【作者】 唐薇婷；

【导师】 肖波；

【作者基本信息】 中南大学 ， 神经病学， 2010， 博士

【摘要】 目的：观察TRPC蛋白在匹罗卡品致痫大鼠海马中的动态表达,探讨其在突触重建中的作用。方法：6-8周龄健康雄性SD大鼠108只,随机分为实验组(n=90)和对照组(n=18)。实验组采用氯化锂-匹罗卡品腹腔注射法建立颞叶癫痫模型；对照组大鼠腹腔注射等量无菌生理盐水。实验组按癫痫持续状态(SE)后1天、7天、15天、30天、60天分为5个亚组,每亚组18只大鼠。以上各亚组再分为3个小组,每小组6只大鼠,分别进行：①Western blot方法检测TRPC及突触重建标志蛋白Synaptophysin在海马中的表达；②Timm染色观察海马苔藓纤维出芽并评分；③免疫荧光双标方法检测海马BDNF与TRPC的表达,尼氏染色观察海马病理改变。对照组随机分为3个亚组,各亚组6只大鼠,分别进行以上三种检测。结果：1.实验组大鼠SE诱发成功率为88.9%,死亡率为22.2%,模型成功率为66.7%。2.实验组大鼠TRPC蛋白表达：①TRPC1蛋白表达量在SE后1d较对照组显著降低(P<0.01),7d降至最低,15d开始恢复但仍然低于正常,30d时显著上调(P<0.05),60d时达高峰(P<0.01)。②TRPC3蛋白表达量从SE后7d起进行性下降(P<0.01),60d时降至最低(P<0.01)。③TRPC4蛋白表达量在SE后1d显著增加达峰值(P<0.01),7d仍高于正常(P<0.01),15d显著下调(P<0.01),60d降至最低(P<0.01)。④TRPC5蛋白表达量从SE后1d起进行性下降(P<0.01),60d时降至最低(P<0.01)。⑤TRPC6蛋白表达量在SE后1d达高峰(P<0.01),其他时间点均显著高于正常(P<0.01)。3. Synaptophysin蛋白表达量在SE后7d、15d、30d、60d显著增加(7d,P<0.05；15d、30d、60d,P<0.01),30d达峰值(P<0.01)。4.实验组大鼠海马神经元缺失以SE后7d和60d最为明显。除齿状回颗粒细胞缺失相对较轻(P<0.05)外,CA区锥体细胞和门区神经元均显著减少(P<0.01)。5.实验组大鼠齿状回内分子层在SE后7d出现Timm颗粒,并呈进行性增加。6.两组各时间点均可检测到BDNF与TRPC1、TRPC3、TRPC4、TRPC5、TRPC6在大鼠海马各区共表达。结论：TRPC可能参与了苔藓纤维出芽,BDNF则可能介导了这一过程。目的：探讨干预BDNF对匹罗卡品致痫大鼠海马TRPC的表达及苔藓纤维出芽的影响。方法：6-8周龄健康雄性SD大鼠270只,随机分为K252a+Pilo组(n=90只),NS+Pilo (n=90)和K252a+NS组(n=90只)。K252a+Pilo组采用氯化锂-匹罗卡品腹腔注射法建立颞叶癫痫模型,在匹罗卡品注射前3小时予侧脑室注射K252a进行干预；NS+Pilo组大鼠在匹罗卡品注射前3小时予侧脑室注射无菌生理盐水；K252a+NS组大鼠侧脑室注射K252a,3小时后予腹腔注射无菌生理盐水。三组均选取腹腔注射后后1天、7天、15天、30天、60天为研究时间点,各时间点18只大鼠,分别进行：①Western blot方法检测TRPC及突触重建标志蛋白Synaptophysin在海马中的表达；②尼氏染色观察海马病理改变；③Timm染色观察海马苔藓纤维出芽并评分。结果：1. K252a+Pilo组SE诱发成功率(83.3%)低于NS+Pilo组(90.7%),SE诱发成功后生存状态好,死亡率(8%)同样低于NS+Pilo组(19.3%)。K252a+Pilo组SE诱导所需平均时间(55.84±17.44 min)较NS+Pilo组(27.80±13.58min)长,致痫所需Pilo平均剂量(34.84±9.44 mg/kg)较NS+Pilo组(24.80±5.58 mg/kg)大,而Ⅲ-Ⅴ级发作的平均持续时间(12.84±5.44 min)较NS+Pilo组(28.20±4.58min)短,终止发作所需水合氯醛平均总量(3.24±0.44 ml/kg)较NS+Pilo组(3.80±0.54 ml/kg)低,以上各差异均具有统计学意义(p<0.01)。2.与K252a+NS组相比：K252a+Pilo组TRPC1蛋白表达量在SE后1d显著上调(P<0.01),呈进行性上升,60d达高峰；TRPC3蛋白表达量在SE后1d显著下调(P<0.05),呈进行性下降,60d时降至最低；TRPC4蛋白表达量在SE后7d、15d、30d、60d显著下调(P<0.01),15d达最低值；TRPC5蛋白表达量在SE后1d显著上调(P<0.01),呈进行性升高,60d达峰值；TRPC6蛋白表达量在SE后1d显著性上调(P<0.01),但上调幅度逐渐减少,30d仍高于K252a+NS组(P<0.01),60d出现明显下调(P<0.01)。与NS+Pilo组相比：K252a+Pilo组各时间点TRPC1、TRPC4.TRPC5蛋白表达量均显著下调(P<0.01);TRPC3蛋白表达量在SE后1d显著下调至最低(P<0.01),随后逐渐恢复,在SE后7d、15d、30d仍下调(P<0.05或P<0.01),至60d显著上调(P<0.01)；而TRPC6在SE后1d显著上调(P<0.05),呈进行性上升,30d达高峰(P<0.01),60d显著下调(P<0.01)。3.与K252a+NS组相比,K252a+Pilo组Synaptophysin蛋白在SE后15d表达显著增多(P<0.01),呈进行性上升,至60d达最高峰(P<0.01)。与NS+Pilo组相比,K252a+Pilo组Synaptophysin蛋白的表达在SE后1d显著上调(P<0.01),呈进行性上升,至60d达最高峰(P<0.01)。4. K252a+Pilo组可见海马神经元缺失,以SE后7d和60d最为明显。除齿状回颗粒细胞缺失相对较轻(P<0.05)外,CA区锥体细胞和门区神经元均较K252a+NS组显著减少(P<0.01)。而K252a+Pilo组海马神经元缺失程度较NS+Pilo组则明显减轻,尤以SE后7d开始差异明显,具有统计学意义(p<0.05或p<0.01)。5. K252a+Pilo组海马齿状回内分子层在SE后15d开始出现Timm颗粒,随后进行性增加至本研究终点,与NS+Pilo组相应时间点相比出芽程度显著减轻(p<0.05)。结论：1.干预BDNF可减轻痫性发作的程度。2.干预BDNF可能通过改变TRPC的表达抑制苔藓纤维出芽。更多还原

【Abstract】 Objective To observe the dynamic changes of TRPC expression in the rat hippocampus after pilocarpine-induced seizures, and to investigate its involvement in synapse remodeling.Method 108 healthy male Sprague-Dawley (SD) rats were divided randomly into experimental group (n=90) and control group (n=18). The temporal lobe epilepsy model was established by intraperitoneal injection of lithium and pilocarpine, while the controls were injected with equal dose of saline (NS). The experimental rats were equally divided into 5 subgroups at time points 1 day、7 days、15 days、30 days and 60 days after status epileptics (SE). Each subgroup was subsequently divided into 3 panels (6 rats in each panel) for the following tests respectively:①expression of TRPC and Synaptophysin