马索罗酚和丹参酮ⅡA对实验性自身免疫性脑脊髓炎小鼠治疗作用的病理学研究

发布时间：2018-04-04 10:42

本文选题：多发性硬化　切入点：马索罗酚和丹参酮ⅡA　出处：《河北医科大学》2014年硕士论文

【摘要】：目的：中枢神经系统脱髓鞘疾病是部位发生在脑和脊髓，主要特征是髓鞘损伤，而神经元胞体和轴索损害相对较轻的一组疾病。多发性硬化（Multiple sclerosis, MS）是由免疫介导的中枢神经系统慢性炎性脱髓鞘性疾病，病因及发病机制迄今不明，最新研究表明，MS的病因与遗传因素和环境因素有关,而疾病导致的髓鞘脱失和轴索变性是患者表现为神经功能缺损，病程呈现为缓解-复发和病情持续进展的主要原因。实验性自身免疫性脑脊髓炎(Experimental autoimmune encephalomyelitis, EAE)是MS经典的动物模型，其病程上可以表现为复发-缓解，很接近于人类MS的临床表现及病理特征。本研究应用MOG35-55建立EAE动物模型，在EAE小鼠出现神经症状后开始给予马索罗酚和丹参酮IIA治疗，模拟临床的治疗时间，通过HE染色观察两种药物对EAE动物模型是否有抗炎作用，通过罗克沙尔坚牢蓝（Luxol Fast Blue）特殊髓鞘染色和Bielschowsky's神经轴索染色从病理学和形态学的角度观察马索罗酚和丹参酮IIA是否能够减轻CNS髓鞘的脱失，减轻轴索病理损伤，降低EAE小鼠的神经功能评分。方法：本研究采用了45只8-12周龄，健康C57BL/6雌性小鼠，小鼠随机分成三组即EAE组、NDGA组和DANIIA组，以MOG35-55为抗原免疫C57BL/6小鼠建立EAE模型。用生理盐水将抗原MOG35-55稀释成为5mg/ml的液体状态，然后将完全弗氏佐剂（其中包含的结核杆菌H37Ra的终浓度为4mg/ml）按1:1的体积加入并混合均匀，乳化后按每只0.1ml在小鼠脊柱两侧的皮肤下分四点注射，免疫后的当天即第0h及第48h腹腔内注射500ngPTX（1000ng/只）。小鼠发病后，自神经功能评分达到1分以上时开始给予药物治疗，即EAE组小鼠腹腔注射5%DMSO，NDGA组小鼠腹腔注射马索罗酚，DANIIA组小鼠腹腔注射丹参酮，分别于治疗前，治疗10d，治疗20d取材，每组各取5只。取材部位为小鼠脊髓腰膨大处，组织进行石蜡包埋，分别通过HE染色观察病变部位炎性反应程度和血管套形成的情况，罗克沙尔坚牢蓝（Luxol Fast Blue）特殊髓鞘染色和Bielschowsky's神经轴索染色从病理学及形态学的角度观察马索罗酚和丹参酮IIA两种药物能否可以减轻小鼠病变部位的髓鞘脱失，减轻组织的轴索损伤，降低EAE小鼠的神经功能评分起到治疗作用。应用SPSS19.0统计软件进行统计学处理，结果为计量资料则均数±标准差(±S)表示，而多组计量资料均数的比较则采用ANOVA方差分析，若不满足正态分布或方差齐性，则采用秩和检验的方法，P0.05表明差异有统计学意义。结果： 1动物临床症状及评分观察： 1.1临床症状： 45只小鼠全部在10-16d内开始发病，表明EAE动物模型制作成功，可以给予药物干预治疗，EAE组小鼠免疫后全部发病，出现皮毛粗糙、精神萎靡不振、行走困难、丧失食欲、体重减轻等表现。临床表现最严重在发病高峰期，小鼠出现双后肢甚至全部四肢肌力降低甚至全部瘫痪、大便、小便均失禁、不能够自主翻身，个别成垂死状态，并且持续较长时间。治疗组的小鼠免疫后也全部发病，于症状评分达到1分以上时给予药物干预，治疗前与EAE组临床症状评分无显著差异,治疗10天后，此时EAE处于发病高峰期，临床表现严重，而给予丹参酮及马索罗酚治疗的小鼠出现尾部瘫痪或者双后肢轻瘫或全瘫，无大小便失禁及不能自主翻身等症状，治疗20天后，，EAE小鼠随着病程发展，症状有所缓解，但仍遗留尾部瘫痪或后肢瘫痪等症状，给予丹参酮及马索罗酚治疗的小鼠症状缓解较快，临床症状表现更轻，仅表现为尾部轻瘫或者尾部无力等症状。 1.2临床评分观察：计算并比较各组中每只小鼠于治疗10d及治疗20d的临床评分均数。EAE组小鼠神经功能评分高于丹参酮组及马索罗酚组（P0.05）。丹参酮组及马索罗酚组小鼠神经功能评分相比较，差别没有统计学意义（P＞0.05）。 2HE染色： 2.1治疗前三组可见散在炎性细胞浸润，大部分集中在脊髓白质附近。 2.2治疗10天EAE组可见大片状炎性细胞的浸润，甚至形成了“血管袖套”现象，即脊髓多处小血管周围，特别是小静脉四周出现了炎症细胞，大部分为淋巴细胞，病变大多在前角白质，灰白质交界及前正中裂血管周围，丹参酮组及马索罗酚组“血管袖套”形成较少，炎性细胞主要集中于小血管周围，三组HE病理评分有统计学差异。 2.3治疗20天EAE组炎性细胞和“血管袖套”减少，丹参酮组及马索罗酚组炎性细胞更少，“血管袖套”消失，三组HE病理评分有统计学差异。 3LFB染色：三组小鼠在治疗前，髓鞘并无明显的脱失部位，但仍能观察到脊髓白质内部分髓鞘变薄，颜色变浅。EAE组在发病高峰期可观察到脊髓内存在大小不等的片状脱髓鞘区域，病变大多表现在前后角周围内的白质，蓝色髓鞘部位减少，颜色变淡，甚至出现了大片状气球样空泡变性的髓鞘脱失区域，髓鞘脱失部位常伴随着大片炎症细胞浸润，表明髓鞘脱失和炎症浸润有一定关系，在治疗20d后较高峰期脱髓鞘区域内病变无明显改变；而丹参酮组及马索罗酚组在治疗10d后可见部分髓鞘变薄，颜色变浅，存在一些小的髓鞘脱失区，但没有形成大片状髓鞘脱失区域，病灶散在成点灶状，治疗20d较治疗前及治疗10d髓鞘变粗，蓝色髓鞘区域有所增加，三组髓鞘病理评分在治疗10d及治疗20d均具有统计学差异。 4．Bielschowsky's神经轴索染色: 4.1治疗前：三组小鼠均已存在轴索损伤，损伤的轴索表现为形状不规则、排列紊乱、肿胀扭曲。 4.2治疗10天：EAE组处于发病高峰期，轴索损伤更为明显，病灶成大片状，形态更加不规则，直径大小不一，轴索断裂，断端形成卵圆小体，甚至出现轴索的消失，部位与炎性细胞浸润及髓鞘的脱失具有一致性，并且与疾病的严重程度相关，丹参酮组及马索罗酚组轴索也存在损失，但范围及程度与EAE组相比较轻。 4.3治疗20天：EAE组在治疗20d后与治疗10天相比，无明显变化，丹参酮组及马索罗酚组损伤较治疗前及治疗10d较轻。结论： 1马索罗酚和丹参酮ⅡA作为干预药物能改善EAE小鼠的临床症状，为临床治疗多发性硬化奠定实验基础。 2马索罗酚和丹参酮ⅡA对EAE小鼠在抗炎、髓鞘脱失、轴索损伤方面具有治疗作用
[Abstract]:Objective : To study the clinical manifestation and pathological characteristics of the central nervous system inating disease ( CNS ) . The results showed that the etiology of the MS was related to the genetic factors and the environmental factors . The results showed that the etiology of the MS was related to the genetic factors and the environmental factors .

