【作者】 张扬；

【导师】 王亮； 董强； 肖保国；

【作者基本信息】 复旦大学 ， 神经病学， 2010， 硕士

【摘要】 缺血性脑卒中是引起人类死亡和致残的主要疾病之一。神经丝氨酸蛋白酶抑制剂(neuroserpin,NSP)属于serpins超家族,是一种轴突选择性的神经丝氨酸蛋白酶抑制剂,主要在中枢神经系统(Central nervous system,CNS)表达。NSP参与调控CNS细胞外组织型纤溶酶原激活物(Tissue plasminogen activator, tPA)、纤维蛋白溶酶的蛋白水解的活性。研究表明,NSP对缺血的神经元或脑组织存在保护作用。应用NSP治疗急性脑缺血患者可以降低缺血灶的体积,显著减少血脑屏障(Blood-brain barrier,BBB)渗漏、脑水肿程度,减少缺血再灌注所致的细胞凋亡。我们前期的研究,从体外途径证实了NSP可减轻糖氧剥夺(Oxygen-glucose deprivation,OGD)及tPA对神经元的毒性损伤而起到保护神经元的作用,可以抑制缺氧复氧干预引起的小胶质细胞(Microglia,MG)的活化和增殖,并且可以减少MG的白介素-1β(Interleukin-1β,IL-1β)和一氧化氮(Nitric oxide,NO)等自由基和炎性因子的释放。本研究观察星形胶质细胞缺氧复氧后细胞活性的变化,研究NSP预处理对缺氧复氧后的星形胶质细胞活性是否存在影响。最后,探讨NSP对缺氧复氧后的星形胶质细胞的这种影响的可能机制。我们拟通过本研究进一步明确NSP神经保护机制。第一部分星形胶质细胞的培养和OGD后复糖复氧模型的建立目的：构建星形胶质细胞OGD后复糖复氧模型为下一步实验创造条件。方法：体外传代培养SD大鼠皮层星形胶质细胞并鉴定。建立缺氧复氧(hypoxia/reoxygenation、H/R)模型。结果：1、传代培养的星形胶质细胞经GFAP特异性抗体的免疫荧光染色证实纯度达95%以上2、星形胶质细胞在糖氧剥夺6小时以后,细胞活性逐渐下降,及至12小时,星形胶质细胞活性已明显低于正常对照(P<0.05)。星形胶质细胞在糖氧剥夺12小时后再复糖复氧时,随着复氧时间的延续,其活性持续下降。至复糖复氧24小时,星胶细胞活力只有OGD前的一半左右。结论：传代星形胶质细胞缺氧复氧模型有效。第二部分NSP对OGD后复糖复氧星形胶质细胞的影响目的：利用建立的OGD模型,探讨NSP能否影响OGD后复氧星形胶质细胞的活性,并检测其炎症介质释放的变化情况。方法：1、体外传代培养大鼠皮层星形胶质细胞并鉴定。建立星形胶质细胞缺氧复氧模型。2、检测不同浓度NSP对缺氧复氧(Hypoxia/reoxygenation,H/R)星形胶质细胞的影响。细胞随机分成正常组、H／R组(OGD12小时后复糖复氧24小时)NSP+H/R(分为1.25、2.5、5、10、20、40ng／ml五个终浓度预处理星形胶质细胞1小时,然后OGD12小时后复糖复氧24小时)。利用MTT法测定细胞存活率、LDH法测定细胞损伤程度。3、NSP (5ng/ml)预处理对H／R星形胶质细胞的影响。细胞随机分成正常组、H／R组(OGD12小时复糖复氧0、2、4、6、8、10、12、24小时)、NSP+H/R (5ng/mlNSP预处理星形胶质细胞1小时,然后OGD12小时复糖复氧0、2、4、6、8、10、12、24小时)。利用MTT法测定细胞存活率；流式细胞术,行Annexin V-FITC和PI检测各组星形胶质细胞凋亡率。4、NSP (5ng/ml)预处理对H／R星形胶质细胞释放自由基NO及IL-1β、TNF-α等细胞因子的影响。细胞随机分成正常组、H／R组(OGD12小时后复糖复氧0、6、12小时)、NSP+H/R (5ng/ml NSP预处理星形胶质细胞1小时,然后OGD12小时复糖复氧0、6、12小时)。以ELISA对细胞培养上清液NO、IL-1β及TNF-α的含量分别进行测定。结果：1、不同浓度的NSP预处理后,OGD12小时后复糖复氧24小时(OGD12h R24h)的星形胶质细胞的活力较对照组有明显的升高,而细胞毒性明显下降。在NSP较低浓度时,随着其浓度的增加,星胶细胞的活力呈梯度上升,并于5ng／ml时达到其最大效应。2、NSP预处理的星形胶质细胞,OGD12小时后复糖复氧不同时间的星形胶质的活力较相应复氧时间的对照组有明显的提升。流式细胞术结果显示,NSP预处理的星形胶质细胞在OGD12h R24h时细胞凋亡程度比对照组明显减轻。3、NSP预处理的星形胶质细胞,在缺氧复氧过程中释放自由基NO以及IL-1β及TNF-α等炎性介较对照组均有不同程度的减少。结论：1、NSP预处理能够减轻星形胶质细胞在H／R过程中的活性下降,并发挥其抗凋亡作用。2、经NSP预处理,星形胶质细胞对H／R过程中的炎性反应有一定程度的抑制。第三部分Neuroserpin对星形胶质细胞作用的机制目的：探讨NSP预处理对其有丝分裂原激活蛋白酶(Mitogen activation protein kinase,MAPK)、丝／苏氨酸蛋白激酶B (Serine/threonine protein kinase B,Akt)和核转录因子κB (nuclear factor-κB,NF-κB)等各信号通路的变化的影响。方法：体外培养传代大鼠皮层星形胶质细胞并鉴定。细胞随机分为正常组、H／R组(OGD12h R24h)、NSP+H/R组(5ng/mlNSP预处理星形胶质细胞1小时,然后OGD12h R24h)。提取各处理组细胞的蛋白,以免疫印迹法分别检测各处理组MAPK、Akt和NF-κB等各信号通路的主要活性蛋白表达情况。结果：1、星形胶质细胞在H／R后,MAPK信号通路的几个磷酸化蛋白p-ERK1/2, p-JNK,p-P38的表达均比正常明显升高。NSP预处理,并不影响MAPK三条主要信号通路的激活程度。2、星形胶质细胞在H／R后,Akt信号通路的磷酸化蛋白p-Akt表达比正常明显升高。NSP预处理,并不影响Akt信号通路的激活程度。3、星形胶质细胞在H／R后,胞浆p-IKKα/β及胞核NF-κB P65水平显著升高,提示NF-κB信号通路的激活。而NSP预处理组,胞浆p-IKKα/β及胞核NF-κB P65的水平较缺氧复氧对照组明显低,即NF-κB信号通路受抑制。结论：星形胶质细胞在H／R后,MAPK, PI3K/Akt以及NF-κB等几条信号通路均较正常组有不同程度的激活。而NSP预处理组,NF-κB通路激活可受到明显抑制。结论1、NSP预处理能够减轻星形胶质细胞在H／R过程中的活性下降,并发挥其抗凋亡作用。2、NSP预处理后,星形胶质细胞在H／R过程中炎性反应受到一定程度的抑制。3、星形胶质细胞在缺氧复氧过程中,包括MAPK、PI3K/Akt、和NF-κB等主要的细胞存活、凋亡相关的信号通路均被有不同程度的激活。4、经NSP预处理的星形胶质细胞,NF-κB通路激活可受到明显抑制。更多还原

