摘要
背景和目的
脑出血(ICH)是一种威胁人类健康的急性疾病。许多研究认为：细胞凋亡参与了神经元的继发性损伤,细胞凋亡是脑出血后神经元损伤的重要因素之一。与非糖尿病患者相比；患糖尿病的脑出血患者在住院期间病情以及预后的状况都相对较差。高血糖可能会促进神经元的凋亡并且已经在缺血性脑血管病中得到证实,而目前这方面在脑出血中的研究相对较少。
本实验通过建立高血糖大鼠脑出血动物模型,利用免疫组织化学和TUNEL方法,观察血肿周围脑组织Bax、Bcl-2的表达和神经细胞的凋亡；探讨高血糖对血肿周围脑组织Bax、Bcl-2表达和神经细胞凋亡的影响,为脑出血的临床治疗提供理论依据。
材料与方法
健康成年雄性SD大鼠96只,随机分为4.组：假手术组、正常血糖组、高血糖组以及胰岛素干预组。每组设4个时间点：6h、24h、72h、7d。
通过腹腔注射链脲菌素(STZ)制作高血糖动物模型,血糖值大>11.1mmol/L,认为该模型制作成功。
在立体定向仪下于右侧尾状核区注入未凝自体动脉血建立脑出血动物模型。
组织切片的制备：成功模型在相应时间点灌注、取材,以注射针道为中心做冠状切片。
指标检测：Bax、Bcl-2的检测是采用免疫组织化学染色。应用图像分析系统测其免疫染色阳性区平均积分光密度,取其平均值；神经细胞凋亡采用TUNEL法染色。凋亡指数(AI)=凋亡神经元/总神经元×100%。
统计学方法：所有数据均用均数±标准差(x±s)表示,采用SPSS13.0软件对数据进行统计分析,多组均数间的比较采用单因素方差分析,均数间的两两比较采用LSD检验,Bax与TUNEL的相关性采用直线相关分析,检验水准a=0.05。
结果
1 Bax和Bcl-2表达的动态变化
在假手术组纹状体Bax和Bcl-2有少量表达,从6h至7d变化相对较小。在正常血糖组、高血糖组以及胰岛素干预组血肿周围脑组织不同时间点均有Bax和Bcl-2的表达,且从6h逐渐增高,24h达峰,7d仍有表达。高血糖组大鼠脑血肿周围组织Bax和Bcl-2的表达在相应时间点明显高于其它组(P<0.05),胰岛素干预组上述指标在相应时间点均低于高血糖组(P<0.05),而高于假手术组,与脑出血组无明显差别(P>0.05)。
2神经细胞凋亡的动态变化
在假手术组纹状体仅见少量的凋亡细胞,从6h至7d变化相对较小。在正常血糖组、高血糖组以及胰岛素干预组血肿周围脑组织不同时间点均有凋亡细胞的表达,且从6h逐渐增高,24h达峰,7d仍有持续存在。高血糖组大鼠脑血肿周围组织凋亡细胞在相应时间点明显明显高于其它组(P<0.05),胰岛素干预组上述指标在相应时间点均低于高血糖组(P<0.05),而高于假手术组,与脑出血组无明显差别(P>0.05)。
3血肿周围细胞凋亡与Bax表达的相关性结果
高血糖组同一时间点的凋亡指数与Bax表达呈正相关性(r=0.80,P<0.05)。
结论
1.高血糖能促进大鼠脑血肿周围组织Bax、Bcl-2的表达以及神经元的凋亡。
2.高血糖组的细胞凋亡与Bax表达呈正相关性。
3.胰岛素适当控制血糖可以减少神经元的凋亡
Background and Objective
Intracerebral hemorrhage (ICH) is an emergent disease. At present, there are many research results reveal that cell apoptosis participates in the secondary injury of neurocytes. So cell apoptosis is an important factor of neurocytes injury of intracerebral hemorrhage. Compared with non-diabetics, diabetics have a worse hospital and long-term outcome after intracerebral hemorrhage. Hyperglycemia may promote the apoptotosis of neurons, which has been verified in the ischemic cerebrovascular disease. At present, it was researched rarely in the ICH.
