第一部分
干细胞移植治疗神经系统疾病的文献计量学分析
目的：缺血性脑卒中和帕金森病是干细胞移植治疗神经系统疾病的两个热点领域。文献计量学应用数学和统计学方法对文献情报进行定量分析，其在医学领域的应用日益广泛。本研究利用文献计量学对干细胞移植治疗神经系统疾病进行直观定量分析，以期勾勒出该领域发展的整体图景，为制定干细胞发展策略提供依据。
方法：本研究以Web of Science核心合集数据库为数据源，时间限制为1999年至2018年，分别对干细胞治疗缺血性脑卒中和帕金森病相关研究文献进行检索。利用HistCite、VOSviewer和Medical Text Indexer等软件或在线工具对文献特征和医学主题词随时间变化情况进行分析。
结果：干细胞治疗缺血性脑卒中领域：年度发文量呈波动上升趋势；Stroke的发文量和共被引次数均排名第一，是本研究领域影响力最大的期刊；Michael Chopp和Cesario V. Borlongan是发文量最大、最具学术影响力的两位研究作者；美国和中国是学术贡献最多的两个国家，相互之间合作密切，发文量排名前十的研究机构均来自这两个国家；神经干细胞（neural stem cells，NSCs）和间充质干细胞（mesenchymal stem cells，MSCs）是研究最多的细胞类型，内皮祖细胞的研究数量正快速增加。干细胞治疗帕金森病领域：年度发文量逐渐增多；哈佛大学、卡罗林斯卡学院、京都大学和隆德大学是研究活跃的机构；美国的发文量稳居世界第一，远超其他国家；中国的发文量较本世纪初期有了明显增加，目前居世界第二；诱导多能干细胞（induced pluripotent stem cells，iPSCs）、NSCs和胚胎干细胞（embryonic stem cell，ESCs）是研究最广泛的细胞类型，其中iPSCs的临床转化处于领先地位，NSCs的临床前数据已经有丰富积累，ESCs由于伦理障碍和畸胎瘤风险而面临临床转化受阻。
结论：在干细胞治疗缺血性脑卒中和帕金森病这两个热点领域，美国是研究贡献最多的国家，中国在近二十年内取得巨大进步。目前，在缺血性脑卒中领域，MSCs和NSCs是研究最广泛的细胞类型；在帕金森病领域，iPSCs和NSCs是研究最多的细胞类型，具有良好的临床转化前景。
 

第二部分
缺血性脑卒中食蟹猴模型的行为学和影像学研究
目的：灵长类动物与人类在进化上亲缘关系较近，是研究缺血性脑卒中的理想实验动物。光化学法制作缺血性脑卒中模型具有操作微创、可重复性好的优势。本研究从行为学和影像学两个方面评价光化学法制作的缺血性脑卒中灵长类模型的合理性。
方法：本研究采用成年雄性食蟹猴为实验动物，利用光化学法诱导局灶皮层梗死，制作缺血性脑卒中食蟹猴动物模型。在造模后第1天和第3天对食蟹猴进行改良Kito量表评分和多模态磁共振扫描（3D-T1、3D-T2和BOLD序列）。比较实验动物在缺血性脑卒中不同时期的行为学和影像学表现，并进行统计分析。
结果：根据改良Kito评分结果，在造模后第1天和第3天食蟹猴均表现出明显的神经功能障碍，运动系统评分在第3天显著高于第1天（P = 0.024）。在造模后第1天，病灶体积与面部感觉评分（r = 0.791, P = 0.034）、手运动评分（r = 0.805, P = 0.029）和总评分（r = 0.786, P = 0.036）存在显著正相关关系；在造模后第3天，病灶体积与意识评分（r = 0.749, P = 0.005）、感觉系统评分（r = 0.637, P = 0.026）和总评分（r = 0.777, P = 0.003）存在显著正相关关系。以双侧F1区为感兴趣区进行脑功能连接分析，其结果显示，在造模后第1天，左侧F1区与右侧小脑之间的功能连接减弱；右侧F1区与右侧V1区、右侧Brodmann 5区的功能连接减弱，与右侧F7区之间功能连接增强；在造模后第3天，左侧F1区与右侧F1区的功能连接减弱，右侧F1区与左侧黑质的功能连接减弱。
结论：在超急性期和急性期，均可见Kito量表评分与病灶体积存在显著相关性，表明本研究所制备缺血性脑卒中食蟹猴模型的合理性。功能影像结果提示，该动物模型可以表现出小脑交叉失联络、双侧初级运动皮层功能连接减弱等改变，较好的模拟了人类脑梗死后的部分神经网络改变。
 

