【作者】 周敬华；

【导师】 孙圣刚；

【作者基本信息】 华中科技大学 ， 神经病学， 2006， 博士

【摘要】 第一部分脑出血大鼠血肿周围组织水孔蛋白-4的表达分析目的：研究脑出血（intracerebra hemorrhage，ICH）后血肿周围组织水孔蛋白-4（aquaporin-4，AQP4）表达的变化，探讨AQP4在脑水肿形成过程中的作用。方法：运用立体定向技术，向大鼠尾壳核内注射自体尾动脉血建立脑出血模型。采用免疫组织化学技术对出血周围组织及皮质AQP4蛋白进行动态检测。结果：与正常组比较，脑出血6h后血肿周围组织AQP4蛋白表达开始增高（P＜0.01），在出血第3天达高峰（P＜0.01），以后逐渐下降，到第7天仍高于正常水平（P＜0.01），与脑出血后脑水肿的变化规律相似。在大脑皮质AQP4蛋白表达亦相应增加，但不如血肿周围组织明显。单纯尾壳核内注射凝血酶后，注射部位邻近脑组织AQP4蛋白表达增高主要集中在注射后第1～3天。结论：脑出血时血肿周围AQP4蛋白表达增加，与水肿进程关系密切。AQP4蛋白表达可能主要由凝血酶所诱导，上调的AQP4蛋白可能参与脑出血后脑水肿的形成。第二部分凝血酶对大鼠原代星形胶质细胞水孔蛋白-4表达的影响目的：研究不同浓度凝血酶（thrombin，TB）对体外培养大鼠原代星形胶质细胞水孔蛋白-4（aquaporin-4，AQP4）表达的影响，探讨脑出血后星形胶质细胞水肿机制。方法：新生SD大鼠大脑皮质分离星形胶质细胞，传代培养纯化至98％以上。分别用0.5 U／ml，1 U／ml，100 U／ml，200 U／ml浓度的凝血酶对星形胶质细胞处理24 h后，采用RT-PCR及免疫组织化学技术检测星形胶质细胞AQP4 mRNA和AQP4蛋白表达，TUNEL方法检测细胞凋亡情况，并对星形胶质细胞进行形态学及细胞活性观察。结果：正常星形胶质细胞AQP4 mRNA和AQP4蛋白呈微弱表达，高浓度的凝血酶（100 U／ml，200 U／ml）处理后，AQP4 mRNA和AQP4蛋白表达明显升高（P＜0.01），星形胶质细胞肿胀，TUNEL阳性细胞数显著增多。低浓度的凝血酶（0.5 U／ml，1 U／ml）处理后，AQP4 mRNA和AQP4蛋白表达出现下调（P＜0.05），星形胶质细胞形态无明显变化，细胞凋亡数与对照组比较并没有显著增加。结论：高浓度凝血酶能诱导AQP4 mRNA及AQP4蛋白高表达，使星形胶质细胞胞体肿胀，细胞活性下降，并发生凋亡。第三部分水孔蛋白-4与血脑屏障通透性关系及其调节的实验研究目的：探讨精氨酸加压素及V_1a受体(V_1aR)拮抗剂对脑出血后水孔蛋白-4（aquaporin-4，AQP4）表达及血脑屏障（blood-brain barrier，BBB）通透性的影响。方法：运用立体定向技术，向大鼠尾壳核内注射自体尾动脉血建立脑出血模型。模型制作成功后，治疗组侧脑室注射V_1aR拮抗剂，ICH组仅注射等量人工脑脊液。采用免疫组织化学技术对血肿周围组织AQP4蛋白进行检测。通过检测渗出到脑血管外的伊文氏蓝（Evans Blue，EB）的含量来定量观察BBB的通透性。结果：脑出血6 h后尾壳核血肿周围组织AQP4蛋白表达开始增高，在脑出血第1天达高峰（P＜0.01），持续到第3天以后逐渐下降，到第7天仍高于正常水平。而用V_1aR拮抗剂脑室注射后，AQP4蛋白表达在各时间点表达明显降低。BBB通透性在脑出血6h后开始升高，1d达高峰（P＜0.01），3d后回落，AQP4蛋白表达与BBB通透性呈显著正相关。侧脑室注入V_1aR拮抗剂后，BBB通透性与对照组比较明显下降（P＜0.05）。 结论：脑出血时AQP4表达增加参与了BBB的破坏，V_1aR拮抗剂能抑制AQP4蛋白的表达，保护BBB，减轻脑出血后脑水肿。 第四部分 精氨酸加压素对原代培养星形胶质细胞水孔蛋白-4表达的影响 目的：研究精氨酸加压素（arginine vasopressin，AVP）对体外培养星形胶质细胞水孔蛋白-4（aquaporin-4，AQP4）表达的影响，探讨精氨酸加压素对星形胶质细胞容积调节的机制。 方法：新生SD大鼠大脑皮质分离星形胶质细胞，传代培养纯化至98％以上。星形胶质细胞经500 nM的AVP及其与V_1a受体(V_1aR)拮抗剂共同处理1、6、12、24 h后，采用免疫组织化学技术及RT-PCR对AQP4蛋白和AQP4 mRNA进行检测，并对星形胶质细胞进行形态学观察。并对脑微血管内皮细胞进行培养，AVP处理后倒置显微镜下动态观察形态变化。 结果：500 nM的AVP处理6 h后，AQP4 mRNA表达开始升高（P＜0.01），到12 h达高峰（P＜0.01），24 h后仍维持在较高的水平（P＜0.05）。AVP处理后AQP4蛋白表达亦出现相似的变化。V_1aR拮抗剂干预后，AQP4蛋白及AQP4 mRNA表达与对照组比较未出现升高（P＞0.05）。此外，AVP干预24 h后星形胶质细胞出现胞体肿胀，突起回缩，加用V_1aR拮抗剂处理后该现象未出现。AVP干预对脑微血管内皮细胞形态没有影响。 结论：高水平的AVP能通过激活V_1aR而诱导AQP4 mRNA和AQP4蛋白高表达，导致星形胶质细胞胞体肿胀，突起回宿，失去正常星形形态。AVP对AQP4表达有调控作用，借此可对星形胶质细胞体积进行调节，但对脑微血管内皮细胞无此作用。第五部分精氨酸加压素诱导水孔蛋白-4表达及细胞凋亡机制的研究目的：探讨在精氨酸加压素（arginine vasopressin，AVP）在上调AQP4（aquaporin-4，AQP4）表达过程中，p38 MAPK信号通路的作用及对星形胶质细胞凋亡的作用。方法：大鼠大脑皮质分离星形胶质细胞，传代培养纯化至98％以上。星形胶质细胞经分别用AVP、V_1a受体(V_1aR)拮抗剂和SB 203580进行处理，采用免疫组织化学技术及RT-PCR对AQP4 mRNA进行检测，Western blot检测p38 MAPK信号通路在AVP诱导AQP4表达中的活化程度，及Caspase-3 P20活性片段。同时对星形胶质细胞进行形态学和细胞活性观察。结果：500 nM的AVP处理6 h后，AQP4 mRNA表达开始升高（P＜0.01），到12 h达高峰（P＜0.01），24 h后仍维持在较高的水平（P＜0.05）。加入p38 MAPK抑制剂SB 203580干预后，AQP4 mRNA表达水平与对照组比较差异不显著（P＞0.05）；AVP处理15 min后p38 MAPK磷酸化水平开始增加，30 min达高峰，持续到60 min开始下降。V_1aR拮抗剂处理后p38 MAPK磷酸化水平整个时间段均未出现明显变化；AVP作用后6h Caspase-3蛋白表达开始增加（P＜0.01），12 h时表达达高峰（P＜0.01），到24 h仍高于正常水平。此外，AVP干预后细胞活性下降，凋亡细胞增多，SB 203580及V_1aR拮抗剂对AVP诱导的细胞凋亡均有抑制作用。结论：AVP通过激活V_1aR引起p38 MAPK信号通路活化从而诱导AQP4 mRNA高表达，从基因水平对AQP4进行调节，并增加Caspase-3活性，使星形胶质细胞发生凋亡。p38 MAPK抑制剂及V_1aR拮抗剂抑制AQP4 mRNA的表达，对星形胶质细胞具有保护作用。更多还原

