摘要
目的 观察多聚ADP-核糖聚合酶(PARP)与钙依赖性蛋白激酶(Calpain)在脑缺血再灌流损伤中的表达以及凋亡的形态学特征，探讨PARP和Calpain在细胞凋亡中的作用。
方法 成年健康雌性Wistar36只大鼠，应用线栓法建立大脑中动脉闭塞再灌流模型(MCAO)。随机分为假手术组和实验组。用原位TUNEL和原位杂交技术探求脑缺血后细胞凋亡发生的意义和动力学各方面的变化以及PARP和Calpain在大鼠脑缺血再灌流不同时间点的表达。
结果 缺血再灌流组凋亡细胞主要位于缺血周围区，坏死细胞主要集中于缺血中心区，再灌流2h即出现凋亡细胞，随着再灌流时间的延长逐渐增多，至24h达高峰，2d开始下降，至14d时仍高于假手术组，各相邻时间点比较差异显著(P＜0.05)。缺血中心区凋亡细胞数量较少，其变化规律与缺血周围区相似，除再灌流2h，7d，14d外，其余各时间点无显著性差异(P＞0.05)。PARP的表达在脑缺血再灌流2h、6h、12h、2d、3d、7d后神经元数皮质区与假手术组比较，相差非常显著(P＜0.01)。再灌流后2h明显升高，6h达高峰，然后下降，14d降至最低点，与假手术组相比也有统计学意义(p＜0.05)。纹状体与假手术组比较，再灌流2h、6h、12h、2d、3d、7d相差非常显著(P＜0.01)。再灌流后2h已达高峰，然后逐渐下降，14d降至最低点，不过与假手术组相比仍有统计学意义(p＜0.05)Calpain的表达在脑缺血1h再灌流2h、6h、12h、2d、3d、7d后神经元数在皮质区与假手术组比较，相差非常显著(P＜0.01)。再灌流后2h已达高峰，6h下降，12h又明显升高，然后逐渐下降，14d降至最低点。但14d与假手术组相比，有统计学意义(p＜0.05)。纹状体与假手术组比较，相差非常显著(P＜0.01)．再灌流后2h明显升高，6h反而下降，12h又升高至高峰，然后逐渐下降，14d降至最低点。但14d与假手术组相比，有统计学意义(p＜0.05)。
结论 脑缺血后细胞凋亡主要位于缺血周围区，PARP与Calpain在脑缺血细胞凋亡中起重要的作用。PARP与Calpain抑制剂对缺血性脑损伤的防治具有非常重要的意义。
Aim The study is to explore the expression of poly (ADP-ribose) polymerase (PARP) and Calpain in focal cerebral ischemia with reperfusion injury and observe morphological features of apoptotic cells and the role of PARP and Calpain in apoptosis.
Methods 36 adult female Wistar rats were subjected to middle cerebral artery occlusion (MCAO) and different time of reperfiision. Rats were randomly divided into MCAO groups and sham group that were same as operation, but no MCAO. To explore the implication and the kinetics of such event in apoptosis induced insult induced by MCAO in situ TUNEL technic. To observe the expression of PARP and Calpain at the different time points and then assess their function in apoptosis induced by MCAO in situ hybridization technic
Results Apoptotic cells lie in the boundary zone to the ischemic core. The number of apoptotic cells in the ischemic core was less than ischemic penumbra (p<0.05) except for 2 h, 7 d, 14 d. Situ hybridization demonstrated the evident expression of PARP at 2 h. The peak of PARP was first detected at 2 h in the corpus striatum and then cerebral cortex at 6 h . With increasing length of survival, the expression of PARP began to decrease, then dropped a very low level at 14 d. But there are difference between MCAO group and sham control (p<0.05). Situ hybridization demonstrated the evident expression of Calpain in the cerebral cortex and corpus striatum at 2 h. The study was detected a bimodal pattern of Calpain with an initial increase at 2 h followed by a more prominent secondary increase at 12 h after reperfusion. After 24 h Calpain began to decrease, then dropped a very low level at 14 d. But there are difference between MCAO group and sham control (p<0.05).
