大鼠脑缺血后神经细胞自身修复机制的初步研究
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摘要
脑缺血是一种严重威胁人类健康的疾病，缺血后神经细胞的存亡与否取决于神经元损伤程度和自身修复能力之间的平衡。因此，研究各种措施在缺血后是如何加强神经元修复、减轻神经元损伤已成为当今的热点。血管内皮生长因子(VEGF)作为一种重要的神经营养因子，脑缺血后表达增加；且外源性VEGF具有神经保护作用。然而对于内源性VEGF是否具有神经保护作用，其机理如何，目前还不清楚。因此，我们首先研究了内源性VEGF是否具有神经保护作用，进一步探讨这种作用是否同影响神经细胞的DNA修复作用有关。与此同时，越来越多的研究结果表明成年脑内持续存在着神经元再生；脑缺血可上调大脑皮层和海马的神经元再生，这可能是一种重要的神经保护机制。因此，在第二部分工作中，我们研究了脑缺血后纹状体内是否存在神经元再生；如果有的话，新生神经元来自何处及电刺激是否可以上调缺血脑的神经元再生。
第一部分：VEGF对缺血损伤脑的保护作用及其机制分析
血管内皮生长因子(VEGF)是一种重要的神经营养因子，缺血后其脑内的表达可显著上调；给予外源性VEGF时可减轻缺血性神经元损伤的程度。研究表明：这种外源性VEGF的神经保护作用一方面可能同其促血管生成或避免血管内皮细胞凋亡有关；另一方面，则可能是通过激活与VEGF受体偶联的磷脂酰肌醇-3-激酶(PI3-K)／Akt信号转导途径，促进了抑凋亡蛋白的表达并下调激活型caspase-3的含量。对于缺血诱导的内源性VEGF高表达是否同样具有神经保护作用，目前还不清楚。
脑缺血后一系列的病理变化导致神经细胞DNA损伤。如果DNA损伤得不到及时修复，则会发生神经元凋亡，进一步加重缺血性神经元损伤。然而缺血在导致DNA损伤的同时，还激活自身的DNA修复系统。核苷酸剪切修复(NER)就是细胞内一种重要的DNA修复方式，它可修复一系列大块的DNA损伤，如紫外线、化学药物及氧化剂导致的DNA损伤。进一步的研究表明在NER中存在着优先转录修复(TCR)，即可转录基因的转录链被优先修复。剪切修复耦联因子6(ERCC6)是TCR的重要因子，脑缺血后在缺血区呈时间依赖性表达。右美沙芬及褪黑素等药物的神经保护作用部分是通过上调ERCC6的表达来实现的。
在本部分的研究中，我们首先应用VEGF反义寡聚脱氧核苷酸(antisense ODN)抑制脑缺血诱导的内源性VEGF高表达，在明确了VEGF antisense ODN在动物脑内应用的有效性后，观察VEGF antisense ODN对缺血性神经元损伤的影响以了解内源性VEGF是否具有神经保护作用，以及VEGF的神经保护作用
复旦大学博士学位论文
大鼠脑缺血后神经细胞自身修复机制的初步研究
是否同DNA修复因子ERCC6 mRNA表达水平的变化有关。
研究结果:
1.脑缺血再灌注即刻、1天、2天时侧脑室注射VEGF antisense ODN，可以抑
制缺血再灌3天时内源性VEGF的表达。与此同时，VEGF antisense ODN可
显著增大脑梗塞体积，加重缺血性神经元损伤的程度。
2.缺血再灌注3天时，VEGF antisense ODN可显著增加缺血侧脑内TUNEL阳
性细胞的数量。
3.缺血再灌注3天时，VEGF可与DNA修复因子ERCC6 mRNA共存于同一神
经元中。双标细胞的形态基本正常，其数量从缺血中心区到缺血周边区逐渐
增多。侧脑室内注射VEGF antisnese ODN后，缺血周边区的ERCC6 mRNA
阳性细胞的数量显著减少，而缺血中心区的ERCC6 mRNA阳性细胞的数量
没有显著变化。
小结:
1.脑缺血后高表达的内源性VEGF具有神经保护作用。
2.内源性VEGF的神经保护作用可能是通过影响DNA修复因子ERCC6 mRNA
的表达实现的。
第二部分:成年大鼠缺血损伤脑的神经元再生修复及电刺激的上调作用
脑缺血后新生神经元的数量显著增加，不仅在神经元生发区(S VZ和SGZ)，
在诸如海马CAI区、纹状体、大脑皮层中也存在新生神经元。有证据表明:脑
缺血后海马CAI区的新生神经元来自于侧脑室室旁区和海马内的神经前体细
胞，尽管有研究认为纹状体中的新生神经元可能来源于SVZ，但却缺乏直接的
证据予以证实。由于纹状体内的神经元投射到苍白球及黑质构成基底节运动环
路，同时经常为脑缺血损伤所累及，因此，了解脑缺血后纹状体内新生神经元的
来源，进而选择合适的手段增强神经元再生，就可为缺血后脑保护提供一个新的
研究方向。
在本部分研究中，我们在成年大鼠脑缺血前将荧光染料DII注射到侧脑室中，
以选择性标记侧脑室室旁区的细胞;在脑缺血后腹腔内注射BrdU以标记新生细
胞;应用免疫组化双标结合激光共聚焦显微观察明确纹状体内是否存在着新生神
经元及神经元迁移、以及给予电刺激后对此的影响。
结果:
复旦大学博士学位论文
大鼠脑缺血后神经细胞自身修复机制的初步研究
1.缺血再灌2周时，缺血侧纹状体内BrdU阳性细胞的数量显著增加，主要位
于纹状体内侧，并表达幼稚神经元的标志蛋白CRMP一4和成熟神经元的标志
蛋白MAP一2，这些结果表明脑缺血后纹状体内有新生神经元产生。
2.荧光染料DII可以原位标记侧脑室室旁区的细胞，。双标结果显示这些细胞可
表达星型胶质细胞的标志蛋白GFA卫和神?
Stroke has been considered to be a vital disease to human being. The fate of ischemic neurons is determined by the balance of neuronal damage and its pretection. Therefore, much more attention has been paid on how to augment neuronal protection and alleviate neuronal damage. Vascular endothelial growth factor (VEGF), an important neurotrophic factor, could be up-regulated in ischemic brain. It has been proved that exogenous VEGF has neuroprotective role. However, it is still unclear whether ischemia-induced endogenous VEGF expression