In the study, using immunohistochemistry and TUNEL methods, the neuron apoptosis and the expression of Bax and Bcl-2 were explored in rat ICH model and hyperglycemia model, which may provide strategies for clinical treatment on cerebral hemorrhage.
Materials and methods
Ninety-six healthy adult male SD rats were randomly divided into 4 groups: sham group, normal plasma glucose group, hyperglycemia group and insulin intervention group. Each group was divided into 4 various stages:6h,24h,3d,7d.
Hyperglycemia model was induced by the injection of streptozotocin (STZ) into abdominal cavity by injection syringe. The model was successful until blood glucose was higher thanl1.1mmol/L.
ICH model was induced by the injection of autologous unaggregated artery blood into the right basal ganglia of rats by stereotaxic apparatus.
Preparation of the tissue section:The successful models were perfused and took tissue at the according time. The tissue was taken coronary sections before and behind the pin hole.
Examine parameter:Bax and Bcl-2 were studied with immunohistochemistry. Positive area average integral optical density value was measured with image analysis system, its mean value was average integral photodensity. Nerve cell apoptosis was measured by terminal-deoxynucleotidyltransferase mediated nick end label (TUNEL) method. Apoptotic index (AI)=apoptotic neurons/total neurons×100 %.
Statistic analysis:All data were expressed as mean±SD, and analyzed with the SPSS13.0 software, the means of the samples were compared using one-way analysis of variance followed by Least Significant Difference (LSD) for multiple comparisions. Correlation between Bax and TUNEL was judged by linear regression. The significant difference was judged by a=0.05.
Results
1. The dynamic changes of Bax and Bcl-2
The expression of Bax and Bcl-2 in the striatum of sham group rats was low and changed a little from 6h to 7d. The expression of Bax and Bcl-2 in the perihematomal areas at different time points after ICH in ICH group, hyperglycemia group and insulin intervention group began at 6h and increased gradually, ultimate in 24h, still can be detected at 7d. The expression of Bax and Bcl-2 in the hyperglycemia group was significantly higher than that in the other groups at the same time point (P<0.05), the above-mentioned index of insulin intervention group were lower than that in hyperglycemia group and higher than that in the sham group at the same time point (P<0.05), and had no obvious difference with that in the normal glucose group at the same time point (P>0.05).
2. The dynamic changes of neuron apoptosis
Apoptotic neurons were few in the striatum of of sham group rats, and AI had small change from 6h to 7d. AI in the perihematomal areas at different time points after ICH in ICH group, hyperglycemia group and insulin intervention group began at 6h and increased gradually, ultimate in 24h, still can be detected at 7d. AI in the hyperglycemia group was significantly higher than that in the other groups at the same time point (P<0.05), the above-mentioned index of insulin intervention group were lower than that in hyperglycemia group and higher than that in the sham group at the same time point (P<0.05), and had no obvious difference with that in the normal glucose group at the same time point (P>0.05).
3. The result of the relationship analysis of apoptotic and expression of Bax
AI positively correlates with the expression of Bax in hyperglycemia group at the same time point(r=0.80, P<0.05).
Conclusion
1. Hyperglycemia increases the apoptotic neurons and stimulates the expression of Bax and Bcl-2 in perifocal tissue of intracerebral hemorrhage in rats.
2. Apoptotic index positively correlates with the expression of Bax in hyperglycemia group at the same time point.
3. Control of blood glucose level with insulin can reduce neuron apoptosis.
引文
[1]Kimura K, Iguchi Y, Inoue T, et al. Hyperglycemia independently increases the risk of early death in acute spontaneous intracerebral hemorrhage [J]. J Neurol Sci,2007,263(1-2): 228-229.
[2]Xue Mengzhou, Mare R, Del Bigio. Intracerebral injection of autologous whole blood in rats:time course of inflammation and death [J]. Neuroscience Letters,2000,283(3): 230-232.
[3]Bederson JB, Pitts LH, Tsuji M, et al. Rat middle cerebral artery occlusion:Evaluation of the model and development of a neurologic examination [J]. Stroke,1986,17(3):472-476.
[4]Sinar EJ, Mendelow AD, Graham DI, et al. Experimental intracerebral hemorrhage: effects of a temporary mass lesion [J]. J Neurosurg,1987,66(4):568-576.