第三部分
神经干细胞移植治疗缺血性脑卒中的临床前研究
目的：干细胞治疗动物实验的安全性和有效性数据是决定其能否进入临床研究阶段的重要依据。尚没有灵长类动物模型用于神经干细胞（neural stem cells，NSCs）治疗缺血性脑卒中的临床前评价。在本研究中，我们将评价国内自主研发的NSCs治疗缺血性脑卒中食蟹猴模型的安全性和有效性，以期为后续开展临床研究提供前期证据。
方法：将实验动物分为立体定向脑内注射组、经鼻粘膜注射组、经颈动脉注射组和对照组。我们采用血常规、肝肾功能、肿瘤标志物、头部磁共振扫描和组织病理等指标进行安全性评价。我们比较治疗后组间改良Kito量表评分和病灶体积进行有效性评价。
结果：安全性方面：与对照组相比，NSCs治疗组实验动物的体重3月内无明显下降，血常规、肝肾功能和肿瘤标志物在9月内未见异常，解剖肝、脾、肺、肾、睾丸未见肿瘤形成，头部磁共振扫描检查无明显新生肿物。有效性方面：实验动物在接受NSCs治疗后均出现一定程度的神经功能改善，但是与对照组相比，改良Kito量表、病灶体积的差异无显著性。
结论：NSCs移植治疗缺血性脑卒中安全可行，初步研究结果表明其对改善神经功能具有一定的作用。
 

第四部分
神经干细胞移植治疗缺血性脑卒中食蟹猴模型的机制研究
目的：外泌体和多模态脑影像技术在神经系统疾病早期诊断、预后预测、治疗靶点等方面的应用日益广泛。本研究旨在通过外泌体miRNA测序和功能磁共振分析的方法，探索神经干细胞（neural stem cells，NSCs）移植治疗缺血性脑卒中的潜在作用机制。
方法：本研究对不同途径NSCs移植6周后的血浆外泌体进行miRNA测序，与对照组比较发现差异表达的miRNA，然后进行GO功能富集分析和KEGG信号通路分析。以双侧运动相关脑区（F1至F7区）为感兴趣区进行全脑功能连接分析，比较不同途径NSCs移植治疗组和对照组之间脑区功能连接强度的变化。
结果：我们选择差异表达倍数排名前二十的miRNA进行文献回顾，发现大部分外泌体miRNA缺乏神经保护的证据支持，只有三个差异表达外泌体miRNA的变化趋势具有神经保护作用，包括立体定向注射组miR-182下调、经鼻粘膜注射组miR-92a-3p下调和经动脉注射组miR-143-3p下调。GO功能富集分析显示，与全部miRNA分析相比，治疗组差异表达miRNA的靶基因在细胞杀伤、病毒成分、电子载体等富集趋势下降；在细胞聚集、细胞外基质、胶原蛋白等富集趋势增加。KEGG信号通路分析显示，差异表达miRNA靶基因主要位于胞吞作用、肌动蛋白细胞骨架调节等信号通路。与对照组相比，经鼻粘膜注射组右侧F5区与右侧23b区、左侧F6区与左侧24c’区之间功能连接增强；经颈动脉注射组多个脑区之间功能连接增强。
结论：NSCs移植后外泌体miR-182、miR-92a-3p和miR-143-3p表达下调可能是发挥神经保护作用的重要因子。NSCs移植可能通过增加部分运动相关脑区与其他脑区的功能连接强度促进神经功能恢复。
 

Section One: Bibliometric Analysis of Stem Cell Transplantation for Treatment of Nervous System Disorders

Objective: Ischemic stroke and Parkinson’s disease (PD) are two research hotspots in treating neurological diseases with stem cell transplantation. Bibliometrics is used to combine mathematical and statistical methods to analyze bibliographic information quantitatively, and it becomes increasingly popular in the field of medicine. In this study, we used bibliometrics to perform an intuitive and quantitative analysis of stem cell transplantation to treat neurological diseases to demonstrate the landscape of the development of this research field and provide a basis for formulating stem cell-related strategies.

Methods: In this study, the Web of Science core collection database was used as the data source, and the time was limited from 1999 to 2018. The literature search on stem cell treatment of ischemic stroke and PD was conducted. HistCite, VOSviewer, and Medical Text Indexer were used to analyze the changes in literature characteristics and medical subject headings in different periods.

Results: In the field of stem cell treatment of ischemic stroke, the bibliometric findings were as follows: the annual output of publications kept rising in a fluctuating manner; Stroke was the most influential journal in this research field because it ranked first in the number of publications and co-citations; Michael Chopp and Cesario V. Borlongan were the top two researchers with the most significant number of publications and the most potent academic influence; the United States and China were the top two countries with the most academic contributions and close cooperation, and the top ten productive research institutions were all from these two countries; neural stem cells (NSCs) and mesenchymal stem cells (MSCs) were the most studied cell types, and the number of studies on endothelial progenitor cells rapidly increased. In the field of stem cell therapy for PD, the bibliometric findings were as follows: the annual number of publications was gradually increasing; Harvard University, Karolinska Institute, Kyoto University, and Lund University were among the most active research institutions; the number of publications from the United States ranked first in the world, far surpassing others countries; the number of China’s publications has increased significantly since twenty years ago, and China is currently the second most productive country in the world; induced pluripotent stem cells (iPSCs), NSCs and embryonic stem cells (ESCs) were the most widely studied cell types, iPSCs were in a leading position in clinical translation, the preclinical data of NSCs has accumulated rapidly, but the prospect of clinical use of ESCs was hindered due to ethical issues and risk of teratoma formation.