plays a neuroprotective role. In the present study, we investigate whether the induction of endogenous VEGF plays neuroprotective role in ischemic neurons and whether it associates with DNA repair. On the other hand, more and more evidences showed that neurogenesis exists in adult mammalian brain. Ischemia could stimulate neurogenesis in the cerebral cortex and hippocampus which is considered as a very important neuronal protection mechanism. Therefore, in the second part of thi
s study, we further evaluate whether ischemia could induce neurogenesis in the striatum, the most commonly ischemic area; if so, where newborn neurons in the striatum come from and whether electric stimulation could enhance neurogenesis in ischemic injured brain.
The first part: The neuroprotective roles and mechanisms of endogenous VEGF in ischemia-injured brain
Vascular endothelial growth factor (VEGF), an important neural trophic factor, could be up-regulated in ischemic brain. Studies have shown the exogenous VEGF protects neurons against ischemia-reperfusion injury, whose protective mechanism may be related to neoangiogenesis or vessel protection. On the other hand, VEGF may play direct cytoprotective role through its receptor-coupled phosphatidylinositol 3'-kinase (PI3-K)/Akt signal transduction system. Activation of PI3-K/Akt pathway can upregulate antiapoptotic proteins and downregulate activated caspase-3. It's still unknown whether the ischemia-induced endogenous VEGF expression has the same neuroprotective role as the exogenous one does.
The cascade of events following cerebral ischemia could induce neuronal DNA damage. Incomplete repair or nonrepair of DNA damage would induce ischemic neuronal death and further contribute to the expansion of the ischemic lesion. At the same time, cerebral ischemia can activate a complex network of DNA repair to
counteract neuronal DNA damage. Nucleotide excision repair (NER) is the most important and best studied among the DNA repair pathway. In NER, the transcribed strand of transcriptionally active gene is repaired more preferentially and efficiently than the nontranscribed one. ERCC6 has been confirmed to be a transcription-repair coupling factor and to play a crucial role in preferential transcription-repair coupling of NER. Also, several studies have demonstrated that the induction of ERCC6 in ischemic injured neurons contributes to cytoprotection against ischemic neuronal DNA damage.
In the first part of study, we used VEGF antisense oligodeoxynucleotide (ODN) to specially knockdown VEGF induction in ischemic neurons, then analyzed the changes of infarct volume, ischemia-induced TUNEL and ERCC6 mRNA expression to elucidate whether the induced endogenous VEGF plays neuroprotective role probably associated with the expression of ERCC6 mRNA.
