【作者】 邱林；

【导师】 朱长连；

【作者基本信息】 郑州大学 ， 病理学与病理生理学， 2006， 博士

【摘要】 新生儿缺氧缺血(hypoxia ischemia，HI)性脑损伤是严重威胁新生儿健康、导致新生儿死亡和儿童神经系统伤残的主要原因。对于HI，大脑表现为神经损伤与神经保护并存状态。HI后，大量神经细胞死亡，其机制尚未完全清楚，近来研究发现，凋亡诱导因子(apoptosis-inducing factot，AIF)触发的半胱天冬酶(caspase)非依赖凋亡途径以及过量NO所致的亚硝酰基化作用在细胞损伤中发挥重要作用，本研究采用免疫组化、免疫荧光、Western蛋白印迹、酶活性检测等生物学技术探讨AIF以及硝基酪氨酸在未成熟脑缺氧缺血诱导的神经细胞死亡中的作用，进一步明确了神经细胞死亡的发生机制。在大脑的多种适应性反应中，神经干细胞增生且分化产生新的神经细胞将对脑组织损伤后的修复起到重要作用，但在脑发育的不同阶段神经细胞的增生与分化及在HI后的改变尚未见详细报道。本研究通过注射新生细胞标记物Brdu(5-bromo-2—deoxyuridine，5-溴脱氧尿嘧啶核苷)，并采用免疫组化、免疫荧光以及立体测量系统和激光共聚焦成像技术阐述了新生小鼠脑组织发育过程中细胞增生与分化的规律以及HI后的变化，并比较了新生儿期与青少年期不同年龄脑海马区细胞增生与分化的异同，为新生儿缺氧缺血性脑损伤的临床干细胞治疗提供了理论依据。对象：缺氧缺血组Wistar 7日龄新生大鼠147只，随机分为HI后0min、30min、1h、3h、8h、14h、24h、72h(每时间点n=12)，BAF干预组42只，2—亚氨基生物素干预组9只；正常对照组Wistar大鼠60只，随机分为出生后第0d、3d、7d、8d、10d、14d、21d、42d、成年(7d组n=12，余每时间点n=6)。C57／BL6 9日龄新生雄性小鼠45只，随机分为三组，其中缺氧缺血组各10只，正常对照组各5只；21日龄雄性小鼠15只，其中缺氧缺血组10只，正常对照组5只。方法：1缺氧缺血脑损伤模型制作：1.5％—3.5％安氟醚吸入麻醉，结扎左侧颈总动脉，术后1h给予动物吸入湿化氮氧混合气，其浓度和吸入时间在7d大鼠、9d小鼠、21d小鼠不同，分别为7.7％，55min；10％，35min；10％，30min。2给药方法及药物剂量：BAF于HI后2h、12h脑室注射，每次剂量5μl；HI后即刻腹腔注射2—亚氨基生物素，剂量：20mg／kg(10μl／g)；Brdu于不同组间HI后1d、1w、2w开始腹腔注射，每次剂量50mg／kg，每天一次，连续七天。3免疫组化染色：检测AIF，MAP-2，细胞色素C，硝基酪氨酸，活性caspase-3，发夹寡核苷酸探针(HPP)原位杂交，Brdu。4免疫荧光染色：检测AIF-HPP-Hoechst33342，AIF-TUNEL-Hoechst 33342，AIF-细胞色素C-Hoechst 33342，AIF-COX，AIF-MAP-2，硝基酪氨酸-AIF-Hoechst 33342，HPP-硝基酪氨酸-Hoechst 33342，caspase-3-硝基酪氨酸-Hoechst 33342，Brud-NeuN，Brdu-APC，Brdu-Iba1，Brdu-S100B。5 Western蛋白印迹：检测细胞色素C、细胞色素氧化酶Ⅳ(COX)、caspase-3、caspase-9、a-tubulin(a-微管蛋白)、硝基酪氨酸等蛋白表达。6Caspase活性检测：检测caspase-1、2、3、9的活性。7采用激光共聚焦成像技术鉴别新生细胞的分化类型。8细胞计数：高倍视野下在皮层、纹状体、海马、丘脑分别计数免疫组化阳性细胞，海马区Brdu阳性细胞采用立体测量学系统计数。9脑梗塞体积测定：采用Micro Image软件测量MAP-2丢失面积，依据公式计算脑梗塞体积。10统计学处理：两组间比较采用t检验，多组间比较采用ANOVA post Hoc检验，用StatView软件处理，P＜0.05为有显著性差异。结果：缺氧缺血性脑损伤后细胞死亡机制的研究1．凋亡相关蛋白在正常脑组织发育中的变化：脑组织发育期间，AIF水平保持不变。线粒体标记物即线粒体膜结合蛋白细胞色素C氧化酶(COX)以及细胞色素C、caspase-9随脑组织发育表达增高，caspase-3随脑发育高峰期的消退而减少。2．HI后AIF、细胞色素C的释放：HI后即刻AIF跨膜核转移，HI后8h达到峰值，细胞呈凋亡形态学改变，AIF从线粒体的释放早于早于细胞色素C核转移，且AIF核转移早于DNA断裂。3．Caspase广谱抑制剂对AIF的再分布无作用，说明AIF通过caspase非依赖途径发挥作用。4．硝基酪氨酸免疫组化结果：HI后30min，在损伤侧大脑半球(皮层、纹状体、海马和丘脑)即可见到硝基酪氨酸着色明显增强，HI后3h达到峰值，而后下降。其中在皮层，部分阳性细胞在HI后72h再次出现；在室管膜下HI侧和对侧(单纯缺氧侧)均检测到强烈的硝基酪氨酸免疫反应性。5．硝基酪氨酸形成早于AIF的核转移和caspase-3的激活。6．硝基酪氨酸形成早与DNA断裂，硝基酪氨酸阳性细胞位于MAP-2的阴性区(梗塞区)。7．nNOS和iNOS联合抑制剂2—亚氨基生物素在HI后减少硝基酪氨酸形成，降低caspase-3的活性，但对AIF跨膜核转移无作用。正常脑组织发育过程中以及HI脑损伤后细胞增生与分化的研究1．HI对小鼠皮层和纹状体细胞增生的影响：随着脑组织发育，Brdu标记的细胞在皮层和纹状体显著下降。HI损伤显著增加未成熟脑(P9)恢复早期(HI后1w)皮层和纹状体的Brdu阳性细胞数；在青少年期组(P21)，大量的新生细胞仅发现于HI损伤侧的纹状体区。2．HI对小鼠皮层和纹状体新生细胞分化的影响：在未成熟脑皮层恢复早期以及各组的纹状体区仅能检测到少部分Brdu／NeuN双染色细胞，大部分的Brdu标记细胞为神经胶质细胞，神经胶质细胞数随脑组织发育成熟快速降低，未成熟脑皮层和纹状体在HI恢复早期被激发产生大量神经胶质细胞，在青少年期脑纹状体区，HI后Iba1(小胶质细胞)和S100β(星形胶质细胞)阳性细胞分别增加50倍和8倍，但APC(少突细胞)阳性细胞数无显著改变。3．HI对脑发育期海马细胞增生的影响：P9脑组织中Brdu标记细胞数的基线显著高于P21组。HI损伤明显增加P21整个海马区Brdu标记细胞，P9脑组织DG区无变化。4．H工对脑发育期海马新生细胞分化的影响：P9脑组织中神经元细胞再生的基线显著高于P21组。HI后，与正常对照组比较，P21组神经元再生显著增高，而在P9组无明显改变。新生神经元细胞数绝对值在两年龄组间无差别。新生的小胶质细胞和少突细胞在P21组多于P9组。结论：1．AIF核转移是新生大鼠脑缺氧缺血后神经细胞损伤的早期指标，AIF介导的细胞死亡途径在未成熟脑神经细胞损伤中起重要作用。2．硝基酪氨酸是新生大鼠脑缺氧缺血后细胞损伤的早期指标；联合抑制nNOS和iNOS在新生脑缺氧缺血损伤中发挥神经保护作用。3小鼠脑组织细胞增生、分化以及存活与脑组织区域、发育时期以及损伤时程相关。4海马区年龄相关的神经细胞再生和胶质细胞再生的不同导致缺氧缺血损伤后组织修复的不同。更多还原

