MPTP诱导帕金森病小鼠的神经干细胞治疗与干细胞微环境研究

发布时间：2018-05-18 17:32

  本文选题：帕金森氏症 + 人源神经干细胞　； 参考：《北京协和医学院》2014年博士论文

【摘要】：帕金森氏症是常见的中枢神经退行性疾病,在发达国家60岁以上人口中患病率约2%,在我国主要城市老龄人口中患病率相仿。帕金森氏症主要临床表现包括静止性振颤、僵硬、动作迟缓、姿势反射异常等,严重者丧失生活自理能力。相对其他神经退行性疾病,帕金森氏症有一整套包括药物和深部电极刺激手术在内的治疗方案,短期内可改善症状,但均不能逆转神经病理改变,而长期用药带来的异动症等副作用显著。上世纪八十年代起,干细胞与再生医学的发展给该病的治疗带了新的思路,科学家利用人胚胎干细胞和基因工程技术,将能够表达多巴胺的胚胎干细胞移植到神经退变的中脑黑质或者丘脑纹状体区,能够长期改善症状,并已用于临床实验。但由于胚胎干细胞的来源和伦理学限制,未能广泛开展,因而研究的热点转向间充质干细胞、神经干细胞等。在此,我们将体外培养的人类流产胚胎来源神经干细胞系,移植入MPTP诱导的帕金森病小鼠模型纹状体区域,以了解小鼠疾病微环境中此人源神经干细胞系的存活、生长状况,并研究神经干细胞移植对帕金森病小鼠运动功能的影响,并由此建立了一整套应用人源神经干细胞系治疗小鼠神经退行性疾病的研究体系。该研究在移植采用的细胞类型,移植方案的精确度,降低移植副损伤方面相对该领域同期研究有较好的表现。 目的：研究人源神经干细胞移植入帕金森氏症小鼠纹状体后的生存状况；研究疾病微环境中移植神经干细胞的分化、迁移、凋亡等特性；研究移植入神经干细胞与宿主神经网络是否形成联系；研究移植入的神经干细胞,是否能对帕金森病小鼠的运动症状起到改善作用；评估帕金森病小鼠不同运动障碍对人源神经干细胞治疗的敏感性及评估方法；为后期进行不同动物模型及转基因干细胞的移植实验建立可行方案和流程。 研究内容与技术路线： 1、建立稳定的帕金森氏症小鼠模型。采用经典的MPTP帕金森病C57/B16小鼠模型,实验组按照每次MPTP20mg/kg体重(溶于0.9%生理盐水)剂量,共进行腹腔注射4次,每次间隔3小时。对照组腹腔注射相同剂量0.9%生理盐水。 2、行为学评估。采用转棒实验(Rota-Rod)和爬竿实验(Rod Climbing),分别建立帕金森病小鼠的运动障碍评估方法。 3、人源神经干细胞培养,用慢病毒将GFP导入细胞,使细胞带有绿色荧光标记。 4、神经于细胞的立体定向注射。实验分组： 5、评估注入神经干细胞在PD小鼠纹状体微环境内的生存、分化、迁移、凋亡、与宿主神经网络整合等现象。 6、用转棒实验和爬竿实验对比帕金森病小鼠和对照组在治疗前后、不同时期的运动症状变化情况,评估立体定向注射本身和干细胞移植对PD小鼠运动症状的影响。 结果： 1、小鼠以20mg/kg剂量,间隔3小时,分四次注射MPTP后,小鼠3日内死亡率20%,此后未再有死亡。一周后转棒实验持续时间减少74.6%,症状能够在2月内保持稳定。 2、干细胞立体定向注入实验组及对照组小鼠纹状体,术后2日内急性死亡率小于4%,2日后无死亡,一周后伤口愈合好,无感染。 3、观察阴性对照1组(不做立体定向注射)、阴性对照2组(立体定向注射PBS缓冲液),两组在注射后第1,2,3,4,5周时转棒实验运动症状无显著性差异,单纯手术对PD小鼠运动症状无明显影响。 4、对比实验组(立体定向注射神经干细胞)、阴性对照2组(立体定向注射PBS缓冲液),术后前三周实验组转棒持续时间明显改善平均转棒持续时间60.5s,82.5s,98.5s,分别较对照2组提高49.3%,120.1%,94.9%,此后实验组运动能力逐渐下降,显示治疗效果减退,至术后第5周与对照组已无显著性差异。 5、细胞学实验显示,植入人类成体神经干细胞在小鼠纹状体内可以存活,并产生一定迁移,在注入2周后发现最远迁移距离900μm。Double Cortin染色显示部分神经干细胞已分化为新生神经元,2周时比例约为40%,周围大量神经胶质增生。注入2周时部分神经干细胞已分化出轴突,Synaptophysin染色显示部分轴突上已有突触后膜结构,显示移植干细胞已与宿主形成简单神经网络。 6、至干细胞注射后第四周,移植细胞存活率已明显降低,约为第一周时存活细胞的2%,与运动症状治疗效果消退时间平行。 7、注入神经干细胞Ki-67染色,显示已分化的神经元不表达Ki-67,不具备分裂潜能,致瘤可能性低。 结论： 1、MPTP药物可诱导产生短期内症状稳定的帕金森氏症C57/B16小鼠模型。该模型运动症状可以用转棒实验及爬杆实验进行评估。 2、人源神经干细胞注入MPTP帕金森小鼠纹状体区域,可以在4周以内部分改善疾病小鼠的运动障碍,表现为转棒实验持续时间延长,但无法长期保持。 3、人源神经干细胞注入MPTP帕金森小鼠纹状体区域后,能够存活至4周左右。注入2周后已能分化为新生神经元,并在纹状体内具备一定的迁移能力。 4、注入的神经干细胞部分可分化为多巴胺能神经元前体细胞,提示而未能分化为GABA能、谷氨酸能神经元前体细胞,可能与纹状体微环境相关。 5、神经干细胞注入小鼠纹状体后,能够与宿主细胞之间形成突触结构,显示初步具备形成新生神经网络的基础。
[Abstract]:Parkinson's disease is a common degenerative disease of the central nervous system. The prevalence rate is about 2% in the population over 60 years of age in the developed countries. The prevalence rate is similar in the aged population in the main cities of our country. The main clinical manifestations of Parkinson's disease include static tremor, stiffness, slow motion and abnormal postural reflex, and the serious people lose their self-care ability. Relative to them His neurodegenerative disease, Parkinson's disease has a complete set of treatments including drugs and deep electrode stimulation, which can improve symptoms in the short term but can not reverse the neuropathological changes, and the side effects of long-term drug use are significant. Since 80s, the development of stem cells and regenerative medicine gave the