[5]Rosenberg GA, Mun-Bryce S, Wesley M, et al. Collagenase-induced intracerebral hemorrhage in rats [J]. Stroke,1990,21(5):801-807.
[6]Saini KS, Thompson C, Winterford CM, et al. Streptozotocin at low doses induces apoptosis and at high doses causes necrosis in a murine pancreatic beta cell line, INS-1 [J]. Biochem Mol Biol Int,1996,39(6):1229-1236.
[7]Johnson EM Jr, Greenlund LJ, Akins PT, et al. Neuronal apoptosis:Current understanding of molecular mechanisms and potential role in ischemic brain injury [J]. J Neurotrauma, 1995,12(5):843-852.
[8]李玉林主编.病理学[M].北京：人民卫生出版社.2008,17.
[9]李红玲,葛艳萍,郭艳苏,等.实验性脑出血血肿周围组织细胞凋亡的研究[J].中国康复理论与实践,2008,14(3)：206-208.
[10]Xue M, Del Bigio MR. Intracortical hemorrhage injury in rats:relationship between blood fractions and brain cell death [J]. Stroke,2000,31(7):1721-1727.
[11]Matsushita K, Meng W, Wang X, et al. Evidence for apoptosis after intercerebral hemorrhage in rat striatum [J]. J Cereb Blood Flow Metab,2000,20(2):396-404.
[12]Holmin S, Mathiesen T. Intracerebral administration of interleukin-lbeta and induction of inflammation, apoptosis, and vasogenic edema [J]. J Neurosurg,2000,92(1):108-120.
[13]Mayne M, Ni W, Yan HJ, et al. Antisense oligodeoxynucleotide inhibition of tumor necrosis factor-alpha expression is neuroprotective after intracerebral hemorrhage [J]. Stroke,2001,32(1):240-248.
[14]Vaughan PJ, Pike CJ, Cotman CW, et al. Thrombin receptor activation protects neurons and astrocytes from cell death produced by environmental insults [J]. J Neurosci,1995, 15(7 Pt 2):5389-5401.
[15]Xue M, Del Bigio MR. Acute tissue damage after injections of thrombin and plasmin into rat striatum [J]. Stroke,2001,32(9):2164-2169.
[16]Goldstein L, Teng ZP, Zeserson E, et al. Hemin induces an iron-dependent, oxidative injury to human neuron-like cells [J]. J Neurosci Res,2003,73(1):113-121.
[17]Hua Y, Xi G, Keep RF, et al. Complement activation in the brain after experimental intracerebral hemorrhage [J]. J Neurosurg,2000,92(6):1016-1022.
[18]Lavine SD, Hofman FM, Zlokovic BV. Circulating antibody against tumor necrosis factor-alpha protects rat brain from reperfusion injury [J]. J Cereb Blood Flow Metab, 1998,18(1):52-58.
[19]Tsujimoto Y, Finger LR, Yunis J, et al. Cloning of the chromosome breakpoint of neoplastic B cells with the t (14; 18) chromosome translocation [J]. Science,1984, 226(4678):1097-1099.
[20]Apte SS, Mattei MG, Olsen BR. Mapping of the human Bax gene to chromosome 19q13.3-q13.4 and isolation of a novel alternatively spliced transcript, BAX delta [J]. Genomics,1995,26(3):592-594.
[21]Kuwana T, Newmeyer DD. Bcl-2-family proteins and the role of mitochondria in apoptosis [J]. Curr Opin Cell Biol,2003,15(6):691-699.
[22]Cai J, Yang J, Jones DP. Mitochondrial control of apoptosis:the role of cytochrome c [J]. Biochim Biophys Acta,1998,1366(1-2):139-149.
[23]Gajkowska B, Wojewodzka U, Gajda J. Translocation of Bax and Bid to mitochondria, endoplasmic reticulum and nuclear envelope:possible control points in apoptosis [J]. J Mol Histol,2004,35(1):11-19.
[24]Simmen T, Aslan JE, Blagoveshchenskaya AD. PACS-2 controls endoplasmic reticulum-mitochondria communication and Bid-mediated apoptosis [J]. EMBO J,2005, 24(4):717-729.
