【作者】 唐颖馨；

【导师】 王伟；

【作者基本信息】 华中科技大学 ， 神经病学， 2009， 博士

【摘要】 第一部分Connexin43基因敲除小鼠的繁殖鉴定及小鼠局灶性脑缺血模型的建立目的：缝隙连接(gap junction，GJ)是由缝隙连接蛋白(connexin，Cx)组成的细胞间通道。在中枢神经系统中，缝隙连接广泛存在于胶质细胞之间，以及神经元和胶质细胞之间。在脑缺血状态下，这些通道仍然部分开放，在脑缺血的病理生理过程中扮演了重要的角色。在本部分研究中，我们探讨了connexin43基因敲除小鼠的繁殖及鉴定，并用大脑中动脉闭塞及光化学两种造模方法，在该小鼠建立了局灶性脑缺血模型，用于后续研究。方法：connexin43基因敲除小鼠种鼠购自The Jackson Laboratory，独立送回风净化笼中饲养，繁殖。新生小鼠剪尾提取DNA后，PCR鉴定其基因型，分组用于后续实验。大脑中动脉闭塞模型采用线栓法。光化学血栓形成法采用腹腔注射玫瑰红后颅脑局部冷光源照射法。术后进行行为学评分，红四氮唑(TTC)染色观察脑梗死。结果：connexin43基因敲除小鼠种鼠繁殖情况良好。新生小鼠中，CX43-／-小鼠由于心脏右室流出道畸形，出生后即死亡；CX43+／-小鼠及CX43+／+小鼠存活率基本相当，基因型鉴定结果稳定。线栓法大脑中动脉闭塞缺血模型造模成功，缺血后小鼠体征明显，TTC染色提示缺血侧皮层及(或)皮层下局灶性梗死；光化学法皮层局灶性缺血模型造模成功，TTC染色提示冷光源照射处皮层局灶性梗死。结论：本部分研究成功繁殖了connexin 43基因敲除小鼠，基因型鉴定结果稳定。用线栓法和光化学法两种不同的造模方法制作小鼠局灶性脑缺血模型，模型成功率稳定。用于后期的相关机制研究。第二部分缝隙连接通讯对小鼠大脑中动脉闭塞后海马迟发性神经元死亡的影响及机制研究目的：海马(hippocampus)的血供并不来源于大脑中动脉，然而大脑中动脉缺血后却观察到了远隔的、非大脑中动脉供血区海马的迟发性神经元死亡(delayed neuronal death，DND)现象。在中枢神经系统中，广泛存在的缝隙连接(gap junction，GJ)，是小分子物质(如，钙离子)快速扩散和交流传播的通道。本部分实验观察了减少缝隙连接通讯，对大脑中动脉闭塞后海马迟发性神经元死亡的影响，并对其机制进行了初步的探讨。方法：根据基因型鉴定结果，实验小鼠分为假手术组、CX43+／+小鼠脑缺血组和CX43+／-小鼠脑缺血组，建立MCAO模型，术后1d，比较各实验组之间的神经功能评分，部分小鼠处死，TTC染色计算梗死灶体积。其余小鼠分别于缺血后3d、7d处死，断头取脑，利用尼氏染色和TUNEL技术检测海马神经元结构变化及丢失及凋亡细胞百分率；脑片钙成像技术观察海马区细胞内钙离子浓度：荧光定量试剂盒检测海马区钙蛋白酶(calpain)活性。缺血后30d，进行水迷宫测试，观察各组小鼠学习记忆功能(海马相关)有无差别。结果：假手术组小鼠术后1d，无神经功能缺失，TTC染色未见梗死区。缺血后3d、7d海马区均未见神经元凋亡：钙成像观察海马荧光弱：海马钙蛋白酶活性低。缺血30d后水迷宫测试，提示学习记忆功能正常。CX43+／+小鼠脑缺血组术后1d，神经功能缺失明显，TTC染色可见明显缺血侧梗死区。缺血后3d、7d，部分小鼠海马区可见神经元凋亡；钙成像可见海马区荧光强度显著增高：同时海马钙蛋白酶活性显著增高。缺血30d后水迷宫测试，提示学习记忆功能受损明显。相对野生型小鼠，CX43+／-小鼠术后1d，神经功能缺失显著降低，TTC染色提示梗死灶也明显减小。缺血后3d、7d，CX43+／-小鼠出现海马区DND(+)比例明显低于CX43+／+小鼠组，凋亡细胞百分率也低于CX43+／+小鼠组；钙成像的荧光强度较CX43+／+小鼠组弱，但较假手术组明显增强；海马钙蛋白酶活性较假手术组增高，但低于CX43+／+小鼠组。缺血30d后水迷宫测试，提示学习记忆功能受损，但程度较CX43+／+小鼠组轻。结论：减少缝隙连接通讯，减轻了MCAO后小鼠神经功能缺失症状，减小梗死体积，减少海马区的钙离子浓度增加及钙依赖性钙蛋白酶(calpain)活性，降低海马迟发性神经元死亡的发生率及DND(+)细胞百分率，改善缺血后远期的学习记忆功能。提示缝隙连接通讯在局灶性脑缺血后远隔的海马区域的迟发性损伤的形成中起到了一定的作用，可能与钙离子通过缝隙连接的传播和扩散有关。第三部分缝隙连接通讯对小鼠局灶性皮层缺血边缘区神经元凋亡的影响及机制研究目的：观察缝隙连接通讯对小鼠局灶性皮层缺血边缘区神经元凋亡的影响，并初步探讨其信号通路。方法：根据基因型鉴定结果，实验小鼠分为假手术组、CX43+／+小鼠脑缺血组和CX43+／-小鼠脑缺血组，建立光化学血栓形成模型。分别于缺血后2h、6h、12h、1d、3d、7d处死动物，断头取脑，免疫荧光技术观察缺血边缘区神经元内calpain、Bax、caspase12及活化的caspase3蛋白表达，计算阳性细胞百分比。结果：(1)假手术组可见极少量活化的caspase3蛋白，缺血后，CX43+／+小鼠脑缺血组和CX43+／-小鼠脑缺血组活化的caspase3蛋白的表达明显增多，随时间呈逐渐上升趋势，7d时达到顶峰，CX43+／+小鼠脑缺血组的表达多于CX43+／-小鼠脑缺血组，在2h和7d时，两组差异明显。(2)假手术组可见calpain蛋白表达，缺血后，CX43+／+小鼠脑缺血组和CX43+／-小鼠脑缺血组calpain蛋白的表达明显增多，1d时达到顶峰，后又下降，CX43+／+小鼠脑缺血组的表达多于CX43+／-小鼠脑缺血组，在12h和1d时，两组差异明显。(3)假手术组可见caspase12蛋白表达，缺血后2h，CX43+／+小鼠脑缺血组caspase12蛋白表达迅速增高并达到顶峰，随后下降，1d时再次增高，呈双峰型；CX43+／-小鼠脑缺血组caspase12蛋白的表达峰值推后，于缺血后6h达到顶峰，后逐渐下降，7d时稍有上升，略呈双峰型；在2h、6h、12h和1d时，两组差异明显。(4)假手术组可见Bax蛋白表达；缺血后2h，CX43+／+小鼠脑缺血组Bax蛋白表达迅速增高，随后下降，1d时再次增高，并达到顶峰，且核表达增加明显，呈双峰型；CX43+／-小鼠脑缺血组缺血后2h即开始增高，逐渐缓慢上升，峰值出现在缺血后3d；CX43+／+小鼠脑缺血组的表达多于CX43+／-小鼠脑缺血组，在2h、6h、1d、7d时，两组差异明显。结论：局灶性皮层缺血后缺血边缘区进入凋亡的神经元逐渐增多；calpain的激活是神经元凋亡的机制之一：Bax(线粒体通路)和caspase12(内质网通路)参与了calpain相关的信号转导：CX43+／-小鼠脑缺血组Bax和caspase12的表达少于CX43+／+小鼠脑缺血组，差异明显，且峰值表达时间点推后。更多还原