【Abstract】 Neonatal hypoxic-ischemix brain damage is one of the most serious diseasethreatening the life and health of newborn infants and leading to disability in nervoussystem of children. The brain response to HI appears as a balance between theactivation of neurodestructive components and endogenous protective system. Themechanism of cell death after HI is still unknown. Recent study show thecaspase-independent apoptosis triggered by apoptosis-inducing factor (AIF) and theprotein nitrosylation induced by excessive nitric oxide (NO) play an important role inneuronal injury. The immunohistochemisty Staining, immunofluorescence staining,western blot, caspase activity were used as parameters to investigate the effect of AIFand nitrosylation in neonatal hypoxia ischemia brain injury.Among the various adaptative responses of the brain to injury, it has beenrecently reported that new neurons can be generated through the proliferation ofprogenitor cells, and thus might help to repair the brain damage. It has beendemonstrated that there is endogenetic neurogenesis in the normal brain. However,there is no report on neurogenesis in developing brain and effect of HI onneurogenesis. We use the Brdu (5-bromo-2-deoxyuriding), the marker of newgenerated cells, to label the new produced cells, combined withimmunohistochemisty staining, immunofluorescence staining, stereological systemand confocal imaging to study the cell proliferation and differentiation in developingbrain after cerebral hypoxia ischemia.Objects: HI group: After HI injury, 1477-day-old newborn Wistar rats were dividedrandomly into 0min, 30min, 1h, 3h, 8h, 14h, 24h, 72h (each time point n=12) groups,BAF treatment group 42 pups (HI 24h n=9, HI 72h n=33), 2-iminobiotin treatment group 9 pups. Normal control group: 60 Wistar rats were divided randomly into 0d,3d, 7d, 8d, 10d, 14d, 21d, 42, adult groups (each time point n=6, except 7d n=12).Forty five 9-day-old C57/BL6 male mice were divided randomly into threegroups. There were 10 mice in each HI group and 5 mice for each normal control. 1521-day-old C57/BL6 male mice were divided into HI group (n=10), normal control(n=5).Methods: 1. The preparation of the hypoxia-ischemia brain injury model: The Wistarrats/C57/BL6 male mice were anesthetized with 1.5%-3.5% halothane and the leftcommon carotid artery was ligated. After 1h recovery, the animals were givenhumidified oxygen (7.7%, 55min for 7-day-old Wistar rat; 10%, 35min for 9-day-oldC57/BL6 mice; 10%, 30min for 21-day-old C57/BL6). 