【Abstract】 Ischemia Stroke is one of the main diseases that cause unnature death and disability. Neuroserpin(NSP), mainly expressed in centrol nervers system, is one of the members of serpins super family. NSP is a inhibitor of neurogenic tissue plasminogen activator(tPA),and participate in the regulation of the activity of proteolysis of extracellular tPA and plasmase. Some researches show that NSP can protect neuron from death in ischemic stroke. In the early stage of ischemic stroke,the application of NSP can reduce the volume of infarction,the degree of edema,the leakage of the blood-brain barrier(BBB) extremely. And finally alleviate the apopotosis of neuron after reperfusion. In our previous research,we demonstrated that NSP can alleviate the toxic injury of neuron after oxygen and glucose deprivation (OGD) in vitro. We also found that, NSP can inhibit the activation of microglia after OGD,and reduced the secretion of IL-1β，NO. In this research, we observed the change of activity of astrocytes after OGD injury.We also investigated that after NSP pre-disposal,whether the activity of astrocytes changed after OGD and what mechanisms involved. After this research,we want to go to a step further to identify the neuroprotection effect of NSP.PartⅠThe subcultured astrocytes and the build up of the hypoxia/reoxygenation modelObjective:To build up the hypoxia/reoxygenation(H/R) model.Methods:We used subcultured cortical astrocytes of SD rats and verificated it with immunofluorescence double label,then built up the H/R model.Results:1. The purity of subcultured astrocytes reach 95% with the immunofluorescence double label of GFAP. 2. Six hours after OGD,the activity of astrocytes begin to descend gradually. And 12 hours after OGD,the activity below normal group obviously(P<0.05). After OGD 12h, the activity of astrocytes descend gradually along with the time of reoxygenation. And,when the reoxygenation last for 24 hours, the activity of astrocytes remains about 50% compared with the activity of Astrocytes before OGD treatment.Conclusions:The hypoxia/reoxygenation(H/R) model of subcultured cortical astrocytes is effective.PartⅡThe effect of NSP on Astrocytes undergoing Hypoxia/ReoxygenationObjective:To investigate the effect of NSP on the activity and the release of inflammatory mediators of Astrocytes undergoing Hypoxia/Reoxygenation.Methods:1. To subcultured astrocytes and build up the H/R model.2. Investigate the effect of NSP,with different concentration, on the activity of astrocytes after H/R. The cells were randomly divided into three groups.The first group is normal group;The second group is H/R group(OGD12h and then reoxygenation 24h).The third group is H/R with NSP predisposal(This group of cell was treated with NSP for 1 hour and then OGD12h reoxygenation 24h. The final concentration of NSP was 1.25、2.5、5、10、20、40ng/ml.). We tested the activity and the injury of astrocytes with the method of MTT and LDH respectively.3. The effect of NSP(5ng/ml) predisposal on astrocytes after H/R. The cells were randomly divided into three groups. The first group is normal group;The second group is H/R group(OGD12h and then reoxygenation 0、2、4、6、8、10、12、24h);The third group is H/R with NSP predisposal(This group of cell was treated with NSP for 1 hour and then OGD12h reoxygenation 0、2、4、6、8、10、12、24h. The final concentration of NSP was 5ng/ml.).At each time pot,we tested the activity of astrocytes with the method of MTT. And then,we tested the apoptosis rate with Annexin V-FITC/PI(Flow cytometry).4. The effect of NSP(5ng/ml) predisposal on the secretion of NO、IL-1β、TNF-αof