【作者】 李树基；

【导师】 高天明；

【作者基本信息】 南方医科大学 ， 神经生物学， 2009， 硕士

【摘要】 脑血管病发病率高,严重危害人类健康,是当今第三大死亡原因及首位致残原因。脑缺血/缺氧损伤所致的神经元大量死亡是导致中风后神经功能丧失的主要原因,而目前对缺血性脑损伤的机制仍未阐明,临床也无理想的治疗手段。脑中风可引起缺血中心区神经元的急性损伤,以及缺血半暗区的迟发性神经元死亡(Delayed Neuronal Death,DND),半暗区的细胞死亡过程是可以通过治疗干预来逆转的。因此,研究迟发性神经元死亡的发生机制对发现治疗时间窗长的脑保护靶点有重要的意义。钙超载学一直在脑缺血后迟发性神经元死亡发生机理研究中占有统治地位,但针对钙超载学说而研发的NMDA受体阻断剂因其短暂的治疗时间窗(1h之内)及严重的副作用而在临床试验中宣告失败。因此,发现非NMDA受体新型信号通路以及寻找治疗时间窗长和副作用小的神经保护靶点成为十分迫切的课题。我们实验室以往在体和离体研究均提示BK通道活动增强是海马神经元缺血/缺氧损伤的重要机制之一。本研究在氧葡萄糖剥夺(oxygenand glucose deprivation,OGD)诱导的离体培养海马神经元死亡模型上,进一步探讨了BK通道活动增强的机制及BK通道阻断剂神经保护作用的时间窗。实验取原代培养12天海马神经元建立氧葡萄糖剥夺模型,缺氧缺糖4h,恢复正常培养条件后继续培养48h,OGD可诱导约20%神经元死亡。为探讨缺血/缺氧后BK通道活动增强的机制,我们检测了复氧后BK通道在细胞膜上水平的变化。于复氧后0h,1h,3h,6h,18h取全细胞做Biotinylation assay提取细胞膜蛋白,SDS-PAGE电泳及Western blot,分别检测总蛋白和膜蛋白中BK通道表达的变化。与正常培养神经元相比,OGD刚结束时(0h)BK通道总表达水平下降,差别有统计学意义(P＜0.05),1h后恢复正常表达水平,之后BK通道总表达水平无明显变化(P=0.093)。与此相反,复氧后BK通道膜上的表达水平呈持续升高(P＜0.05),复氧后6h,18h时,分别增加为对照组的2.03和2.30倍,差别有统计学意义(P＜0.05)。OGD复氧同时培养基内给予不同浓度(1μM、5μM、10μM和20μM)BK通道特异性阻断剂paxilline,48h后用DAPI染核检测神经元凋亡率或MTT比色法检测细胞活性。Paxilline呈浓度依赖性保护OGD诱导的神经元死亡,10μM paxilline达到最大保护作用。为了进一步探讨paxilline神经保护的时间窗,分别在复氧后0h,6h,12h加入BK通道阻断剂paxilline(10μM),复氧48h用MTT法检测细胞的存活率,不同时间点给药均能提高细胞的存活率,对OGD诱导的神经元死亡有完全保护作用,但不同时间点给药组间没有统计学差异(P＞0.05)。进一延迟给药时间至18h,24h,与OGD组相比,各时间点给药均可减少缺氧复氧诱导的神经元死亡50%左右,18h和24h给药仍有保护作用。BK通道特异性开通剂NS1619能诱导正常培养神经元死亡,与对照组相比,死亡率高出20%(P＜0.05),NS16196处理4h和48h存活率均有降低(P＜0.05)。为了进一步证明paxilline的作用靶点是BK通道,我们用BK通道特异性开通剂NS1619与paxilline共同作用于细胞,NS1619(20μM)本身并不能加重缺氧复氧后神经元的损伤但可完全阻断paxilline(10μM)对缺氧复氧所致神经元损伤的保护作用(P＜0.05)。综上所述,BK通道在氧葡萄糖剥夺后膜上的表达水平增加,BK通道特异性阻断剂paxilline能保护氧葡萄糖剥夺诱导的海马神经元死亡,且复氧后24小时给药仍有保护作用,而开通剂NS1619则能诱导正常培养海马神经元死亡且可完全阻断paxilline的神经保护作用。所以,BK通道可能是脑中风治疗的一重要潜在神经保护靶点。更多还原