protein in rats’hippocampus by Western blot;②Timm staining and score;③co-expression of TRPC and BDNF in rats’hippocampus by double immunofluorescence and hippocampal pathology by Nissl staining. The control rats were equally divided into 3 subgroups and received the aforementioned tests. Results1. The lithium-pilocarpine model of TLE:SE rate reached 88.9%, total mortality rate was 22.2% and the succeful rate was 66.7%.2. The expression of TRPC protein in the hippocampus of experimental rats compared with the control rats:①The expression of TRPC1 protein markedly decreased from 1d after SE (P＜0.01), and reached a minimum on 7d. On 15d, it rebounded gradually yet was still lower than normal. Until 30d, it significantly increased and reached the peak on 60d (P<0.01).②The expression of TRPC3 protein gradually decreased from 7d after SE (P<0.01) and reached a minimum on 60d.③The expression of TRPC4 protein was markedly up-regulated and reached the peak on 1d after SE. Thereafter, it gradually decreased but maintained higher than in control on 7d(P<0.01), but the protein level on 15d after SE was lower than in control group(P<0.05), and lowest on 60d after SE.④The expression of TRPC5 was markedly decreased from the time point of 1d after SE (P<0.01), then gradually decreased, reaching its lowest on 60d after SE.⑤The expression of TRPC6 was markedly increased and reached its peak on 1d after SE (P<0.01), and it was higher than normal at all the other time points (P<0.01).3. The expression of Synaptophysin in the experimental groups rats compared with the control group rats:The expression of Synaptophysin was markedly up-regulated on 15d、30d、60d after SE (P＜0.05 or P<0.01), and reached the peak on 30d after SE. 4. The loss of hippocampal neurons in the experimental group was most evident on 7d and 60d after SE. Significant loss of hilar neurons and pyramidal neurons was present in area CA1 (P<0.01), while the loss of granular cells in dentate gyrus was relatively slight (P＜0.05).5. There were Timm granules in molecular layer of gyrus dentatus since 7d after SE in the hippocampus of experimental rats, then they gradually increased.6. In the experimental group, BDNF and TRPC1、TRPC3、TRPC4、TRPC5、TRPC6 co-expression was observed all the time in rat’s hippocampus.ConclusionTRPC may play a potential role in mossy fiber sprouting, in which BDNF may involve. Objective To observe the effect of intervention in BDNF on TRPC expression and mossy fiber sprouting in rat’s hippocampus after pilocarpine-induced seizures.Method 270 healthy male Sprague-Dawley (SD) rats were divided randomly into 3 groups:the K252a+Pilo group (n=90), the NS+Pilo group (n=90) and the K252a+NS group (n=90). Rats in K252a+Pilo group were treated by intraperitoneal injection of lithium and pilocarpine, with intracerebroventricular injection of K252a 3h before pilocarpine injection; the NS+Pilo group rats received intracerebroventricular injection of sterile saline 3h before pilocarpine injection; and the K252a+NS group rats received intraperitoneal injection of sterile saline 3h after intracerebroventricular injection of K252a. These three groups all selected 5 time points (1d、7d、15d、30d、60d after intraperitoneal injection) for investigation. The rats of each time point (n=18) received the detection below respectively:①expression of TRPC and Synaptophysin protein in rats’hippocampus by Western blot;②Timm staining and score;③hippocampal pathology by Nissl staining.Results1. SE ratio of the K252a+Pilo group (83.3%) was lower than that of the NS+Pilo group (90.7%), and