Conclusion Apoptosis lie in the boundary zone to the ischemic core. PARP and Calpain play an important role in apoptosis. The inhibition of PARP and Calpain can prevent and cure cerebral ischemic damage.
引文
1 李敬诚，董为伟。脑缺血后细胞凋亡及其基因研究 脑血管疾病基础与临床研究进展 重庆，重庆大学出版社，1998：18～31
2 Chopp M, Li y, Powers C, et al. Apoptosis and expression after focal cerebral ischemia in rats. [J]Cereb Blood Flow Metab, 1997, 17 (Supply 1):S253
3 杨其伟。降低PARP酶活性对外源基因稳定整合的影响。遗传，1997，05．20；19(3)
4 Eguchi Y, Shimizu S, Tsujimoto Y. Intracellular ATP levels determine cell death fate by apoptosis or necrosis [J]. Cancer Res, 1997,57(100): 1835-1840.
5 Bartus RT, Dean RL, Mennerick S, et al. Temporal ordering of pathogenic events following transient global ischemia. Brain Res 1998, Apr 20; 790(1-2): 1-13
6 Cookson MR, Inc PG, Usher PA, et al. Poly ADP-ribose) polymerase is found in both the nucleus and cytoplasm of human CNS neurons. [J].Brain Res, 1999,834(1-2): 182-185
7 Aoufouchi S, Shall S. Regulation by phosphorylation of Xenopus laevis poly (ADP-ribose) polymerase enzyme activity during oocytc maturation. Biochem [J], 1997,325:543-551.
8 Kato N,Morita H,Sugiyama T, et al. Evalrattion of the poly(ADP-ribose) polymerase gene in human stroke.Atherosclerosis, 1999,148(2):345-352
9 Szabo C, Dawson VL. Role of poly(ADP-Ribose) synthetase in inflammation and ischemia-reperfusion. Trends pharmacol Sci, 1998,19(7):287-298.
10 Davidovic L,Vodenicharov M,Affar EB. et al. Importance of poly(ADP-ribose)glycohydrolase in the control of poly (ADP-ribose)metabolism. Exp Cell Res, 2001,268(1):7-13.
11 Bums TF, Bernhard EJ,EI-Deiry WS.Tissre specific expression of p53 target genes suggests a key role for KILLER/DR5 in p53-dependent apoptosis in vivo.Oncogene,2000,113 (pt6):955-961.
12 KimJ W, Won J, SohnS, et al. DNA-binding activity of the N-terminal cleavage product of poly (ADP-ribose)polymerase is required for UV mediated apoptosis. J of cell sci, 2000,113(pt6):955-961.
13 Le Rhun Y. Celluar responses to DNA damage in the absence of Poly(ADP-ribose) polymerase.Biochem Biophys. Res Commun, 1998,245:1-10.
14 Griffin RJ.The role of inhibitors of Poly(ADP-ribose)polymerase as resistance
modifying agents in cancer therapy.Biochimie,1995,77:408-422.
15 Jackson SP. The recognition of DNA damage. Curr Opin Genet Dev,1996,6:19-25
16 毛力真。．PARP与缺血性神经元损伤后DNA的修复。中国临床神经科学。1999，4(7)：246-248
17 罗玉敏，Chen Jun，秦震．脑缺血与DNA损伤。国外医学脑血管疾病分册。2002，5(8)：259-261
18 Bonfoco E, Krainc D, Ankarcrona M, et al. Apoptosis and necrosis: two distinct events induced, respectively, by mild and intense insults with N -methyl-D-aspartate or nitric oxide superoxide in cortical cell cultures. Proc Natl Acad Sci USA, 1995, 92: 7162-7166.