2. Drug administration: (1)BAF was given by intracerebroventicular (ICV) injection 2h and 12h after HI,respectively. The total does of BAF was 5μl (1μl 100nM BAF, 4μl PBS, pH 7.4). (2)2-iminobiotin was given by intraperitoneal injection immediately after HI. The doesis 20mg/kg(10μl/g). (3) Brdu was given by intraperitoneal injection and started theinjection from 1d (group1), 1w(group2), 2w(group3) respectively after HI. The doeswas 50mg/kg/day for seven days. 3. immunohistochemisty staining: To detect AIF,MAP-2, Cyt c, nitrotyrosine, active caspase-3, HPP, Brdu. 4. Immunofluorescentstaining: To detect AIF-HPP-Hoechst 33342, AIF-TUNEL-Hoechst 33342, AIF-Cytc-Hoechst 33342, AIF-COX, AIF-MAP-2, nitrotyrosine-AIF-Hoechst 33342,HPP-nitrotyrosine-Hoechst 33342, caspase-3-nitrotyrosine-Hoechst 33342,Brdu-NeuN, Brdu-APC, Brdu-S100β. 5. Western blot: To detect the proteinexpression of Cyt c, COX, caspase-3, caspase-9, a-tubulin, nitrotyrosine. 6. Thedetermination of caspase activity: To detect the activity of caspase-1, 2, 3, 9. 7. Theconfocal imaging system: To identify the phenotype of the new generated cells. 8.Cell counting: Cell counting was performed in the cortex, striatum, hippocampus,thalamus and Positive cells were counted at 400×magnification. The Brdu positivecells in hippocampus were counted by stereological system. 9. Evaluation of braindamage: Using Micro Image software to measure the MAP-2 negative area. Theinfart volume was calculated by the formula. 10. Statistics: All the data wereexpressed as mean±SD. Unpaired t-test was used when compared two groups.ANOVA with Fisher’s post-hoc test was used when comparing more than two groups.Statview software was used to analysis the data. Significance level was assigned at p＜0.05.Results: To study the mechanism of cell death after HI brain damage. 1. Thechanges of apoptosis-related protein in developing brain: The total levels of AIF werevirtually unchanged duing normal brain development from postnatal day to adult. Themitochondrial marker cytochrome c oxidase (COX) displayed an increase, and so didcytochrome c and caspase-9. Caspase-3 decreased as the brain growth spurt leveledout. 2. AIF and cytochrome c translocation after HI: AIF was translocated to nucleiimmediately following HI and reached peak at 8h post-HI. The AIF positive cellsstaining grew increasingly stronger and more condensed during reperfusion,eventually outlining only pyknotic nuclei. The redistribution of AIF is earlier thanthat of cytochrome c. 3. AIF nuclei translocation precedes the DNA damage. 4. TheAIF redistribution was no changed after BAF treatment which indicated AIFmediated caspase-independent cell death passway. 5. Nitrotyrosine immunoreactivity:The time course of nitrotysine immuneoreactivity in ipsilateral hemisphere (cortex,striatum, hippocampuss and thalamus) was detected already 30min post-HI andincreased peak at 3h and decreased afterwards. In the cortex, there appeared to be asecond increase at 72h post-HI. The nitrotyrosine immuneoreactivity was particularlystrong in the subependymal layer, where stem cells and progenitors reside. 