expression of Ku70 started to decrease at 0.5h of reperfusion in the caudate putamen and at 2h in the cortex (P<0.05), The number of Ku70 positive cells was decreased remarkably in the entire damaged area as early as 6
hours after reperfusion and remained reduced until 48 hours. IPC (MCAO 20min) or RSM (15g/kg i.p.) significantly suppressed the early reduction of Ku70 expression in ischemic cortex and caudate putamen, compared with controls, the number of Ku70 positive cells was significantly higher at different reperfusion time (P<0.01).
(3) There were no TUNEL positive cells in the nomal control group, sham operation group and controlateral nonischemic hemisphere. TUNEL positive cells begin to rise from 6h after reperfusion, reached peak at 24h and 48h (P<0.01). IPC (MCAO 20min) or RSM (15g/kg i.p.) reduced significantly TUNEL positive cells in ischemic cortex and caudate putamen at different reperfusion time after MCAO 2h (P<0.01).
(4) The number of Ku 70 positive cells was decreased remarkably in the entire damaged area as early as 6 hours after ischemia/reperfusion and neuronal apoptosis occurred after the reduction of Ku expression.
Our results demonstrate:
(1) Expression of DNA repair protein Ku70 decreased remarkably in the damaged regions at early stage of cerebral ischemia/reperfusion, which results in DNA DSB unrepaired or incompletely repaired, It may involve the mechanism of subsequent neuronal apoptosis.
(2) The neuroprotective mechanism of IPC is associated with upregulating expression or activity of DNA repair
引文
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54. Ramana CV, Boldogh I, Izumi T, Mitra S. Activation of apurinic/apyrimidinic endonuclease in human cells by reactive oxygen species and its correlation with their adaptive response
to genotoxicity of free radicals. Proc Natl Acad Sci U S A. 1998 Apr 28;95(9):5061-5066.
55. Fujimura M, Morita-Fujimura Y, Narasimhan P, Copin JC, Kawase M, Chan PH. Copper-zinc superoxide dismutase prevents the early decrease of apurinic/apyrimidinic endonuclease and subsequent DNA fragmentation after transient focal cerebral ischemia in mice. Stroke. 1999;30(11):2405-2415.
56. Shen MR, Zdzienicka MZ, Mohrenweiser H, Thompson LH, Thelen MP. Mutations in hamster single-strand break repair gene XRCC1 causing defective DNA repair. Nucleic Acids Res. 1995;26(4): 1032-1037.
57. Fujimura M, Morita-Fujimura Y, Sugawara T, Chan PH. Early decrease of XRCC1, a DNA base excision repair protein, may contribute to DNA fragmentation after transient focal cerebral ischemia in mice. Stroke. 1999;30(11):2456-2463.
58. Masson M, Niedergang C, Schreiber V, Muller S, Menissierde Murcia J, de Murcia G. XRCC1 is specifically associated with poly(ADP-ribose) polymerase and negatively regulates its activity following DNA damage. Mol Cell Biol. 1998; 18(6):3563-3571.
59. Wu A, Liu Y. Effects of deltamethrin on nitric oxide synthase and poly(ADP-ribose) polymerase in rat brain. Brain Res. 1999;850(1-2):249-252.
60. Szabo C, Zingarelli B, O'Connor M, Salzman AL. DNA strand breakage, activation of poly (ADP-ribose) synthetase, and cellular energy depletion are involved in the cytotoxicity of
macrophages and smooth muscle cells exposed to peroxynitrite. Proc Natl Acad Sci USA. 1996;93(5):1753-1758.
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67. Ferguson DO, Sekiguchi JM, Chang S, Frank KM, Gao Y, DePinho RA, Alt FW. The nonhomologous end-joining pathway of DNA repair is required for genomic stability and the suppression of translocations. Proc Natl Acad Sci USA. 2000;97(12):6630-6633.
68. Jackson SP. Sensing and repairing DNA double-strand breaks. Carcinogenesis. 2002;23(5):687-696.
69. Pastwa E, Blasiak J. Non-homologous DNA end joining. Acta Biochim Pol. 2003;50(4):891-908.
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71. Longa EZ, Weinstein PR, Carlson S, Cummins R. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke. 1989 Jan;20(1):84-91.
72. Chan PH. Reactive oxygen radicals in signaling and damage in the ischemic brain. J Cereb Blood Flow Metab. 2001; 21(1):2-14.