【Abstract】 Part One To make photothrombosis and middle carotid arteryocclusion model in heterozygous connexin 43 null miceObjective: Gap junctions assemble astrocytes into syncytia, allowing exchange ofmetabolites, catabolites, and second-messenger molecules. Connexin 43 is the predominantconnexin of astrocytic gap junctions. Astrocytic gap junctions remain open during ischemicconditions. Gap junctions therefore may link ischemic astrocytes in an evolving infarct withthe surroundings. In this part, we used the heterozygous connexin 43 null mice, whichexhibit reduced cx43 expression, to made photothrombosis and MCAO model.Materials and Methods: Heterozygous connexin 43 null mice mice were buyed fromThe Jackson Laboratory. The genotypes of the newborns were judged by protocols fromThe Jackson Laboratory. According to their genotype, mice were divided into 2 groups:cx43+/+ group and cx43+/- group. Middle carotid artery occlusion （MCAO） model andphotothrombosis model were made. Behavior test were carried out 24h after ischemia andthe infarct volume were calculated by TTC staining.Results: According to protocol from The Jackson Laboratory, the genotypes ofnewborn mice were judged successfully. Although cx43-/- die immediately after birth,heterozygous cx43 null mice cx43+/- are viable. Middle carotid artery occlusion （MCAO）model and photothrombosis model are successfully made in heterozygous cx43 null miceand the wildtyp ones. The infracted tissue are white with TTC staining.Conclusion: Middle carotid artery occlusion （MCAO） model and photothrombosismodel are successfully made in heterozygous cx43 null mice. Part Two Effect of gap junction communication on delayedneuronal cell death in mouse hippocampus after MCAOObjective: Delayed neuronal death （DND） was found in hippocampus, which is notsupplied by middle carotid artery, after middle carotid artery occlution （MCAO）. In brain,astrocytes are coupled extensively by gap junctions, which are highly conductive channelsthat allow the direct transfer of intracellular messengers such as Ca²⁺ and inositoltriphosphate （IP3） between interconnected cells. In this part, we observed the effect ofblocking gap junction communication after MCAO on the delayed neurnal death inipsilateral hippocampus and explored the mechanism.Materials and Methods: Mice were divided into CX43+/+ sham group, CX43+/-sham group, CX43+/+ MCAO group and CX43+/- MCAO group, according to theirgenotypes, 1d after surgery, behavior test and