【Abstract】 Part IAnalysis of the expression of aquaporin-4 in experimental intracerebralhemorrhage ratsObjective: To study the expression of AQP4（aquaporin-4） in order to find the pathologic effect of AQP4 protein on brain edema formation after hematoma formation.Methods: Intracerebral hemorrhage （ICH） models were established by infusing autologous caudate artery blood into caudate-putamen nucleus （CPu）, in addition, 15 U thrombin was injected into CPu. the expression of AQP4 protein was detected by immunohistochemical technique.Results: 6 hour after ICH, the expression of AQP4 protein in astrocytic end-feet adjacent to capillaries around the hematoma obviously increased（P<0.01）, especially on the 1th day and 3th days（P<0.01）, and went down to the level which is above the norml level gently on the 7th day（P<0.01）. In thrombin treated rats, AQP4 protein expression increased occurred at 6th hour, especially on 1th day and the 3th day （P<0.01）. From the 5th day, AQP4 protein expression decreased to normal level （P>0.05）.Conclusions: AQP4 protein played a very important role in the formation of brain edema after ICH, expression of AQP4 protein may be induced by thrombin.Part II Thrombin induced aquaporin-4 expression in rat primary astrocytesObjective: To study the biologic effects of various concentrations of thrombin on the aquaporin-4（AQP4） expression in rat primary cultured astrocytes, and explore the regulation mechanism of transmembrane water transportation in astrocytes after intracerebral hemorrhage （ICH）.Methods: Primary cultured astrocytes were incubated in culture medium containing various concentrations of thrombin for 24 h and harvested for determination of AQP4 mRNA and AQP4 protein expression by reverse transcription polymerase chain reaction （RT-PCR） and immunohistochemical technique. Cell apoptosis was detected by TdT-mediated dUTP nick end labeling （ TUNEL ） technique. Cell morphology was observed by phase contrast microscope, and cell viability was assayed by MTT.Results: AQP4 mRNA and protein showed a weak expression in normal astrocytes. The expression of AQP4 mRNA and AQP4 protein significantly increased in astrocytes following the higher concentrations of thrombin （100 U/ml, 200 U/ml）treatment, which resulted in markedly astrocyte swelling. The number of TUNEL positive cells