astrocytes after H/R. The cells were randomly divided into three groups. The first group is normal group;The second group is H/R group(OGD12h and R0、6、12h);The third group is H/R with NSP predisposal(This group of cell was treated with NSP for 1 hour and then OGD12h R0、6、12h. The final concentration of NSP was 5ng/ml.). At each time pot,we tested the concentration of NO、IL-1βand TNF-a in the culture Supernatant of astrocytes.Results:1. After the predisposal of NSP with different concentration,the activity of astrocytes(H/R) enhance extremely, while the toxic substances release of astrocytes reduced. At the low concentration stage, the activity of astrocytes enhance with the increase of the concentration of NSP. And, at the concentration of 5ng/ml, NSP exhibit the best protection effect.2. After the predisposal of NSP, the activity of astrocytes elevated compared with the control group at each time pot of reoxygenation. As to the results of Flow cytometry, we found that the apoptosis rate of astrocytes(H/R) reduced after the predisposal of NSP.3. After the predisposal of NSP, the secretion of NO、IL-1βand TNF-αof astrocytes reduced at the the stage of reoxygenation.Conclusions:1. The NSP predisposal enhance the activity and exhibit the anti-apoptosis effect of astrocytes undergoing H/R.2. The NSP predisposal inhibits the inflammatory reaction of astrocytes undergoing H/R.PartⅢThe mechanisms of the protective effect of neuroserpinObjective:To study the signal transduction mechanism of astrocytes and to investigate the possible mechanism of the protective effects of NSP by H/R which can activate Astrocytes.Methods:We used subcultured astrocytes and built the H/R model. The cells were randomly divided into three groups:normal group, H/R group (OGD12h reoxygenation 24h), and NSP+H/R group (This group of cell was treated with NSP for 1 hour and then OGD12h reoxygenation 24h. The final concentration of NSP was 5ng/ml). Expression of MAPK,AKT and NF-κB was determined by Western-blot.Results:1. The western-blot show that small quantity expression of phospho-ERK, phosphor-JNK and phospho-P38 in normal astrocytes and the level increased obviously under the stimulation of H/R. While,the activation of MAPK was not influenced by the predisposal of NSP.2. The western-blot show that small quantity expression of p-AKT in normal astrocytes and the level increased obviously under the stimulation of H/R. While,the activation of MAPK was not influenced by the predisposal of NSP.3. The western-blot show that small quantity expression of p-IKKα/βand NF-κB P65 (nuclear) in normal astrocytes and the level increased obviously under the stimulation of H/R. After the predisposal of NSP, the activation of NF-κB was inhibited extremely.Conclusions:The signal transduction of MAPK, PI3K/Akt and NF-κB of astrocytes was activated after H/R. And the predisposal of NSP inhibite the activation of NF-κB extremely.Conclusions:1. The NSP predisposal enhance the activity and exhibit the anti-apoptosis effect of astrocytes after H/R.2. The NSP predisposal inhibit the inflammatory reaction of astrocytes after H/R.3. The signal transduction of MAPK, PI3K/Akt and NF-κB of astrocytes was activated after H/R. And the predisposal of NSP inhibite the activation of NF-κB extremely.更多还原