RESULTS:
1. Immunostaining was used to examine the expression of VEGF to prove the validity of VEGF antisense ODN. Compared with random ODN treatment, VEGF antisense reduced the number of VEGF positive neurons in the ipsilateral striatum and frontoparietal cortex at 3 d reperfusion after ischemia. Cresyl violet staining and image processing and analysis system were used to determine the infarct volume. They showd that VEGF antisense ODN significantly increased infarct volume at 3 d reperfusion after ischemia.
2. In situ TUNEL staining was used to detect DNA double-strand breaks in the ischemic brains. TUNEL positive cells were found in ipsilateral striatum and frontoparietal cortex, but not in
第一部分：VEGF对缺血损伤脑的保护作用及其机制分析
血管内皮生长因子(VEGF)是一种重要的神经营养因子，缺血后其脑内的表达可显著上调；给予外源性VEGF时可减轻缺血性神经元损伤的程度。研究表明：这种外源性VEGF的神经保护作用一方面可能同其促血管生成或避免血管内皮细胞凋亡有关；另一方面，则可能是通过激活与VEGF受体偶联的磷脂酰肌醇-3-激酶(PI3-K)／Akt信号转导途径，促进了抑凋亡蛋白的表达并下调激活型caspase-3的含量。对于缺血诱导的内源性VEGF高表达是否同样具有神经保护作用，目前还不清楚。
脑缺血后一系列的病理变化导致神经细胞DNA损伤。如果DNA损伤得不到及时修复，则会发生神经元凋亡，进一步加重缺血性神经元损伤。然而缺血在导致DNA损伤的同时，还激活自身的DNA修复系统。核苷酸剪切修复(NER)就是细胞内一种重要的DNA修复方式，它可修复一系列大块的DNA损伤，如紫外线、化学药物及氧化剂导致的DNA损伤。进一步的研究表明在NER中存在着优先转录修复(TCR)，即可转录基因的转录链被优先修复。剪切修复耦联因子6(ERCC6)是TCR的重要因子，脑缺血后在缺血区呈时间依赖性表达。右美沙芬及褪黑素等药物的神经保护作用部分是通过上调ERCC6的表达来实现的。
在本部分的研究中，我们首先应用VEGF反义寡聚脱氧核苷酸(antisense ODN)抑制脑缺血诱导的内源性VEGF高表达，在明确了VEGF antisense ODN在动物脑内应用的有效性后，观察VEGF antisense ODN对缺血性神经元损伤的影响以了解内源性VEGF是否具有神经保护作用，以及VEGF的神经保护作用
复旦大学博士学位论文
大鼠脑缺血后神经细胞自身修复机制的初步研究
是否同DNA修复因子ERCC6 mRNA表达水平的变化有关。
研究结果:
1.脑缺血再灌注即刻、1天、2天时侧脑室注射VEGF antisense ODN，可以抑
制缺血再灌3天时内源性VEGF的表达。与此同时，VEGF antisense ODN可
显著增大脑梗塞体积，加重缺血性神经元损伤的程度。
2.缺血再灌注3天时，VEGF antisense ODN可显著增加缺血侧脑内TUNEL阳
性细胞的数量。
3.缺血再灌注3天时，VEGF可与DNA修复因子ERCC6 mRNA共存于同一神
经元中。双标细胞的形态基本正常，其数量从缺血中心区到缺血周边区逐渐
增多。侧脑室内注射VEGF antisnese ODN后，缺血周边区的ERCC6 mRNA
阳性细胞的数量显著减少，而缺血中心区的ERCC6 mRNA阳性细胞的数量
没有显著变化。
小结:
1.脑缺血后高表达的内源性VEGF具有神经保护作用。
2.内源性VEGF的神经保护作用可能是通过影响DNA修复因子ERCC6 mRNA
的表达实现的。
第二部分:成年大鼠缺血损伤脑的神经元再生修复及电刺激的上调作用
脑缺血后新生神经元的数量显著增加，不仅在神经元生发区(S VZ和SGZ)，
在诸如海马CAI区、纹状体、大脑皮层中也存在新生神经元。有证据表明:脑
缺血后海马CAI区的新生神经元来自于侧脑室室旁区和海马内的神经前体细
胞，尽管有研究认为纹状体中的新生神经元可能来源于SVZ，但却缺乏直接的
证据予以证实。由于纹状体内的神经元投射到苍白球及黑质构成基底节运动环
路，同时经常为脑缺血损伤所累及，因此，了解脑缺血后纹状体内新生神经元的
来源，进而选择合适的手段增强神经元再生，就可为缺血后脑保护提供一个新的
研究方向。
在本部分研究中，我们在成年大鼠脑缺血前将荧光染料DII注射到侧脑室中，
以选择性标记侧脑室室旁区的细胞;在脑缺血后腹腔内注射BrdU以标记新生细
胞;应用免疫组化双标结合激光共聚焦显微观察明确纹状体内是否存在着新生神
经元及神经元迁移、以及给予电刺激后对此的影响。
结果:
复旦大学博士学位论文
大鼠脑缺血后神经细胞自身修复机制的初步研究
1.缺血再灌2周时，缺血侧纹状体内BrdU阳性细胞的数量显著增加，主要位
于纹状体内侧，并表达幼稚神经元的标志蛋白CRMP一4和成熟神经元的标志
蛋白MAP一2，这些结果表明脑缺血后纹状体内有新生神经元产生。
2.荧光染料DII可以原位标记侧脑室室旁区的细胞，。双标结果显示这些细胞可
表达星型胶质细胞的标志蛋白GFA卫和神?