【Abstract】 Stroke has a high incidence and is harmful to human health,it is a leading cause of death and the third cause of disability.The loss of neurological functions following stroke is caused by massive loss of neurons resulting from hypoxic-ischemie insults. The mechanism underlying the brain ischemic injury is unclear and there is no effective clinical treatment yet.Up to now,several hypotheses have been put forward, for example,Ca²⁺ overload,free radical and so on.The acute neuronal damage is followed by a second round of neuronal injury that occurs hours to days after brain ischemia in the neighboring areas,which called delayed neuronal death（DND）.So it’s very important to study the mehanism underlying DND for prolonging the timescale of clinical treatment.Most recently,it is suggested that over-activation of K+ channel may be involved in neuronal death induced by different insults.We have previously shown that the enhancement of BK channel activation is an important mechanism mediating hippocampal neuronal damage both in vivo and in vitro.The present study focuses on the memchanism underlying enhancement of BK channel activation after hypoxic-ischemic and the neuroprotective timescale of BK channel antagonist.Cultured neurons were prepared from neonatal Sprague-Dawley rats.Neurons cultured for 12 days in vitro were subjected to OGD for 4 hours and then reoxygenated for untill being treated,and cell death was assessed by the fuorescent dye DAPI or MTT cell viability assay.Statistical comparisons of pooled data were performed independent samples t-tests and one-way analysis of variance（ANOVA） （SPSS 13.0）,statistical differences were evaluated by one-way analysis of variance followed by LSD-t test or S-N-K for multiple comparison.The results showed that OGD induced about 20%of neuronal death at 48 h after reoxygenation（P＜0.05）. Treatment with EBSS containing 1g/L glucose didn’t affect the survial rate of normal cultured neurons.To investigate the memchanism underlying enhancement of BK channel activation after hypoxic-ischemic,membrane protein was extracted by biotinylation assay and performed western blot with whole cell lysis together.The total BK channel expression decreased just after reoxygenation and returned to normal level after 1 hour（P=0.028）,while the surface level increased by about 2 folds 6 hours after reoxygenation(（P＜0.05）.Application of a specific BK channel blocker,paxilline（5μM、10μM and 20μM） after reoxygenation showed completly protective effect in a concentration-dependent manner（P＜0.05） and achieved maximal protective effect at 10μM.Paxilline didn’t affect the survial rate of normal cultured neurons.To investigate the neuroprotective timescale of paxilline, paxilline（10μM） was added at different time points（0h,6h,12h ） after reoxygenation. Data showed that paxilline could completly protect nerons from death induced by OGD.Further prolonging the timescale,paxilline showed neuroprotective effect at 24h after reoxygenation.Treatment with a specific opener of BK channel,NS 1619（20μmol/L） for 4 hours or 48 hours,both caused about 30%normal cultured cells died （P＜0.05）.In order to make sure that paxilline tageted at BK channel,paxilline was added to medium with NS1619.Our data showed that NS1619 could counteract the neuroprotective effect of paxilline but didn’t aggravate OGD-induced cell damage.In conclusion,The total BK channel expression decreased just after reoxygenation and return to normal 1h later,while the surface level remained increased throughout the 24h time period after reoxygenation.Application of a specific BK channel blocker,paxilline protected neuronal death completely and toke effect even 24h after reoxygenation.A specific opener of BK channel,NS1619, caused about 30%normal cultured cells died and it could also counteract the protective effect of paxilline..Accordingly,the BK channel is a potential molecular target for neuroprotective therapy in stroke.更多还原