significantly increased. Otherwise, the downregulated AQP4 mRNA and AQP4 protein occurred in the low concentrations thrombin（0.5 U/ml, 1 U/ml） treated astrocytes, cell volume did not increase in astrocytes. Few TUNEL positive cells were observed.Conclusions: The AQP4 overexpression induced by high concentrations of thrombin causes a increase in astrocytic membrane permeability to water. On the contrary, the decreased AQP4 expression refers to the neuroprotective mechanism of low concentrations of thrombin, and avoids atrocyte volume increase and apoptosis in the cases of the brain insults. Part IIIaquaporin-4 expression and the permeability of blood-brain barrierObjective: To investigate the relationship between aquaporin-4（AQP4） expression and the permeability of blood-brain barrier （BBB）, explore the possibility for V_1a receptor (V_1aR antagonist)antagonist in therapy of brain edema .Methods: Intracerebral hemorrhage （ICH）models were established by injection of autologous caudate atery blood into the caudate-putamen nucleus （CPu）. After successful surgergy, in treated group 0.1μg V_1aR antagonist was injected into lateral cerebral ventricle. In ICH group, the rats received intracerebroventricular injection of the equivalent artificial CSF. Immunohistochemistry method was adopted to analyse the expression of AQP4 protein around the microvessels. The BBB permeability was evaluated quantitatively by measuring Evans blue dye extravasations.Results: After experimental intracerebral hemorrhage, the AQP4 protein performed significant enhanced level in the ICH group, especially at 1 d, 3 d after ICH（P<0.01）. In addition, the expression of AQP4 was positively correlated with the permeability of BBB. When the rats received V_1aR antagonist treatment, AQP4 protein showed a lower expression than that in the control group（P<0.01）. The BBB permeability showed a significant decrease at every time point in the treated group comparing with the control group（P<0.05）.Conclusions: There is a close relationship between AQP4 expression and permeability of BBB. Furthermore V_1aR antagonists may protect the BBB and reduce brain edema in the perihematoma region after ICH by downregulating the expression of AQP4 protein. Part IVEffect of arginine vasopressin on aquaporin-4 expression in primarycultured astrocytesObjective: To investigate the effects of arginine vasopressin （AVP） on the aquaporin-4（AQP4） expression and cell volume in primary cultured astrocytes.Methods: Astrocytes were purified to >98% by passage from new-born rat cerebral cortex. Primary cultured astrocytes were incubated in culture medium containing 500 nM AVP or 500 nM V_1a receptor (V_1aR) antagonist for 1, 6, 12, 24 