mortality rate was lower as well (8% and 19.3%, respectively). The mean time required for inducing SE in the K252a+Pilo group (55.84±17.44min) was longer than that in the NS+Pilo group (27.80±13.58min). The dosage of pilocarpine required for inducing SE in the K252a+Pilo group (34.84±9.44 mg/kg) was larger than that in the NS+Pilo group (24.80±5.58 mg/kg). The mean duration of grade III-V seizure in the K252a+Pilo group (12.84±5.44 min) was shorter than in the NS+Pilo group (28.20±4.58 min). And the average total amount of chloral hydrate required for ending seizures in the K252a+Pilo group (3.24±0.44 ml/kg) was lower than that in the NS+Pilo group (3.80±0.54 ml/kg). All the differences above were of statistic significance (P<0.01).2. The expression of TRPC protein in the K252a+Pilo group rats compared with the K252a+NS groups:①The expression of TRPC1 protein was markedly up-regulated since 1d after SE(P<0.01), then gradually increased, and reached the peak on 60d after SE.②The expression of TRPC3 protein markedly decreased since 1d after SE (P＜0.05), then gradually decreased and reached the lowest on 60d.③The expression of TRPC4 protein markedly decreased on 7d、15d、30d、60d after SE (P<0.01), and reached the lowest on 15d.④The expression of TRPC5 protein was markedly up-regulated since 1d after SE (P<0.01), then gradually increased and reached the peak on 60d.⑤The expression of TRPC6 protein was markedly up-regulated since 1d after SE (P<0.01), but the extent of increase declined, on 30d it was still higher than that of the K252a+NS group (P<0.01), but became lower than normal on 60d (P<0.01). Compared with the K252a+NS group:At each time point, the expression of TRPC1、TRPC4 and TRPC5 protein were all down-regulated greatly (P<0.01); The expression of TRPC3 protein was significantly down-regulated since 1d after SE and reached the lowest (P<0.01); Thereafter, it gradually increased but was still lower on 7d、15d、30d (P<0.01 or P<0.05), until it was significantly up-regulated on 60d (P＜0.01).However, the expression of TRPC6 protein was markedly up-regulated since 1d after SE(P<0.05), then gradually increased, and reached the peak on 30d after SE(P<0.01), but markedly decreased on 60d.3. The expression of Synaptophysin protein in the K252a+Pilo group compared with that in the K252a+NS group:it was markedly up-regulated on 15d after SE (P<0.01), inceased gradually, and then reached the peak on 60d (P＜0.01). Compared with that in the NS+Pilo group:it was markedly up-regulated since 1d after SE (P<0.01), inceased gradually, and then reached the peak on 60d (P<0.01).4. In the K252a+Pilo group, hippocamapal neuron loss can be observed, extremely obvious on 15d and 60d after SE. Compared with the K252a+NS group, significant hilar neuron loss and pyramidal neuron loss was present in area CA1-3 (P＜0.01), while the loss of granular cells in dentate gyrus was relatively slight (P<0.05). Compared with the NS+Pilo group, the degree of hippocampal neuron loss in the K252a+Pilo group was significantly sligter, especially since 7d after SE, with significant statistic differences (P<0.05).5. There were Timm granules in molecular layer of gyrus dentatus since 15d after SE in the K252a+Pilo group. The Timm score gradually increased and reached the peak on 60d. Compared with the NS+Pilo group, the degree of sprouting in the K252a+Pilo group was markedly slighter (P＜0.01).Conclusion1. Intervention in BDNF can decrease the severity of seizure.2. Intervention in BDNF can inhibit mossy fiber sprouting, possibly through regulating the expression of TRPC.更多还原