19 Tokime T, Nozaki K, Sugino T, et al. Enhanced poly(ADP-dbosyl) ation after focal ischemia in rat brain. J Cereb Blood Flow Metab 1998 Sep; 18(9): 991-7
20 Zhang J, Dawson TM, SNYDER sh. Nitric oxide activation of poly (ADP-dbose) synthetase in neurotoxiciy [J]. Science, 1994, 263(514):687-689.
21 Yang J, Klaidman L, Chang M, Kem S, et al. Nicotinamide therapy protects against both necrosis and apoptosis in a stroke model.Pharmacol Biochem Behav 2002 Nov;73(4):901
22 Ou BR, Forsberg NE. Determination of skeletal muscle, calpain and calpainstatin activities during maturation. Am [J] Physiol,1991,261(6):677-683
23 肖小华，黄如训．卡配因及其抑制剂在脑缺血中的作用．国外医学脑血管疾病分册 1999，4(7)：195-196
24 邓志宽，董为伟．卡配因与缺血性脑损伤．国外医学脑血管疾病分册 2002，3(10)：223-224
25 Bartus RT, Baker KL, HeisterAD, et al. Postischemia akministration of AK275,a calpain inhibitor, provides substantial protection against focal ischemic brain damage. [J] Cereb Blood Flow Metab,1994,14:537-544
26 Neumar RW, Meng FH, Mills AM,et al.Calpain activity in the rat brain after transient forebrain ischemia. Exp Neurol 2001 ,Jul; 170(1):27-35
27 Markgraf,-C-G; Velayo,-N-L; Johnson,-M-P et al. Six-hour window of opportunity for calpain inhibition in focal cerebral ischemia in rats.Stroke.1998,29(1):152-8
28 Robinson A.. Inhibiting calpain, rescuing cells. Can Med Assoc J, 1996, 154(2): 193-195
29 Rheaume E. The large subunit of replication factor C is a substrate for Caspase-3 in vitro and is cleaved by a Caspase-3-1ike protease during Fas-mediated apoptosis.
EMBO J, 1997,16(21): 6346.
30 Schulze-osthoff K, Ferrari D, Los M, et al. Apoptosis signaling by death receptors. Eur J Biochem, 1998, 254(3): 439-459.
31 Bergrnarm A, Agapite J, Steller H. Mechanism and control of programmed cell death in invertebrates. Oncogene, 1998, 17(25): 3215-3223.
32 Hara H, Friedlander RM, Gagliardini V, et al. Inhibition ofinterleukin 1β converting enzyme family proteases ischemic and excitotoxic neuroal damage. Proc Natl Acad Sci U S A, 1997, 94(5): 2007-2012.
33 Himi T, Ishizaki Y, Murota S. A caspase inhibitor blocks ischemiainduced delayed neuronal death in the gerbil. Eur J Neurosci, 1998, 10(2): 777-781.
34 Binienda Z. A fetal rat model of acute perinatal ischemic-hypoxia. Ann N Y Acad Sci, 1995, 72(1): 28-38.
35 Zhong LT, Sarafian T, Kane DJ, et al. Bcl-2 inhibits death of central neural cells induced by multiple agents. Proc Natl Cad Sci USA, 1993, 90(10): 4533-4537.
36 Vaux DL. Toward an understanding of the molecular mechanisms of physiological cell death. Proc Natl Acad Sci USA, 1993, 90(3):786-789.
37 Reed J C. Bcl-2 family proteins and mitochondria. Biochim Biophys Acta, 1998, 1366(1-2): 127-137.
38 Oltvai Z N, Milliman C L, Korsmeyer S J. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death, Cell, 1993, 74(4): 609-619.
39 Chao D T, Korsmeyer S J. Bcl-2 family: Ragulators of cell death. Ann Rev Immun, 1998,16(2): 395-419.
40 Carlos PC, Donald LP, Lee JM. Excitoxic neuronal death in the immature brain is an apoptosis-necrosis morphological continuum. J Comp Neurol, 1997,387(1): 70-87.