Stroke has been considered to be a vital disease to human being. The fate of ischemic neurons is determined by the balance of neuronal damage and its pretection. Therefore, much more attention has been paid on how to augment neuronal protection and alleviate neuronal damage. Vascular endothelial growth factor (VEGF), an important neurotrophic factor, could be up-regulated in ischemic brain. It has been proved that exogenous VEGF has neuroprotective role. However, it is still unclear whether ischemia-induced endogenous VEGF expression plays a neuroprotective role. In the present study, we investigate whether the induction of endogenous VEGF plays neuroprotective role in ischemic neurons and whether it associates with DNA repair. On the other hand, more and more evidences showed that neurogenesis exists in adult mammalian brain. Ischemia could stimulate neurogenesis in the cerebral cortex and hippocampus which is considered as a very important neuronal protection mechanism. Therefore, in the second part of thi
s study, we further evaluate whether ischemia could induce neurogenesis in the striatum, the most commonly ischemic area; if so, where newborn neurons in the striatum come from and whether electric stimulation could enhance neurogenesis in ischemic injured brain.
The first part: The neuroprotective roles and mechanisms of endogenous VEGF in ischemia-injured brain
Vascular endothelial growth factor (VEGF), an important neural trophic factor, could be up-regulated in ischemic brain. Studies have shown the exogenous VEGF protects neurons against ischemia-reperfusion injury, whose protective mechanism may be related to neoangiogenesis or vessel protection. On the other hand, VEGF may play direct cytoprotective role through its receptor-coupled phosphatidylinositol 3'-kinase (PI3-K)/Akt signal transduction system. Activation of PI3-K/Akt pathway can upregulate antiapoptotic proteins and downregulate activated caspase-3. It's still unknown whether the ischemia-induced endogenous VEGF expression has the same neuroprotective role as the exogenous one does.
The cascade of events following cerebral ischemia could induce neuronal DNA damage. Incomplete repair or nonrepair of DNA damage would induce ischemic neuronal death and further contribute to the expansion of the ischemic lesion. At the same time, cerebral ischemia can activate a complex network of DNA repair to
counteract neuronal DNA damage. Nucleotide excision repair (NER) is the most important and best studied among the DNA repair pathway. In NER, the transcribed strand of transcriptionally active gene is repaired more preferentially and efficiently than the nontranscribed one. ERCC6 has been confirmed to be a transcription-repair coupling factor and to play a crucial role in preferential transcription-repair coupling of NER. Also, several studies have demonstrated that the induction of ERCC6 in ischemic injured neurons contributes to cytoprotection against ischemic neuronal DNA damage.
In the first part of study, we used VEGF antisense oligodeoxynucleotide (ODN) to specially knockdown VEGF induction in ischemic neurons, then analyzed the changes of infarct volume, ischemia-induced TUNEL and ERCC6 mRNA expression to elucidate whether the induced endogenous VEGF plays neuroprotective role probably associated with the expression of ERCC6 mRNA.
RESULTS:
1. Immunostaining was used to examine the expression of VEGF to prove the validity of VEGF antisense ODN. Compared with random ODN treatment, VEGF antisense reduced the number of VEGF positive neurons in the ipsilateral striatum and frontoparietal cortex at 3 d reperfusion after ischemia. Cresyl violet staining and image processing and analysis system were used to determine the infarct volume. They showd that VEGF antisense ODN significantly increased infarct volume at 3 d reperfusion after ischemia.
2. In situ TUNEL staining was used to detect DNA double-strand breaks in the ischemic brains. TUNEL positive cells were found in ipsilateral striatum and frontoparietal cortex, but not in
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