73. Love S. Oxidative stress in brain ischemia. Brain Pathol. 1999;9(1): 119-131.
74. Kim GW, Copin JC, Kawase M, Chen SF, Sato S, Gobbel GT, Chan PH. Excitotoxicity is required for induction of oxidative stress and apoptosis in mouse striatum by the mitochondrial toxin, 3-nitropropionic acid. J Cereb Blood Flow Metab. 2000;20(1):119-129.
75. Tamura T, Ishihara M, Lamphier MS, Tanaka N, Oishi I, Aizawa S, Matsuyama T, Mak TW, Taki S, Taniguchi T. An IRF-1-dependent pathway of DNA damage-induced apoptosis in mitogcn-activated T lymphocytes. Nature. 1995; 376(6541): 596-599.
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77. Dirnagl U, Simon RP, Hallenbeck JM. Ischemic tolerance and endogenous neuroprotection. Trends Neurosci. 2003; 26(5): 248-254.
78. Baek SH, Kim JY, Choi JH, Park EM, Han MY, Kim CH, Ahn YS, Park YM. Reduced glutathione oxidation ratio and 8 ohdG accumulation by mild ischemic pretreatment. Brain Res. 2000;856(1-2):28-36.
79. Kitagawa K, Matsumoto M, Tagaya M, Hata R, Ueda H, Niinobe M, Handa N, Fukunaga R, Kimura K, Mikoshiba K. 'Ischemic tolerance' phenomenon found in the brain. Brain Res. 1990;528(1):21-24.
80. Barone FC, White RF, Spera PA, Ellison J, Currie RW, Wang X, Feuerstein GZ. Ischemic preconditioning and brain tolerance: temporal histological and functional outcomes, protein synthesis requirement, and interleukin-1 receptor antagonist and early gene expression. Stroke. 1998;29(9): 1937-1951.
81. Chen J, Graham SH, Zhu RL, Simon RP. Stress proteins and tolerance to focal cerebral ischemia. J Cereb Blood Flow Metab. 1996;16(4):566-577.
82. Shimizu S, Nagayama T, Jin KL, Zhu L, Loeffert JE, Watkins SC, Graham SH, Simon RP. bcl-2 Antisense treatment prevents induction of tolerance to focal ischemia in the rat brain. J Cereb Blood Flow Metab. 2001;21(3):233-243.
83. Sugawara T, Noshita N, Lewen A, Kim GW, Chan PH. Neuronal expression of the DNA repair protein Ku 70 after
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85．刘军，匡培根．丹参对大鼠脑缺血再灌注损伤保护机制的实验研究．中国神经免疫学和神经病学杂志．1998；5(2)：77-82．
86. Kuang P, Zhou X, Xu B, et al. The protective effect of radix salviae miltiorrhizae composita in cerebral ischemia. J Tradit Chin Med. 1983;3(3): 193-198.
87．吴卫平，匡培根．脑缺血再灌注后脑组织C—fos基因表达与丹参的影响．临床神经科学．1995；3(2)：63-65．
88. Kuang P, Xiang J. Effect of RSM on EAA and IAA during cerebral ischemia in gerbils—a microdialysis study. J Tradit Chin Med. 1994;14(1):45-50.
89. Wu W, Kuang P, Li Z. Protective effect of radix salviae miltiorrhizae on apoptosis of neurones during focal cerebral ischemia and reperfusion injury. J Tradit Chin Med. 1997; 17(3):220-225.
90．张金涛，李义召，赵书平，王树新，王利群，刘赛男．脑缺血再灌注后ICE、Bcl-2的表达及丹参的神经保护作用研究．卒中与神经疾病，2001；8(1)：26-28．
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52. Kawase M, Fujimura M, Modta-Fujimura Y, Chan PH. Reduction of apurinic/apyrimidinic endonuclease expression after transient global cerebral ischemia in rats: implication of the failure of DNA repair in neuronal apoptosis. Stroke. 1999;30(2):441-449.
53. Walton M, Lawlor P, Sirimanne E, Williams C, Gluckman P, Dragunow M. Loss of Ref-1 protein expression precedes DNA fragmentation in apoptotic neurons. Brain Res Mol Brain Res. 1997;44(1): 167-170.
