【作者】 李战永；

【导师】 张涛；

【作者基本信息】 南开大学 ， 生物信息学， 2012， 博士

【摘要】 目的通过在体实验观察外源性硫化氢（H₂S）对大鼠海马CA1区锥体细胞和大鼠胶质瘤细胞在缺血损伤时的保护作用，并探讨相关机制。方法⑴40只雄性wistar大鼠随机分成4组：假手术组、缺血组、H₂S组、缺血+H₂S组，每组10只。采用双侧颈总动脉永久结扎（2VO）的方法建立慢性脑缺血大鼠模型；H₂S组和缺血+H₂S组大鼠给予腹腔注射NaHS（5.6mg/kg/day）处理。利用Morris水迷宫实验（MWM）观察比较各组大鼠空间学习、记忆能力；各组大鼠在MWM测试后分别断头取脑，测定海马内H₂S含量；通过组织学方法（HE染色）观察各组大鼠海马CA1区锥体细胞的形态结构。⑵32只雄性wistar大鼠随机分为4组，每组8只，分组及处理方法同⑴。在体记录各组大鼠海马CA3、CA1区的局部场电位，用计算神经生物学方法分析大鼠海马CA3、CA1区神经网络的振荡模态（包括θ波段和γ波段）并进行比较；分别在体记录各组大鼠海马CA1区的长时程增强（LTP），并用免疫组织化学法观察各组大鼠海马CA1区生长相关蛋白（GAP-43）的表达情况。⑶40只雄性Sprague-Dawley大鼠随机分成4组：假手术组，H₂S组，肿瘤组，肿瘤+H₂S组，每组10只。选用大鼠C6胶质瘤细胞系，通过纹状体内微量细胞注射的方法建立大鼠恶性胶质瘤（GBM）动物模型，H₂S组和肿瘤+H₂S组大鼠在脑内注射1周后给予腹腔注射NaHS（5.6mg/kg）1次；脑内注射3周后，各组大鼠分别断头取脑，肿瘤侧半脑用于制备冰冻切片，HE染色观察瘤内细胞形态，并计算瘤体体积；免疫组化染色观察瘤内血管内皮标记蛋白CD34、缺氧诱导因子-1α（HIF-1α）和金属基质蛋白酶-2（MMP-2）的阳性表达情况，评估瘤体内微血管密度（MVD）；肿瘤对侧半脑用于脑内H₂S含量测定。结果⑴慢性脑缺血大鼠海马内H₂S含量显著降低（缺血组vs.假手术组，P＜0.05）；经外源性H₂S处理后，慢性脑缺血大鼠海马内的H₂S含量显著升高（缺血+H₂S组vs.缺血组，P＜0.05），但仍低于假手术组（缺血+H₂S组vs.假手术组，P＜0.05）。⑵MWM结果表明，与假手术组大鼠相比，缺血组大鼠的逃避潜伏期明显延长（P＜0.01），目标象限百分比显著减少（P＜0.001），穿越平台次数明显减少（P＜0.001）；经外源性H₂S腹腔注射处理后，缺血+H₂S组大鼠的逃避潜伏期（秒）明显缩短（定位巡航第5天，缺血组与缺血+H₂S组相比为：15.91±2.51vs.8.09±1.50，P＜0.01），目标象限百分比（%）明显增加（缺血组与缺血+H₂S组相比为：27.27±3.37vs.37.25±3.92，P＜0.05），穿越平台次数（次）显著增加（缺血组与缺血+H₂S组相比为：1.83±0.27vs.3±0.46，P＜0.05）。四组大鼠的平均游泳速度无显著性差别（P＞0.05）。⑶假手术组和H₂S组的CA1区锥体细胞形态正常。缺血组海马的CA1区锥体细胞出现明显的核固缩现象，胞浆染色不均，难以分辨正常的胞体形态，细胞周围可见明显水肿，细胞分布散乱不均；缺血+H₂S组海马的CA1区锥体细胞也可见核固缩现象，但明显少于缺血组，细胞分布趋于均匀，胞周水肿也明显减少，胞浆染色也较均匀，可见多数锥体细胞的形态趋于正常。⑷对局部场电位的计算神经生物学分析结果表明，缺血会导致CA3与CA1之间的θ节律和γ节律相位同步性显著减弱，给予H₂S后，两个脑区减弱的相位同步得到显著恢复。缺血导致CA3-CA1通路θ与γ节律上的信息流单方向指数c2的显著下降，而给予H₂S后，这种下降趋势得到恢复。⑸与假手术组相比，缺血组的场兴奋性突触后电位（fEPSPs）斜率百分比显著降低（123.33±2.62vs.112.35±2.52，P＜0.001），在使用外源性H₂S处理后，缺血+H₂S组大鼠的fEPSPs斜率百分比得到明显提高（缺血+H₂S组vs.缺血组：124.99±3.64vs.112.35±2.52，P＜0.001）。⑹GAP-43主要表达于锥体细胞的胞体及其附近，缺血组的GAP-43阳性表达最低，缺血+H₂S组的GAP-43阳性表达介于假手术组和缺血组之间。缺血组与假手术组比较，P＜0.001；缺血组与缺血+H₂S组比较，P＜0.001。⑺一次性给予低剂量NaHS（5.6mg/kg）可促进荷瘤鼠GBM瘤体的生长，表现为瘤体体积明显增大（P＜0.001），瘤体组织出现空洞，坏死、出血明显增多，新生血管明显增多。⑻肿瘤+H₂S组与肿瘤组大鼠脑内H₂S的含量均高于假手术组（P＜0.05），肿瘤+H₂S组H₂S含量比肿瘤组显著提高（P＜0.05）；肿瘤+H₂S组大鼠脑内的CD34的阳性表达明显高于肿瘤组（P＜0.001）；肿瘤组和肿瘤+H₂S组瘤内的MVD（条/mm2）测定结果分别为41.2±7.9和97.0±10.8,具有极显著性差异，P＜0.001；与肿瘤组相比，肿瘤+H₂S组大鼠瘤体内HIF-1α和MMP-2的阳性表达量均显著增多（P＜0.001）。结论⑴外源性H₂S可提高缺血大鼠模型海马内H₂S的含量；⑵外源性H₂S可改善慢性脑缺血大鼠的空间认知障碍，其机制与H₂S可改善慢性脑缺血导致的大鼠海马锥体细胞的形态结构异常有关；⑶慢性脑缺血可造成海马内慢γ振荡与θ振荡模态的改变；外源性H₂S可使慢性脑缺血导致的θ与γ模态改变得到一定程度的恢复，这有可能是H₂S对抗慢性脑缺血认知损伤的一个新的机制；⑷H₂S可增强慢性脑缺血时大鼠海马CA1区神经元LTP，这是H₂S改善脑缺血导致的大鼠空间认知障碍的神经电生理机制，这种电生理作用与H₂S促进脑缺血大鼠海马CA1区GAP-43的表达有关；⑸一次性给予低剂量（5.6mg/kg）的外源性H₂S可促进GBM瘤体的血管新生，促进瘤体的生长，其机制与外源性H₂S促进瘤体的HIF-1α和MMP-2表达有关；⑹H₂S对缺血的海马锥体细胞和缺血的胶质瘤细胞都具有神经保护作用。更多还原

