不同来源的间充质干细胞对自身免疫性脑脊髓炎模型小鼠的疗效及其机制研究

发布时间：2018-04-23 06:49

本文选题：脂肪间充质干细胞 + 鞘氨醇激酶1　；参考：《郑州大学》2017年硕士论文

【摘要】：背景和目的:多发性硬化(multiple sclerosis,MS)是一种以中枢神经系统白质炎性脱髓鞘为主要病理特点的自身免疫疾病,最常累及部位为脑室周围白质、视神经、脊髓等。其病因及发病机制迄今不明,目前观点认为,携带先天遗传易感基因的个体,在后天环境中,因病毒感染、外伤等外因的作用下,诱导中枢髓鞘成分发生异常自身免疫应答而致病。目前传统的治疗方法如糖皮质激素、干扰素β等,虽然可以减缓MS的进展,但是,长期服用这些药物往往会导致严重的副作用。因此,寻找疗效好,副作用低的药物迫在眉睫。间充质干细胞(mesenchymal stem cells,MSCs)是一种多能、具有抗炎和再生能力的成体干细胞,普遍存在于骨髓和其它组织中,如脐带(UC),胎儿肝脏和脂肪组织等。近年来,随着对间充质干细胞的研究不断深入,发现其不仅具有突破谱系屏障分化为神经细胞的功能,而且还可以通过抑制免疫反应、促进髓鞘再生和诱导神经损伤修复来治疗一些神经退行性疾病。本实验通过建立自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)小鼠模型,分别采用不同来源的间充质干细胞(mesenchymal stem cells,MSC)对EAE模型小鼠进行治疗,观察各组小鼠在治疗过程中病情轻重的变化,并通过一些实验室检查来探究其可能的机制,从而为MS的治疗提供新的思路和方法。方法:1.采用髓鞘少突胶质细胞糖蛋白(MOG35-55)作为抗原与完全弗氏佐剂混合免疫C57BL/6小鼠制作EAE模型。在小鼠发病后随机分为对照组,模型组,UCMSC组、ADMSC组和UCMSC-SPK1组,通过尾静脉给予不同来源的间充质干细胞治疗,从发病开始后每隔七天注射一次,共3次;正常对照组和模型组用相同剂量的生理盐水替代治疗。2.采用双盲法每天对各组小鼠进行神经功能缺损评分,持续33天。3.免疫后第33天处死小鼠,取出脑和脊髓放入㧟80℃冰箱冻存。4.通过电子显微镜观察各组小鼠脊髓中髓鞘脱失情况。5.通过蛋白印迹法来检测各组小鼠脊髓中GFAP和MBP的表达。6.研磨脾脏细胞收集脾脏细胞,通过流式细胞仪检测脾脏中调节性T细胞(CD4+CD25+和foxp3+)和NK(NK1.1+CD3-)细胞的比例。7.通过携带SPK1基因的腺病毒(Ad-SPK1)转染UCMSC,运用westernblot检测UCMSC、ADMSC和UCMSC-SPK1中SPK1的表达。结果:1.MOG35-55和完全弗氏佐剂混合免疫成功制备了EAE小鼠模型。2.Westernblot结果显示,相比UCMSC,ADMSC中SPK1的表达量更高,而经过携带SPK1基因的腺病毒(Ad-SPK1)的转染,UCMSC-SPK1中SPK1的表达量比ADMSC更高。3.双盲法对各组小鼠神经功能缺损评分结果显示,与模型组相比,UCMSC组、ADMSC组和UCMSC-SPK1组小鼠的神经功能缺损评分明显降低;将这三个细胞治疗组进行比较发现,ADMSC组比UCMSC组的神经评分降低的更加明显,而相较ADMSC组,UCMSC-SPK1组小鼠的神经缺损评分下降的趋势更为显著。4.电镜下观察小鼠脊髓髓鞘脱失情况,结果表明:与正常对照组小鼠相比,模型组中小鼠脊髓的髓鞘大面积脱失;而经过细胞治疗后髓鞘脱失面积明显较少,程度显著减轻;对比三个细胞治疗组之间髓鞘脱失情况发现,相比UCMSC组,ADMSC组和UCMSC-SPK1组小鼠脊髓的改善更加明显,而这种趋势在UCMSC-SPK1组中表现的更加显著。5.Westernblot检测小鼠脊髓中星形胶质细胞活化标记蛋白GFAP的表达发现,与对照组相比,模型组小鼠脊髓中GFAP的表达明显增加,而与模型组相比,UCMSC组,ADMSC组和UCMSC-SPK1组小鼠脊髓中GFAP的表达明显降低;将这三组细胞治疗组进行比较发现,相比UCMSC组,ADMSC组和UCMSC-SPK1组中GFAP的表达降低的更加明显,而相较ADMSC组,UCMSC-SPK1组小鼠脊髓中GFAP的表达减少的更加显著。6.Westernblot检测各组小鼠脊髓中MBP的表达结果显示,与正常组相比,模型组小鼠脊髓中MBP的表达明显减少,而经过MSC治疗后,各治疗组小鼠脊髓中MBP的表达明显增高;将这三个细胞治疗组进行比较发现,ADMSC组小鼠脊髓中MBP的表达要明显多于UCMSC组,而这种趋势在UCMSC-SPK1组中表现的更为显著。7.流式细胞仪对各组小鼠脾脏中的调节性T细胞和NK细胞的比例进行测定,结果显示,相较对照组,模型组小鼠脾脏中调节性T细胞的比例明显降低,而NK细胞的比例却显著升高;经过细胞治疗后,UCMSC组,ADMSC组和UCMSC-SPK1组小鼠脾脏中调节性T细胞的比例显著升高,而NK细胞的比例显著降低;相较UCMSC组,ADMSC组和UCMSC-SPK1组中这种趋势的表现更为明显,而与ADMSC组相比,UCMSC-SPK1组中调节性T细胞升高和NK细胞降低的表现更为显著。结论:1.不同来源的间充质干细胞治疗都可以显著减轻EAE模型小鼠神经功能缺损评分,其机制可能是通过调节免疫反应,抑制神经系统炎症反应和促进髓鞘再生来实现的。2.经过SPK1基因的转染,UCMSC-SPK1在治疗EAE中表现出了比ADMSC更好的疗效,而原始的UCMSC却比ADMSC疗效差。3.不同来源的干细胞,其表达的SPK1的量不同,这可能导致了它们在治疗EAE模型小鼠疗效上的差异。
[Abstract]:Background and objective: multiple sclerosis (MS) is a kind of autoimmune disease characterized by white plasma demyelinating in the central nervous system, which is most often involved in the white matter, optic nerve, and spinal cord around the ventricle. Its etiology and pathogenesis are not so far. Individuals, in the environment, induced by external causes such as virus infection and trauma, induce abnormal autoimmune responses in the central myelin sheath. Traditional therapies such as glucocorticoid and interferon beta can slow the progress of MS, but long-term use of these drugs often leads to serious side effects. Therefore, Mesenchymal stem cells (MSCs) is a kind of pluripotent stem cells with anti-inflammatory and regenerative ability, which is commonly found in bone marrow and other tissues, such as umbilical cord (UC), fetal liver and adipose tissue. It is found that it not only has the function of breaking through the pedigree barrier to differentiate into nerve cells, but also can be used to treat some neurodegenerative diseases by inhibiting the immune response, promoting the regeneration of myelin sheath and inducing the repair of nerve injury. This experiment is based on the establishment of autoimmune encephalomyelitis (experimental autoimmune encephalomyelitis, EAE) mice Mesenchymal stem cells (MSC) was used to treat EAE model mice respectively. The changes of the severity of the disease during the treatment were observed, and some laboratory tests were used to explore the possible mechanisms, so as to provide new ideas and methods for the treatment of MS. Methods: 1. the myelin sheath was less. The EAE model of C57BL/6 mice was immunized with MOG35-55 as antigen and complete Freund adjuvant. The mice were randomly divided into the control group, the model group, the UCMSC group, the ADMSC group and the UCMSC-SPK1 group, and the caudal vein was given to different sources of mesenchymal stem cells, and the injection was given every seven days from the onset of the onset of the disease. 3 times, the normal control group and the model group were treated with the same dose of saline replacement therapy for.2. with double blind method of nerve function defect score in each group every day. After 33 days of.3. immunization, the mice were killed and the brain and spinal cord were taken out, and the cryopreservation.4. at 80 C was used to observe the loss of myelin sheath in the spinal cord of each group through the electron microscope. .5. was used to detect the expression of GFAP and MBP in the spinal cord of mice by Western blot, and.6. was used to grind spleen cells to collect spleen cells. The proportion of T cells (CD4+CD25+ and foxp3+) and NK (NK1.1+CD3-) cells in the spleen was detected by flow cytometry, and.7. was transfected by adenovirus carrying SPK1 gene (Ad-SPK1). Results: the expression of SPK1 in CMSC, ADMSC and UCMSC-SPK1. Results: 1.MOG35-55 and complete Freund adjuvant immunization successfully prepared the EAE mouse model.2.Westernblot results, and the expression of SPK1 was higher in ADMSC than UCMSC, and the expression of the SPK1 was higher than that of the adenovirus carrying the SPK1 gene. The neurological deficit scores of each group showed that the neurological deficit scores of the UCMSC, ADMSC and UCMSC-SPK1 groups were significantly lower than those in the model group; compared with the three cell groups, the ADMSC group was significantly lower than the UCMSC group, compared with the ADMSC group and the UCMSC-SPK1 group. The demyelination of the spinal cord in mice was observed under the.4. electron microscope. The results showed that the myelin sheath of the mouse spinal cord in the model group was greatly lost in comparison with the