[25]朱玉山,陈佺.线粒体与细胞凋亡调控[J].生命科学,2008,20(4)：506-513.
[26]Oltvai ZN, MIlliaman CL, Korsmcyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates cell death [J]. Cell,1993,74(4):609-619.
[27]Wu C, Fujihara H, Yao J, et al. Different expression patterns of Bcl-2, Bcl-xl, and Bax proteins after sublethal forebrain ischemin in C57Black/Crj6 mouse straitum [J]. Stroke, 2003,39(7):1803-1808.
[28]Zha J, Harada H, Yang E, et al. Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-X(L) [J]. Cell,1996,87(4): 619-628.
[29]Berridge MJ, Lipp P, Bootman MD. The versatility and universality of calcium signaling [J]. Nat Rev Mol Cell Biol,2000,1(1):11-21.
[30]Pinton P, Ferrari D, Rapizzi E, et al. The Ca2+ concentration of the endoplasmic reticulum is a key determinant of ceramide-induced apoptosis:significance for the molecular mechanism of Bcl-2 action [J]. EMBO J,2001,20(11):2690-2701.
[31]Murphy AN, Bredesen DE, Cortopassi G, et al. Bcl-2 potentiates the maximal calcium uptake capacity of neural cell mitochondria [J]. Proc Natl Acad Sci,1996,93(18): 9893-9898.
[32]Kluck RM, Bossy-Wetzel E, Green DR, et al. The release of cytochrome c from mitochondria:a primary site for Bcl-2 regulation of apoptosis [J]. Science,1997, 275(5303):1132-1136.
[33]Yang J, Liu X, et al. Bhalla K, et al. Prevention of apoptosis by Bcl-2:release of cytochrome c from mitochondria [J]. Science,1997,275(5303):1129-1132.
[34]Chinnaiyan AM, O'Rourke K, et al. Interaction of CED-4 with CED-3 and CED-9:a molecular framework for cell death [J]. Science,1997,275(5303):1122-1126.
[35]Fogelholm R, Murros K, Rissanen A, et al. Long term survival after primary intracerebral haemorrhage:a retrospective population based study [J]. J Neurol Neurosurg Psychiatry, 2005,76(11):1534-1538.
[36]Stollberger C, Exner I, Finsterer J, et al. Stroke in diabetic and non-diabetic patients: course and prognostic value of admission serum glucose [J]. Ann Med,2005,37(5): 357-364.
[37]Gong C, Hoff JT, Keep RF. Acute inflammatory reaction following experimental intracerebral hemorrhage in rat [J]. Brain Res, Gong C,871(13):57-65.
[38]Esposito K, Marfella R, Giugliano D. Stress hyperglycemia, inflammation, and cardiovascular events [J]. Diabetes Care,2003,26(5):1650-1651.
[39]陆士奇,程晓娟,易晓净,等.蛛网膜下腔出血继发脑血管痉挛患者凝血.纤溶的动态变化研究[J].中国急救医学,2005,25：720-721.
[1]Heiss WD, Kracht LW, Thiel A, et al. Penumbral probability thresholds of cortical flumazenil binding and blood flow predicting tissue outcome in patients with cerebral ischaemia [J]. Brain,2001,124(Pt 1):20-29.
[2]Carhuapoma JR, Wang PY, Beauchamp NJ, et al. Diffusion-weighted MRI and proton MR spectroscopic imaging in the study of secondary neuronal injury after intracerebral hemorrhage [J]. Stroke,2000,31(3):726-732.
[3]贾建平主编.神经病学[M].北京：人民卫生出版社.2008.132-133.
[4]魏巍,吕发金,彭国光.功能磁共振对高血压脑出血患者血肿周边“半暗带”的研究[J].中国神经精神疾病杂志,2008,34(3)：145-148.
[5]Johnson EM Jr, Greenlund LJ, Akins PT, et al. Neuronal apoptosis:current understanding of molecular mechanisms and potential role in ischemic brain injury [J]. J Neurotrauma, 1995,12(5):843-852.
[6]李玉林主编.病理学[M].北京：人民卫生出版社.2008.17.