【Abstract】 Objective⑴To investigate whether exogenous hydrogen sulfide （H₂S） attenuates theneuronal injury in the hippocampal CA1region induced by chronic brain ischemia.⑵To address the neuroprotective effect of H₂S on aberrant glial cells inglioblastoma （GBM）.⑶To explore the mechanism of the neuroprotective effect of H₂S on ischemicneurons in hippocampus as well as glial cell in GBM.MethodsIn the present study, experiments were divided into3parts. In the first andsecond parts, a global model of cerebral ischemia was established by two vesselocclusion （2VO）, and the experimental rats were divided into4groups: sham group,H₂S group （NaHS injected,5.6mg/kg/day, i.p.）,2VO group and2VO-H₂S group（NaHS injected post2VO,5.6mg/kg/day, i.p.）. For the first part experiment, theperformances of learning and memory were examined by the Morris water maze（MWM）. As an H₂S donor, NaHS was administered intraperitoneally （5.6mg/kg/day,i.p.）. The morphology of neurons in hippocampus was determined by hematoxylinand eosin （HE） staining, and H₂S content in hippocampus was evaluated. In thesecond part of the present experiments, LTP from hippocampus Schaffer collaterals toCA1region was performed. Before the LTP induction, the local field potentials （LFPs）were recorded. Growth-associated protein-43（GAP-43） expression in thehippocampus of each rat was analyzed by immunohistochemical staining. In addition,a novel general partial directed coherence （gPDC） algorithm was employed todetermine the coupling interaction between CA3and CA1in hippocampus in twofrequency bands （theta rhythm and gamma rhythm）. In the third part of the presentstudy, in vivo GBM model was conducted using adult rats with intracerebral injectionof rat C6glioma cell line, and another4groups were involved: sham group, H₂Sgroup, GBM group and GBM-H₂S group. To observe the H₂S enhance effect onangiogenesis in GBM, an intraperitoneal injection of NaHS was administrated. HE staining, angiogenesis examination and immunohistochemical analysis of thehypoxia-inducible factor-1alpha （HIF-1α）, matrix metalloproteinase-2（MMP-2） andCD34expressions were performed, respectively. Each section stained withCD34-related antigen was also to highlight the blood vessels for evaluating the tumormicrovessel density （MVD-CD34）.Results⑴Exogenous H₂S significantly improved spatial learning and memory deficitsinduced by brain ischemia （P＜0.01）. Exogenous H₂S inhibited the edema aroundpyramidal neurons and the nuclear