【作者】 魏海亮；

【导师】 胡福广；

【作者基本信息】 河北医科大学 ， 外科学， 2008， 硕士

【摘要】 目的:应用髓鞘碱性蛋白(myelin basic protein ,MBP)主动免疫出血性卒中大鼠,进行行为学观察,及免疫组化研究,探讨胶质纤维酸性蛋白( Glial fibrillary acid protein,GFAP )和神经微丝蛋白200(Neurofilament protein,NF200)在血肿周围脑组织的阳性表达,并应用电镜观察血肿周围脑组织的超微结构变化,研究MBP对出血性卒中大鼠继发性脑损害的神经保护作用。方法:健康成年雌性SD大鼠,用自体动脉血注射法制备大鼠脑出血模型后12小时,进行行为学评分,以2分者作为纳入标准。入选84只大鼠,采用随机数字表法,分为MBP、卵白蛋白(Ovalbumin,VOA)、PBS和空白对照四组(n=21)。用不完全福式佐剂(Incomplete Freund’s Adjuvant,IFA)乳化的MBP(300μg/只)、VOA(200μg/只)、PBS(200μg/只)分别免疫三组SD大鼠,对照组只做脑出血模型,于免疫后1、2、3、5、7、14、21天进行行为学评分,并在免疫后1,3,5,7,14,21天处死动物,脑组织切片行GFAP及NF200免疫组织化学染色,于免疫后第7天每组大鼠取3只行电镜检查。结果:(1)行为学评分:MBP免疫组大鼠在免疫后第1～3天行为学评分明显优于VOA、PBS免疫组与对照组(P<0.05);(2)GFAP免疫组织细胞化学染色观察:脑出血免疫后第1～5天,MBP组血肿周围GFAP阳性细胞数与VOA、PBS及对照组相比有显著性差异(P﹤0.05),后三组相比无统计学意义(P>0.05);脑出血免疫后第7～21天,各组GFAP阳性细胞数相比无明显差异(P>0.05)。(3)NF200免疫组织细胞化学染色观察:MBP组NF200阳性细胞数在免疫后第3～14天与后三组相比有显著性差异(P﹤0.05),后三组之间相比无明显差异(P>0.05)。(4)透射电镜观察:在脑出血免疫后第7天,MBP免疫组神经元细胞和有髓神经纤维的亚细胞器较丰富,神经微丝排列整齐,未发现血管内皮细胞水肿。而其他三组神经元细胞和有髓神经纤维的亚细胞器变形或消失,神经微丝排列紊乱,同时可见血管内皮细胞水肿。结论:(1)MBP主动免疫能促进GFAP和NF200的阳性表达;(2)MBP免疫组行为学观察及血肿周边超微结构的改变明显好于其他组。MBP免疫治疗能阻断出血性卒中的继发性脑损害,进而促进神经症状的改善,即血肿周围神经元的恢复;(3)MBP主动免疫激活自身效应性T细胞透过血脑屏障,聚集在血肿周围,在改善神经元的微环境,阻断脑出血后继发性脑损害及对神经元功能的恢复中发挥了重要作用。目的:研究大鼠出血性卒中后,MBP免疫治疗对BDNFmRNA及蛋白表达的影响。探讨MBP在大鼠中枢神经系统损伤后对抗继发性脑损害——即自身免疫神经保护的机制。方法:(1)健康成年雌性SD大鼠,用自体动脉血注射法制备大鼠脑出血模型后12小时,进行行为学评分,参照Longa方法制定评分标准,以2分者作为纳入试验的标准。入选72只,采用随机数字表法,分为MBP、卵白蛋白VOA、PBS和空白对照四组(n=18)。用不完全福式佐剂乳化的MBP(300μg/只)、VOA(200μg/只)、PBS(200μg/只)分别免疫三组SD大鼠,于免疫后1、2、3、5、7、14、21天进行行为学评分,在免疫后1,3,5,7,14,21天处死动物,脑组织切片行脑源性神经营养因子(brain derived neurotrophi factor,BDNF)免疫组织化学染色。(2)同法制备36只SD大鼠出血模型,随机分为MBP组、对照组(N=18)。脑出血后12h用IFA乳化的MBP(300μg/只)免疫MBP组大鼠。于出血后1、3、5、7、14、21天采用RT-PCR法检测两组大鼠BDNFmRNA在血肿周围脑组织的表达。结果:(1)行为学观察:MBP免疫组大鼠神经学评分明显优于VOA、PBS免疫组及对照组;BDNF的免疫组化检测;BDNF主要由小胶质细胞表达,免疫后第1天,MBP组BDNF阳性细胞数与VOA组、PBS组、空白对照组相比有显著性差异(P﹤0.01),免疫后第3天MBP组BDNF阳性细胞数与后三组相比有明显差异(P﹤0.05),后三组之间相比无统计学意义(P>0.05);免疫后第5～21天,四组大鼠血肿周围BDNF阳性细胞数进一步减少,各组相比无显著性差异(P>0.05)。(3)BDNFmRNA RT-PCR结果显示: MBP组各时间点BDNFmRNA在血肿周围脑组织中的表达均高于对照组,各时间点间两组间比较均有明显差异(P<0.05),尤其免疫后第5,7天两组间有显著性差异(P<0.01). MBP组在第1,14天有两个高峰,免疫后第1天分泌BDNFmRNA最高,免疫后第7天最低,第14天再次达到高峰。结论:(1)大鼠脑出血后MBP主动免疫能上调BDNF蛋白及BDNFmRNA的表达,改善神经元周围微环境,促进神经元的存活及功能恢复;(2)MBP免疫治疗通过激活自身效应性T细胞聚集在血肿周围,阻断中枢神经系统损伤后继发性脑损害的发生,促进中枢神经系统恢复。更多还原