Methods : Forty - five mice of 8 - 12 weeks old and healthy C57BL / 6 female mice were randomly divided into three groups , i.e . , the rats were randomly divided into three groups , i.e . , the rats were injected with 5 % DMSO and 5 mg / ml .

Results :

1 Animal Clinical Symptoms and Score Observations :

1.1 Clinical symptoms :

Forty - five mice were randomly divided into two hind limbs .

1.2 Observation of clinical score :

The scores of neurological function were higher in all groups than in tanshinonegroup ( P > 0.05 ) .

2HE staining :

2.1 Before treatment , three groups were scattered in inflammatory cells , mostly in the vicinity of white matter in the spinal cord .

2.2 During the treatment of 10 days , the infiltration of large flam - shaped inflammatory cells was seen and even the phenomenon of " blood vessel cuff " was formed , that is , there were inflammatory cells around the small vessels around the spinal cord , especially in the periphery of the small vein . Most of them were lymphocytes . Most of the lesions were in the anterior horn white matter , the gray white matter junction and the anterior median fissure blood vessel , the tanshinone group and the horse sorool group " blood vessel cuff " were formed less , the inflammatory cells were mainly concentrated around the small blood vessels , and the HE pathological scores were statistically different .

2.3 There were fewer inflammatory cells and " blood vessel cuff " in the 20 days of treatment , and there were fewer inflammatory cells in the group of tanshinone and mansorool , and the " blood vessel cuff " disappeared , and the HE pathological scores of the three groups were statistically different .

3LFB staining :

There was no significant loss of myelin sheath in the spinal cord before and after treatment .
After 10 days of treatment , the partial myelin sheath became thinner and the color became lighter . There were some small myelinated depigmentation areas . However , there was no area of large flake myelination , and the lesions were scattered in the focal point . The lesions were treated at 20 days before and after treatment . The myelin sheath became thicker , the blue myelin area increased , and the three groups of myelinated pathological scores had statistical difference in the treatment of 10d and 20d .

4 . Bielschowsky ' s neuroaxonal staining :

4.1 Prior to treatment : all three groups of mice had axonal injury , and the injured axons were characterized by irregular shape , disorder of arrangement , and swelling and distortion .

4.2 Treatment for 10 days : the group was in the peak period , axonal injury was more obvious , the focus became larger , the shape was more irregular , the diameter was small , the axon was broken , the broken end formed the small body of the oval , and even the disappearance of the axon appeared , the site was related to the severity of the disease , there was also the loss of the axonal injury of the tanshinone group and the mansorool group , but the extent and extent were lighter than that of the experimental group .

4.3 Treatment for 20 days : After 20 days of treatment , there was no significant change in the treatment period compared with 10 days after treatment .

Conclusion :

Massorool and tanshinone 鈪 can improve the clinical symptoms and lay the foundation for clinical treatment of multiple sclerosis .

2 - Massorool and tanshinone 鈪 have therapeutic effects on anti - inflammatory , myelinating and axonal injury in mice .

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965;R744.51

【引证文献】