[7]Hail N Jr, Carter BZ, Konopleva M, et al. Apoptosis effector mechanisms:a requiem performed in different keys [J]. Apoptosis,2006,11(6):889-904.
[8]Grimm S, Brdiczka D. The permeability transition pore in cell death [J]. Apoptosis,2007, 12(5):841-855.
[9]王海燕,王来栓.细胞凋亡通路研究进展[J].国外医学·生理、病理科学与临床分册,2003,23(5)：490-492.
[10]Tsujimoto Y. Cell death regulation by the Bcl-2 protein family in the mitochondria [J]. J Cell Physiol,2003,195(2):158-167.
[11]Kuwana T, Newmeyer DD. Bcl-2-family proteins and the role of mitochondria in apoptosis [J]. Curr Opin Cell Biol,2003,15(6):691-699.
[12]Yi X, Yin XM, Dong Z. Inhibition of Bid-induced apoptosis by Bcl-2. tBid insertion, Bax translocation, and Bax/Bak oligomerization suppressed [J]. J Biol Chem,2003,278(19): 16992-16999.
[13]Cai J, Yang J, Jones DP. Mitochondrial control of apoptosis:the role of cytochrome c [J]. Biochim Biophys Acta,1998,1366(1-2):139-149.
[14]Gajkowska B, Wojewodzka U, Gajda J. Translocation of Bax and Bid to mitochondria, endoplasmic reticulum and nuclear envelope:possible control points in apoptosis [J]. J Mol Histol,2004,35(1):11-19.
[15]Simmen T, Aslan JE, Blagoveshchenskaya AD. PACS-2 controls endoplasmic reticulum-mitochondria communication and Bid-mediated apoptosis [J]. EMBO J,2005, 24(4):717-729.
[16]朱玉山,陈佺.线粒体与细胞凋亡调控[J].生命科学,2008,20(4)：506-513.
[17]Leu JI, Dumont P, Hafey M, et al. Mitochondrial p53 activates Bak and causes disruption of a Bak-Mcll complex [J]. Nat Cell Biol,2004,6(5):443-450.
[18]Chipuk JE, Kuwana T, Bouchier-Hayes L, et al. Direct activation of Bax by p53 mediates mitochondrial membrane permeabilization and apoptosis [J]. Science,2004,303(5660): 1010-1014.
[19]Richter C, Schweizer M, Cossarizza A, et al. Control of apoptosis by the cellular ATP level [J]. FEBS Lett,1996,378(2):107-110.
[20]Shamima Islam, Ferdaus Hassan, Gantsetseg Tumurkhuu, et al. Lipopolysaccharide prevents apoptosis induced by brefeldin A, an endoplasmic reticulum stress agent, in RAW 264.7 cells [J]. Biochemical and Biophysical Research Communications,2006,340(2): 589-596.
[21]Szegezdi E, Logue SE, Gorman A M, et al. Mediators of endoplasmic reticulum stress-induced apoptosis [J]. EMBO Rep,2006,7(9):880-885.
[22]Marciniak SJ, Ron D. Endoplasmic reticulum stress signaling in disease [J]. Physiol Rev, 2006,86(4):1133-1149.
[23]Zhang K, Kaufman RJ. The unfolded protein response:a stress signaling pathway critical for health and disease [J]. Neurology,2006,66 (2 suppl):S102-S109.
[24]Schroder M, Kaufman RJ. The mammalian unfolded protein response [J]. Annu. Rev Biochem,2005,74:739-789.
[25]Schroder M, Kaufman R J. ER stress and the unfolded protein response [J]. Mutat Res, 2005,569(1-2):29-63.
[26]Faitova J, Krekac D, Hrstka R, et al. Endoplasmic reticulum stress and apoptosis[J]. Cell Mol Biol Lett,2006,11(4):488-505.
[27]Berridge MJ Lipp P, Bootman MD. The versatility and universality of calcium signalling [J]. Nat Rev Mol Cell Biol,2000,1(1):11-21.
[28]Bin Liang, Xuhong Song, Gefei Liu, et al. Involvement of TR3/Nur77 translocation to the endoplasmic reticulum in ER stress-induced apoptosis [J]. Experimental Cell Research, 2007,313(13):2833-2844.