54. Ramana CV, Boldogh I, Izumi T, Mitra S. Activation of apurinic/apyrimidinic endonuclease in human cells by reactive oxygen species and its correlation with their adaptive response
to genotoxicity of free radicals. Proc Natl Acad Sci U S A. 1998 Apr 28;95(9):5061-5066.
55. Fujimura M, Morita-Fujimura Y, Narasimhan P, Copin JC, Kawase M, Chan PH. Copper-zinc superoxide dismutase prevents the early decrease of apurinic/apyrimidinic endonuclease and subsequent DNA fragmentation after transient focal cerebral ischemia in mice. Stroke. 1999;30(11):2405-2415.
56. Shen MR, Zdzienicka MZ, Mohrenweiser H, Thompson LH, Thelen MP. Mutations in hamster single-strand break repair gene XRCC1 causing defective DNA repair. Nucleic Acids Res. 1995;26(4): 1032-1037.
57. Fujimura M, Morita-Fujimura Y, Sugawara T, Chan PH. Early decrease of XRCC1, a DNA base excision repair protein, may contribute to DNA fragmentation after transient focal cerebral ischemia in mice. Stroke. 1999;30(11):2456-2463.
58. Masson M, Niedergang C, Schreiber V, Muller S, Menissierde Murcia J, de Murcia G. XRCC1 is specifically associated with poly(ADP-ribose) polymerase and negatively regulates its activity following DNA damage. Mol Cell Biol. 1998; 18(6):3563-3571.
59. Wu A, Liu Y. Effects of deltamethrin on nitric oxide synthase and poly(ADP-ribose) polymerase in rat brain. Brain Res. 1999;850(1-2):249-252.
60. Szabo C, Zingarelli B, O'Connor M, Salzman AL. DNA strand breakage, activation of poly (ADP-ribose) synthetase, and cellular energy depletion are involved in the cytotoxicity of
macrophages and smooth muscle cells exposed to peroxynitrite. Proc Natl Acad Sci USA. 1996;93(5):1753-1758.
61. Tsurimoto T. PCNA binding proteins. Front Biosci. 1999; 4:D849-858.
62. Tomasevic G, Kamme F, Wieloch T. Changes in proliferating cell nuclear antigen, a protein involved in DNA repair, in vulnerable hippocampal neurons following global cerebral ischemia. Brain Res Mol Brain Res. 1998;60(2): 168-176.
63. Chen J, Uchimura K, Stetler RA, Zhu RL, Nakayama M, Jin K, Graham SH, Simon RP. Transient global ischemia triggers expression of the DNA damage-inducible gene GADD45 in the rat brain. J Cereb Blood Flow Metab. 1998;18(6):646-657.
64. Charriaut-Marlangue C, Richard E, Ben-Ari Y. DNA damage and DNA damage-inducible protein Gadd45 following ischemia in the P7 neonatal rat. Brain Res Dev Brain Res. 1999;116(2):133-140.
65. Featherstone C, Jackson SP. Ku, a DNA repair protein with multiple cellular functions? Murat Res. 1999;434(1):3-15.
66. Difilippantonio M J, Zhu J, Chen HT, Meffre E, Nussenzweig MC, Max EE, Ried T, Nussenzweig A. DNA repair protein Ku80 suppresses chromosomal aberrations and malignant transformation. Nature. 2000;404(6777):510-514.
67. Ferguson DO, Sekiguchi JM, Chang S, Frank KM, Gao Y, DePinho RA, Alt FW. The nonhomologous end-joining pathway of DNA repair is required for genomic stability and the suppression of translocations. Proc Natl Acad Sci USA. 2000;97(12):6630-6633.
68. Jackson SP. Sensing and repairing DNA double-strand breaks. Carcinogenesis. 2002;23(5):687-696.
69. Pastwa E, Blasiak J. Non-homologous DNA end joining. Acta Biochim Pol. 2003;50(4):891-908.
70. Shackelford DA, Tobaru T, Zhang S, Zivin JA. Changes in expression of the DNA repair protein complex DNAdependent protein kinase after ischemia and reperfusion. J Neurosci. 1999; 19(12):4727-4738.
71. Longa EZ, Weinstein PR, Carlson S, Cummins R. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke. 1989 Jan;20(1):84-91.
72. Chan PH. Reactive oxygen radicals in signaling and damage in the ischemic brain. J Cereb Blood Flow Metab. 2001; 21(1):2-14.