normal control group, while the myelin loss area was significantly less and the degree of myelin was significantly reduced after the cell treatment. The myelin sheath was compared between the three cell therapy groups. It was found that the improvement of the spinal cord in the ADMSC and UCMSC-SPK1 groups was more obvious than that in the UCMSC group, and the trend in the group UCMSC-SPK1 was more significant in.5.Westernblot detection of the expression of the astrocyte activation marker protein GFAP in the spinal cord of the mouse spinal cord, and the expression of GFAP in the spinal cord of the model mice was compared with the control group. The expression of GFAP in the spinal cord of the UCMSC, ADMSC and UCMSC-SPK1 groups decreased significantly compared with the model group. Compared with the group UCMSC, the expression of GFAP in the group ADMSC and the UCMSC-SPK1 group decreased more significantly than that in the UCMSC group, and the GFAP expression in the spinal cord of the UCMSC-SPK1 group was less than that in the group of ADMSC. The expression of MBP in the spinal cord of each group of mice by.6.Westernblot showed that the expression of MBP in the spinal cord of the model group decreased significantly compared with the normal group, and the expression of MBP in the spinal cord of each group of mice increased significantly after the treatment of MSC, and the three cell treatment groups were compared to the MBP in the spinal cord of the ADMSC group. The proportion of the regulatory T cells and NK cells in the spleen of each group was measured by the more significant.7. flow cytometer in group UCMSC-SPK1. The results showed that the proportion of the regulatory T cells in the spleen of the model group was significantly lower than that of the control group, while the proportion of NK cells was significantly higher than that of the control group. After cell therapy, the proportion of regulatory T cells in the spleen of the UCMSC, ADMSC and UCMSC-SPK1 groups increased significantly, while the proportion of NK cells decreased significantly; compared with the UCMSC group, the trend in the ADMSC group and the UCMSC-SPK1 group was more obvious, and the regulatory T cells in the UCMSC-SPK1 group and the NK cells in the UCMSC-SPK1 group were higher than those in the ADMSC group. Conclusion: 1. the treatment of mesenchymal stem cells from different sources can significantly reduce the score of neural function defect in EAE model mice. The mechanism may be the expression of.2. through SPK1 gene transfection by regulating the immune response, inhibiting the inflammatory response of the nervous system and promoting the regeneration of myelin sheath, and the expression of UCMSC-SPK1 in the treatment of EAE. The effect of ADMSC was better than that of the original UCMSC, but the stem cells from different sources of.3. were less effective than ADMSC, and the amount of SPK1 expressed differently, which may lead to their difference in the treatment of EAE model mice.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R744.51;R-332

【参考文献】