[29]Pinton P, Ferrari D, Rapizzi E, et al. The Ca2+ concentration of the endoplasmic reticulum is a key determinant of ceramide-induced apoptosis:significance for the molecular mechanism of Bcl-2 action [J]. EMBO J,2001,20(11):2690-2701.
[30]朱玲,罗颂平,许丽绵,等.左归丸对免疫性卵巢早衰小鼠卵巢Fas、Fas-L表达的影响[J].江西中医学院学报,2008,20(1)：52-55.
[31]桑威.Fas/FasL系统参与的几种生物学效应[J].国际免疫学杂志,2006,29(6)：389-392.
[32]Pellegrini M, Bath S, Marsden VS, et al. FADD and caspase-8 are required for cytokine-induced proliferation of hemopoietic progenitor cells [J]. Blood,2005,106(5): 1581-1589.
[1]Tsujimoto Y, Finger LR, Yunis J, et al. Cloning of the chromosome breakpoint of neoplastic B cells with the t (14; 18) chromosome translocation [J]. Science,1984, 226(4678):1097-1099.
[2]Oltvai ZN, MIlliaman CL, Korsmcyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates cell death [J]. Cell,1993,74(4):609-619.
[3]步宏主编.分子病理学进展[M].成都市：四川大学出版社.2006,133-139.
[4]Danial NN, Korsmeyer SJ. Cell death:critical control points [J]. Cell,2004,116(2): 205-219.
[5]Leber B, Lin J, Andrews DW. Embedded together:the life and death consequences of interaction of the Bcl-2[J]. Apoptosis,2007,12(5):897-911.
[6]Youle RJ, Strasser A. The BCL-2 protein family:opposing activities that mediate cell death [J]. Nat Rev Mol Cell Biol,2008,9(1):47-59.
[7]Johnson EM Jr, Greenlund LJ, Akins PT, et al. Neuronal apoptosis:current understanding of molecular mechanisms and potential role in ischemic brain injury [J]. J Neurotrauma,1995,12(5):843-852.
[8]Cai J, Yang J, Jones DP. Mitochondrial control of apoptosis:the role of cytochrome c [J]. Biochim Biophys Acta,1998,1366(1-2):139-149.
[9]Gajkowska B, Wojewodzka U, Gajda J. Translocation of Bax and Bid to mitochondria, endoplasmic reticulum and nuclear envelope:possible control points in apoptosis[J]. J Mol Histol,2004,35(1):11-19.
[10]陈达,刘艳,朱杰,等.依达拉奉对脑出血大鼠神经保护作用的实验研究[J].中国现代医学杂志.2009,19(4)：497-502.
[11]Xue M, Del Bigio MR. Intracerebral injection of autologous whole blood in rats:time course of inflammation and cell death [J]. Neurosci,2000,283(3):230-232.
[12]Holmin S, Mathiesen T. Intracerebral administration of interleukin-lbeta and induction of inflammation, apoptosis, and vasogenic edema [J]. J Neurosurg,2000,92(1): 108-120.
[13]Mayne M, Ni W, Yan HJ, et al. Antisense oligodeoxynucleotide inhibition of tumor necrosis factor-alpha expression is neuroprotective after intracerebral hemorrhage [J]. Stroke,2001,32(1):240-248.
[14]Vaughan PJ, Pike CJ, Cotman CW, et al. Thrombin receptor activation protects neurons and astrocytes from cell death produced by environmental insults [J]. J Neurosci,1995, 15(7 Pt 2):5389-5401.
[15]Xue M, Del Bigio MR. Acute tissue damage after injections of thrombin and plasmin into rat striatum [J]. Stroke,2001,32(9):2164-2169.
[16]Goldstein L, Teng ZP, Zeserson E, et al. Hemin induces an iron-dependent, oxidative injury to human neuron-like cells [J]. J Neurosci Res,2003,73(1):113-121.
[17]Hua Y, Xi G, Keep RF, et al. Complement activation in the brain after experimental intracerebral hemorrhage [J]. J Neurosurg,2000,92(6):1016-1022.
[18]Matsushita K, Meng W, Wang X, et al. Evidence for apoptosis after intercerebral hemorrhage in rat striatum [J]. J Cereb Blood Flow Metab,2000,20(2):396-404.