73. Love S. Oxidative stress in brain ischemia. Brain Pathol. 1999;9(1): 119-131.
74. Kim GW, Copin JC, Kawase M, Chen SF, Sato S, Gobbel GT, Chan PH. Excitotoxicity is required for induction of oxidative stress and apoptosis in mouse striatum by the mitochondrial toxin, 3-nitropropionic acid. J Cereb Blood Flow Metab. 2000;20(1):119-129.
75. Tamura T, Ishihara M, Lamphier MS, Tanaka N, Oishi I, Aizawa S, Matsuyama T, Mak TW, Taki S, Taniguchi T. An IRF-1-dependent pathway of DNA damage-induced apoptosis in mitogcn-activated T lymphocytes. Nature. 1995; 376(6541): 596-599.
76. Gao Y, Ferguson DO, Xie W, Manis JP, Sekiguchi J, Frank KM, Chaudhuri J, Horner J, DePinho RA, Alt FW. Interplay of p53
and DNA-repair protein XRCC4 in tumorigenesis, genomic stability and development. Nature. 2000;404(6780):897-900.
77. Dirnagl U, Simon RP, Hallenbeck JM. Ischemic tolerance and endogenous neuroprotection. Trends Neurosci. 2003; 26(5): 248-254.
78. Baek SH, Kim JY, Choi JH, Park EM, Han MY, Kim CH, Ahn YS, Park YM. Reduced glutathione oxidation ratio and 8 ohdG accumulation by mild ischemic pretreatment. Brain Res. 2000;856(1-2):28-36.
79. Kitagawa K, Matsumoto M, Tagaya M, Hata R, Ueda H, Niinobe M, Handa N, Fukunaga R, Kimura K, Mikoshiba K. 'Ischemic tolerance' phenomenon found in the brain. Brain Res. 1990;528(1):21-24.
80. Barone FC, White RF, Spera PA, Ellison J, Currie RW, Wang X, Feuerstein GZ. Ischemic preconditioning and brain tolerance: temporal histological and functional outcomes, protein synthesis requirement, and interleukin-1 receptor antagonist and early gene expression. Stroke. 1998;29(9): 1937-1951.
81. Chen J, Graham SH, Zhu RL, Simon RP. Stress proteins and tolerance to focal cerebral ischemia. J Cereb Blood Flow Metab. 1996;16(4):566-577.
82. Shimizu S, Nagayama T, Jin KL, Zhu L, Loeffert JE, Watkins SC, Graham SH, Simon RP. bcl-2 Antisense treatment prevents induction of tolerance to focal ischemia in the rat brain. J Cereb Blood Flow Metab. 2001;21(3):233-243.
83. Sugawara T, Noshita N, Lewen A, Kim GW, Chan PH. Neuronal expression of the DNA repair protein Ku 70 after
ischemic preconditioning corresponds to tolerance to global cerebral ischemia. Stroke. 2001;32(10):2388-2393.
84. Ramana CV, Boldogh I, Izumi T, Mitra S. Activation of apurinic/apyrimidinic endonuclease in human cells by reactive oxygen species and its correlation with their adaptive response to genotoxicity of free radicals. Proc Natl Acad Sci USA. 1998;95(9):5061-5066.
85．刘军，匡培根．丹参对大鼠脑缺血再灌注损伤保护机制的实验研究．中国神经免疫学和神经病学杂志．1998；5(2)：77-82．
86. Kuang P, Zhou X, Xu B, et al. The protective effect of radix salviae miltiorrhizae composita in cerebral ischemia. J Tradit Chin Med. 1983;3(3): 193-198.
87．吴卫平，匡培根．脑缺血再灌注后脑组织C—fos基因表达与丹参的影响．临床神经科学．1995；3(2)：63-65．
88. Kuang P, Xiang J. Effect of RSM on EAA and IAA during cerebral ischemia in gerbils—a microdialysis study. J Tradit Chin Med. 1994;14(1):45-50.
89. Wu W, Kuang P, Li Z. Protective effect of radix salviae miltiorrhizae on apoptosis of neurones during focal cerebral ischemia and reperfusion injury. J Tradit Chin Med. 1997; 17(3):220-225.
90．张金涛，李义召，赵书平，王树新，王利群，刘赛男．脑缺血再灌注后ICE、Bcl-2的表达及丹参的神经保护作用研究．卒中与神经疾病，2001；8(1)：26-28．