6.Nitrotyrosine formation preceded AIF translocation to nuclei and caspase-3 activation.7. Nitrotyrosine formation preceded DNA damage and the positive cells located inMAP-2 negative area (infarct area). 8. The nitrotyrosine formation and the activationof caspase-3 were decreased after 2-iminobiotin (the inhibitor of nNOS and iNOS)treatment, but the AIF nuclear translocation was not altered.Cell proliferation and differentiation in developing brain after cerebral HI. 1.Cell proliferation in the cortex and striatum after HI in the developing brain: Thenumber of Brdu labeled cells decreased significantly in both cortex and striatum withbrain development. HI insult increased Brdu positive cells significantly in theipsilateral cortex and striatum at early recovery of the immature brain. In the juvenile,large amount of new born cells was only seen in the ipsilateral striatum. 2. Celldifferentiation in the cortex and striatum after HI in the developing brain: A smallportion of Brdu and NeuN double labeled cells could be detected in the cortex at veryearly recovery in the immature brain and in the striatum of all the groups. Themajority of Brdu labeled cells were neuroglia. The number of the neuroglia cells decreased dramatically with brain maturation. HI insult stimulated to produce a largenumber of neuroglia cells in the ipsilateral cortex and striatum of immature brain atearly recovery after HI. In the juvenile striatum, Iba1 and S-100βpositive cellsincreased 50 and 8 folds after HI, but the number of APC positive cells was nosignificant change. 3. Compare the cell proliferation in neonatal (P9) and juvenile(P21) brain after HI insult: The basal level of Brdu labeled cells and neurogenesis wasmuch higher in the immature brain than that of juvenile. HI insult increased Brdulabeled cells dramatically in the whole juvenile hippocampus, but not in the immaturedentate gyrus (DG). 4. Compare the cell differentiation in neonatal (P9) and juvenile(P21) brain after HI insult: The neurogenesis was increased significantly in thejuvenile compared with the age matched control; however, there was no significantlyincrease in the immature brain. The absolute number of newly generated neuron wasno different between immature and juvenile. More microglia and oligodendrocytewere differentiated in the juvenile compared to immature.Conclusions: 1. AIF translocation is an early marker of DNA damage in neonatalcerebral HI and AIF-mediated cell death may play an important role in HI inducedneuronal loss in the immature brain. 2. Nitrotyrosine is an early marker of neuronaldamage after neonatal HI brain injury. The inhibition of nNOS and iNOS show theeffect of neuronal protection. 3. Cell proliferation, differentiation and survive wasbrain regions, developmental stages and injury time courses related. 4. Thedevelopment related differences in the neurogenesis and gliagenesis respond to HIinjury may underline the differences in tissue restoration in developing brain.更多还原