disease to the disease. With new ideas, scientists have used human embryonic stem cells and genetic engineering techniques to transplant dopamine derived embryonic stem cells into the mesencephalic substantia nigra or the thalamus striatum, which can improve symptoms for a long time and have been used in clinical trials. But because of the origin and ethical limitations of embryonic embryonic stem cells, they have not been widely used. As a result, the focus of the study turned to mesenchymal stem cells, neural stem cells, etc. here, we transplanted the cultured human aborted embryonic stem cell lines into the striatum area of the MPTP induced Parkinson's disease mouse model to understand the survival and growth of this human neural stem cell line in the microenvironment of the mice. The effect of neural stem cell transplantation on the motor function of Parkinson's disease mice was investigated, and a whole set of research system was established to treat the neurodegenerative diseases of mice by using human neural stem cell lines. Good performance.
Objective: To study the survival status of human neural stem cells transplanted into the striatum of Parkinson's disease mice; to study the differentiation, migration and apoptosis of the transplanted neural stem cells in the microenvironment of the disease; to investigate whether the transplanted neural stem cells are associated with the host neural network; to investigate whether the transplanted neural stem cells can be used for PAS. The exercise symptoms of Higginson's disease mice were improved, and the sensitivity and evaluation methods of different motor disorders in Parkinson's disease on human neural stem cells were evaluated, and a feasible scheme and process were established for different animal models and transgenic stem cells in the later period.
Research content and technical route:
1, a stable Parkinson's disease model was established. The classic MPTP Parkinson's disease C57/B16 mouse model was used. The experimental group was injected 4 times in the abdominal cavity for 3 hours each time according to the dose of MPTP20mg/kg body weight (dissolved in 0.9% saline). The control group was injected with the same dose of 0.9% physiological saline.
2, behavioral assessment. Using Rota-Rod and Rod Climbing, Parkinson's disease mice were established to evaluate their movement disorders.
3, human neural stem cells were cultured with lentivirus to transfuse GFP into cells, so that the cells were labeled with green fluorescence.
4, stereotactic injection of neurons.
5, we assessed the survival, differentiation, migration, apoptosis and host neural network integration of neural stem cells injected into the microstripe of PD mice.
6, the changes of motor symptoms in different periods before and after treatment were compared between Parkinson's disease mice and the control group, and the effects of stereotactic injection and stem cell transplantation on the movement symptoms of PD mice were evaluated.
Result锛，

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