[19]Lavine SD, Hofman FM, Zlokovic BV. Circulating antibody against tumor necrosis factor-alpha protects rat brain from reperfusion injury [J]. J Cereb Blood Flow Metab, 1998,18(1):52-58.
[20]王海燕,王来栓.细胞凋亡通路研究进展[J].国外医学·生理、病理科学与临床分册,2003,23(5)：490-492.
[21]Apte SS, Mattei MG, Olsen BR. Mapping of the human Bax gene to chromosome 19q13.3-q13.4 and isolation of a novel alternatively spliced transcript, BAX delta [J]. Genomics,1995,26(3):592-594.
[22]Kuwana T, Newmeyer DD. Bcl-2-family proteins and the role of mitochondria in apoptosis [J]. Curr Opin Cell Biol,2003,15(6):691-699.
[23]Simmen T, Asian JE, Blagoveshchenskaya AD. PACS-2 controls endoplasmic reticulum-mitochondria communication and Bid-mediated apoptosis [J]. EMBO J,2005, 24(4):717-729.
[24]朱玉山,陈佺.线粒体与细胞凋亡调控[J].生命科学,2008,20(4)：506-513.
[25]Nakamura K, Bossy-Wetzel E, Burns K, et al. Changes in endoplasmic reticulum luminal environment affect cell sensitivity to apoptosis [J]. J Cell Biol,2000,150(4): 731-740.
[26]Scorrano L, Oakes SA, Opferman JT, et al. BAX and BAK regulation of endoplasmic reticulum Ca2+:a control point for apoptosis [J]. Science,2003,300(5616):135-139.
[27]Vazquez-Martinez O, Canedo-Merino R, Diaz-Munoz M, et al. Biochemical characterization, distribution and phylogenetic analysis of Drosophila melanogaster ryanodine and IP3 receptors, and thapsigargin-sensitive Ca2+ ATPase[J]. J Cell Sci,2003, 116(Pt 12):2483-2494.
[28]田金周主编.血管性痴呆[M].北京：人民卫生出版社.2003.274-278.
[29]Wu C, Fujihara H, Yao J, et al. Different expression patterns of Bcl-2, Bcl-x1, and Bax proteins after sublethal forebrain ischemin in C57Black/Crj6 mouse straitum [J]. Stroke, 2003,39(7):1803-1808.
[30]Gajewski TF, Thompson CB. Apoptosis meets signal transduction:elimination of a BAD influence [J]. Cell,1996,87(4):589-592.
[31]Zha J, Harada H, Yang E, et al. Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-X(L) [J]. Cell,1996,87(4): 619-628.
[32]Berridge MJ, Lipp P, Bootman MD. The versatility and universality of calcium signaling [J]. Nat Rev Mol Cell Biol,2000,1(1):11-21.
[33]Pinton P, Ferrari D, Rapizzi E, et al. The Ca2+ concentration of the endoplasmic reticulum is a key determinant of ceramide-induced apoptosis:significance for the molecular mechanism of Bcl-2 action [J]. EMBO J,2001,20(11):2690-2701.
[34]Zamzami N, Susin SA, Marchetti P, et al. Mitochondrial control of nuclear apoptosis [J]. J Exp Med,1996,183(4):1533-1544.
[35]Murphy AN, Bredesen DE, Cortopassi G, et al. Bcl-2 potentiates the maximal calcium uptake capacity of neural cell mitochondria [J]. Proc Natl Acad Sci,1996,93(18): 9893-9898.
[36]Kluck RM, Bossy-Wetzel E, Green DR, et al. The release of cytochrome c from mitochondria:a primary site for Bcl-2 regulation of apoptosis [J]. Science,1997, 275(5303):1132-1136.
[37]Yang J, Liu X, et al. Bhalla K, et al. Prevention of apoptosis by Bcl-2:release of cytochrome c from mitochondria [J]. Science,1997,275(5303):1129-1132.
[38]Chinnaiyan AM, O'Rourke K, et al. Interaction of CED-4 with CED-3 and CED-9:a molecular framework for cell death [J]. Science,1997,275(5303):1122-1126.
[39]李玉林主编.病理学[M].北京：人民卫生出版社.2008.17.