h, harvested for determination of AQP4 protein and AQP4 mRNA expression by immunohistochemical technique and reverse transcription polymerase chain reaction （RT-PCR）. Brain microvascular endothelial cells were cultured in vitro and treated with AVP. Cell morphology was observed by a phase contrast microscope.Results: After astrocytes were treated with 500 nM AVP for 6 h, the expression of AQP4 mRNA began to increase（P<0.01）, at 12 h reached expression peak（P<0.01）, at 24 h the AQP4 mRNA still maintained the higher expression（P<0.01）. The similar change was showed for AQP4 protein. AVP treatment resulted in significant astrocyte volume increase at 24 h, V_1aR antagonist inhibited upregulation of AQP4 expression and cell swelling. After AVP treatment, the morphology of endothelial cells remained as the normal.Conclusions: The results suggest high level of AVP may induce overexpression of AQP4 mRNA and AQP4 protein by activation of V_1aR, which increases water permeability of astrocyte membrane, and makes astrocyte swell. The morphology of endothelial cell was not affected by AVP. Part Varginine vasopressin induced expression of aquaporin-4 and astrocyte apoptosis by the way of p38 MAPKObjective: To determine the role of p38 MAPK in the aquaporin-4（AQP4） expression induced by arginine vasopressin（AVP）, investigate the effect of p38 MAPK on apoptosis in cultured rat astrocytes.Methods: Primary cultured astrocytes were treated with AVP V_1aR antagonist and SB 203580, harvested for determination of AQP4 mRNA expression by reverse transcription polymerase chain reaction （RT-PCR）. p38 MAPK phosphorylation and protein expression of Caspase-3 P20 were assessed by Western blot analysis. Cell viability was assayed by MTT, Cell apoptosis was detected by TdT-mediated dUTP nick end labeling （TUNEL ） technique.Results: After astrocytes treated with 500 nM AVP for 6 h, the expression of AQP4 mRNA began to increase（P<0.01）, at 12 h, reached expression peak（P<0.01）, at 24 h, the AQP4 mRNA still maintained the higher expression（P<0.01）. This course were not exhibited after SB 203580 treatment. p38 MAPK phosphorylation increased 15 min after AVP treatment, remained elevated level even at 120min. Protein expression of Caspase-3 were upregulated 6 h after AVP treatment（P<0.01）, it reached the peak at 12 h（P<0.01）. AVP treatment resulted in significantly apoptosis at 6 h、 12 h and 24 h （P<0.01）. V_1aR antagonist and SB 203580 inhibited this change.Conclusions: AVP may induce higher expression of AQP4 mRNA and apopotosis through stimulation of p38 MAPK in astrocytes, SB 203580 and V_1aR antagonist could inhibit upregulation of AQP4 mRNA in the AVP environment, protect astrocyte from apoptosis.更多还原