【Abstract】 Objective: myelin basic protein (myelin basic protein, MBP) active immunization of hemorrhagic stroke in rats, behavioral observation, and immunohistochemical study, GFAP and NF200 of hematoma in the brain tissue around the expression, and application of electron microscopy hematoma in the brain tissue around the ultrastructural changes, the study of hemorrhagic stroke MBP secondary brain damage in the neuroprotective effect.Methods: healthy adult female SD rats, using autologous blood injection method of rat cerebral hemorrhage model after 12 hours, behavioral score to 2 minutes, as the inclusion criteria. Selected 84 rats by using random number table is divided into MBP, egg albumin (Ovalbumin, VOA), PBS and four blank control group (n=21). With incomplete adjuvant IFA Fook-emulsification of the MBP (300μg /), VOA (200μg /), PBS (200μg /) three groups were immune SD rats, the control group only cerebral hemorrhage model, 1,2,3,5,7,14,21 days after vaccination, a behavioral score, and in the days after immunization 1,3,5,7,14,21 animals were killed, brain tissue to glial fibrillary acidic protein (Glial fibrillary acid protein, GFAP) and Neurofilament protein 200 (Neurofilament protein, and NF200) immunohistochemical staining. In the first seven days after immunization of each group will take three electron microscope.Results:⑴behavioral score: MBP immune rats immunized with 1 to 3 days behavioral score was superior to VOA, PBS immune group and the control group (P <0.05);⑵GFAP immunohistochemistry staining of observation: cerebral hemorrhage after immunization 1 ~ 5 days, MBP Group hematoma around GFAP positive cells and VOA, PBS and the control group there were significant differences (P <0.05). After three groups showed no statistical significance (P> 0.05); cerebral hemorrhage after immunization 7 ~ 21 days, the groups of GFAP positive cells showed no significant difference (P> 0.05).⑶NF200 immunohistochemical staining of observation: MBP Group NF200 positive cells in the immune after 3 to 14 days after the three groups compared with a significant difference (P <0.05), after the three groups showed no significant difference (P>0.05).⑷TEM observation: cerebral hemorrhage in the first seven days after immunization, MBP immunohistochemical neuronal cells and myelinated nerve fibers in the cell-rich, Neurofilament neat and found no vascular endothelial cells edema. The other three groups neuronal cells and nerve fibers of the deformation of cells or disappeared, Neurofilament are disordered, and that vascular endothelial cell edema.Conclusion: (1) to promote active immunization MBP and NF200 GFAP expression;(2)MBP immunohistochemical behavioral observation and hematoma surrounding ultrastructural changes clearly better than the other group. MBP immune therapy can block hemorrhagic stroke secondary brain damage, thereby promoting the improvement of neurological symptoms, or hematoma surrounding neurons recovery;(3) MBP own initiative immune activation effect of T cells through the blood-brain barrier, gathered around the hematoma in improving the micro-environment of the neurons, blocked cerebral hemorrhage secondary brain damage and the recovery of neuronal function play an important role.Objective: To study the rats hemorrhagic stroke, immune therapy on the MBP BDNFmRNA and protein expression. MBP study in the rat central nervous system injury confrontation secondary brain damage - that is, autoimmune nerve protection mechanism.Methods:⑴healthy adult female SD rats, using autologous blood injection method of rat cerebral hemorrhage model after 12 hours, behavioral score, in the light Longa method developed standard score to 2 minutes, as included in the pilot standards. 72 selected by random number table is divided into MBP, ovalbumin VOA, PBS and four blank control group (n = 18). With incomplete adjuvant Fook-emulsification of the MBP (300μg /), VOA (200μg /), PBS (200μg /) three groups were immune SD rats after vaccination, 1,2,3,5,7, 14, 21 days behavioral score,In the days after immunization 1,3,5,7,14,21 animals were killed, brain tissue to the brain-derived neurotrophic factor (brain derived neurotrophi factor, BDNF) immunohistochemical staining.⑵Preparation of 36 with SD rats bleeding were randomly divided into MBP group and the control group (N = 18). 12 h after cerebral hemorrhage emulsified with the IFA MBP (300μg /) MBP immune rats. 1,3,5,7,14,21 days after the bleeding by RT-PCR detection of the two groups BDNFmRNA hematoma in the brain tissue around the expression.Results:(1)behavioral observation: MBP immunohist- ochemical rat neural score was superior to VOA, PBS immune group and the control group;⑵BDNF Immunohistochemical detection; BDNF mainly by microglial cells, one day after immunization, MBP Group BDNF positive cells with the VOA group, PBS group, the control group compared with a significant difference (P <0.01) Three days after immunization MBP Group BDNF positive cells compared with the three groups after a significant difference (P <0.05) between the three groups after showed no statistical significance (P> 0.05); Immunity after 5 to 21 days, four groups of rats around the hematoma BDNF positive cells further reduced, the group there was no significant difference (P> 0.05). (3)BDNFmRNA RT-PCR results show that: MBP group at different time points BDNFmRNA hematoma in the brain tissue around the expression of the control group, all time points compared between the two groups were significantly different (P <0.05) and, in particular 5,7 days after immunization between the two groups significant difference (P <0.01). MBP Group 1,14 days in the first two peaks, one day after immunization secretion BDNFmRNA highest, seven days after immunization minimum, 14 days once again reached a peak.Conclusion: (1) MBP rat brain hemorrhage after active immunization can BDNFmRNA increases BDNF protein and the expression of neuronal improve micro-environment, and promote neuronal survival and functional recovery;(2) MBP treatment by activating their immune effector T cells gathered around the hematoma, central nervous system injury after blocking secondary brain damage occurred, and promote restoration of the central nervous system.更多还原