shrink induced by ischemia. Intraperitonealinjection with a certain concentration of NaHS （5.6mg/kg/day） could increase thecontent of H₂S in hippocampus of2VO animals. A subnormal level of H₂S content inhippocampus was existed in2VO rats. The H₂S level in hippocampus in2VO-H₂Sgroup was elevated after treated with NaHS, while it was lower than that in shamgroup. It can be seen that the H₂S level in hippocampus was associated with the dataobtained from behavior and morphology tests.⑵Exogenous H₂S enhanced the LTP in the hippocampus of2VO rats, andpromoted the expression of GAP-43in the CA1region of hippocampus post ischemia.The phase locked values （PLV） were significantly increased in theta and gammarhythms after H₂S treatment in2VO rats. The unidirectional influence from CA3toCA1reduced significantly at theta and gamma rhythms in2VO rats, and enhancedafter H₂S treatment, which were associated with the LTP alterations.⑶After NaHS injection, the clinical symptoms of tumor-bearing rats becamemore serious, and the dramatic distress and obvious weight loss were found. HEstaining showed more evidence of tumor in GBM-H₂S animal brains than that inGBM animal brains. The mean tumor volume became much larger in GBM-H₂S ratsthan that in GBM animals （P＜0.001）. Immunohistochemical analysis exhibited thatthe HIF-1α, MMP-2and CD34expressions were obviously increased after NaHSintraperitoneal injection in GBM-H₂S rats. And the MVD-CD34was markedly higherin GBM-H₂S group.Conclusions⑴the H₂S content in hippocampus could be increased by treating with a certain concentration of exogenous H₂S. H₂S could improve impairment of learning andmemory in2VO rats. The underlying mechanism was associated with improvementof the edema around pyramidal neurons and the nuclear shrink induced by ischemia.⑵Exogenous H₂S enhanced synaptic plasticity in the hippocampus of2VO rats,The underlying mechanism was associated with H₂S promoted the expression ofGAP-43in the CA1region of post-ischemic hippocampus. The changes of oscillatorypattern in hippocampal neurons induced by2VO got retrieved in a degree by H₂Streatment, which might represent a novel recognition to the mechanism of H₂Simproving cognitive impairments.⑶H₂S serves as a stimulator in the angiogenesis of GBM via multiplemechanisms including the increase of HIF-1α and MMP-2.⑷Exogenous H₂S has neuroprotective effects on both pyramidal neurons in theCA1region of hippocampus and glial cell in GBM when ischemic injury existed inthese two kinds of neuron.更多还原