TTC staining were carried out. Then micewere killed at 3d and 7d post ischemia respectively, and the samples were harvested forfurther use. Neuronal cell popotosis were measured by TUENL; the calcium in neurons ofhippocampus were measured by two-photon calcium imaging; the calpain activity weremeasured by Calpain Activity Assay Kit and water maze were carried out 30d afterischemia.Results: Our results showed that compared with CX43+/+ MCAO group, behaviortest scores and infarct volum were low in CX43+/- MCAO group. DND were detected infewer mice in CX43+/- MCAO group and the calcium concentration and calpain activitywere low in this group. 30 days after ishchmia, mice of CX43+/- MCAO groupperformaced better in water maze task than those of CX43+/+ MCAO group.Conclusion: Blocking gap junction communication may reduce the infart volume and incidence rate of delayed neuronal death in hippocampus after middle carotid arteryocclusion. The free exchange of intracellular calcium between dying cells and those inremote region might contribute to expansion of ischemic damage. Part Three Effect of gap junction communication on neuronalcell death in perilesional zone after focal cerebral ischemia in miceObjective: To explore the effect of gap junction communication on neuronal cell deathin perilesional zone after focal cerebral ischemia in mice and the mechanism.Methods: Mice were divided into CX43+/+ sham group, CX43+/- sham group,CX43+/+ ischemia group and CX43+/- ischemia group, according to their genotypes.Photothrombosis were made. Mice were killed at 2h, 6h, 12h, 1d, 3d and 7d post ischemiarespectively, and then the samples were harvested for further use. calpain、Bax、caspase 12、and actived-caspase3 were detected by immunofluorescence.Results: （1） Actived-caspase3 protein increased post ischemia and actived-caspase3positive cells were fewer in CX43+/- ischemia group. （2） Calpain protein increased postischemia and reach the summit at 1 day post ischemia. Calpain-positive cells were fewer inCX43+/- ischemia group. （3） Caspase 12 protein increased post ischemia and reached thesummit at 2h post ischemia in CX43+/+ ischemia group, but the time in CX43+/- ischemiagroup was 6h. Caspasel2-positive cells were fewer in CX43+/- ischemia group. （4） Baxprotein increased post ischemia and reached the summit at 1d post ischemia in CX43+/+ischemia group, but the time in CX43+/- ischemia group was 3d. Bax-positive cells werefewer in CX43+/- ischemia group.Conclusion: Apopotosis neurons increased in prelesional zone post focal ishchemia.Calpain, Bax and caspase12 take part in the neurnal apoptosis. The Bax- andcaspase12-positive cells were fewer in CX43+/- ischemia group.更多还原