Conclusion: In two hot research areas of stem cell treatment of ischemic stroke and PD, the United States has the most research contributions. China has made significant progress in the past two decades. In the field of ischemic stroke, MSCs and NSCs are the most widely explored cell types; in the area of PD, iPSCs and NSCs are the most investigated cell types, both of which have brilliant prospects of clinical translation.

Section Two: Behavioral and Imaging Studies of Cynomolgus Monkey Models of Ischemic Stroke

Objective: The evolutionary relationships between humans and primates are close. Non-human primates are highly recommended as experimental animals for ischemic stroke studies. The ischemic stroke can be induced by photochemical methods, which have advantages of minimal invasiveness and good reproducibility. In this study, we evaluated the rationality of the primate models of ischemic stroke made by photochemical methods in behavior assessment and functional imaging changes.

Methods: In this study, adult male cynomolgus monkeys were used as experimental animals, and focal cortical infarction was induced by photochemical methods. The cynomolgus monkeys were scored according to the modified Kito scale and received multimodal MRI scans including 3D-T1, 3D-T2, and BOLD sequences on the 1st and 3rd day after induced stroke. The behavioral and imaging data of experimental animals in different periods of ischemic stroke were analyzed.

Results: According to the results of the modified Kito scale, cynomolgus monkeys showed apparent neurological dysfunction on the 1st and 3rd day after modeling, and the motor system score on the 3rd day was significantly higher than that on the 1st day (P = 0.024). On the 1st day after modeling, the lesion volume was positively correlated with facial sensory score (r = 0.791, P = 0.034), hand movement score (r = 0.805, P = 0.029) and total score (r = 0.786, P = 0.036); on the 3rd day after stroke, the lesion volume was positively correlated with consciousness score (r = 0.749, P = 0.005), sensory system score (r = 0.637, P = 0.026) and total score (r = 0.777, P = 0.003). The bilateral F1 areas were selected as regions of interest in the seed-based functional connectivity analysis. On the 1st day after stroke, the functional connectivity strength between area F1 of left side and the cerebellum of right side decreased; the functional connectivity strength between area F1 of right side and area V1 of right side as well as Brodmann area 5 of right side was weakened, and the functional connectivity strength between area F1 of right side and area F7 of right side increased. On the 3rd day after stroke, the functional connectivity strength between left area F1 and right area F1 and the functional connectivity strength between right area F1 and left substantia nigra were weakened.

Conclusion: In the hyperacute and acute phases of ischemic stroke, the significant correlation between the Kito scale score and the lesion volume can be observed. The functional imaging results suggest that the animal models can present changes such as crossed cerebellar diaschisis and weakened functional connectivity of bilateral primary motor areas. The validity of the cynomolgus monkey models of ischemic stroke is proved in this study.

Section Three: Preclinical Studies of Neural Stem Cell Transplantation in the Treatment of Ischemic Stroke

Objective: The preclinical data of stem cell therapy in animal models are vital for future clinical trials. There was no preclinical study using primate models to determine the safety and efficacy of neural stem cells (NSCs) in ischemic stroke treatment. In this study, we evaluated the safety and efficacy of the domestically produced NSCs in treating ischemic stroke cynomolgus monkey models, aiming to provide preliminary evidence for future clinical studies.

Methods: The experimental animals were divided into four groups: stereotactic intracerebral injection group, trans-nasal submucosal injection group, intra-carotid artery injection group, and control group. We used critical parameters from blood routine, liver and kidney function, tumor markers, head MRI scan, and histologic examination to evaluate the safety. We compared the modified Kito scale score and lesion volume between groups after NSCs treatment to assess the efficacy.

Results: Compared with the control group, the body weight of the experimental animals in the NSCs treatment group did not show significant decrement within three months, and the blood routine, liver and kidney function, and tumor markers were within the reference ranges within nine months. The histologic examinations of the liver, spleen, lung, kidney, and testis showed no tumor formation. No tumor was found on the brain MRI. Experimental animals showed a certain degree of improvement in neurological function after receiving NSCs treatment. However, there was no significant difference in the modified Kito scale scores and lesion volumes when comparing the treatment groups and the control group.

Conclusion: NSCs transplantation is safe and feasible for the treatment of ischemic stroke. Preliminary results demonstrate improved neurological function in animal models.

Section Four: Potential Mechanism of Neural Stem Cell Transplantation in the Treatment of Ischemic Stroke Cynomolgus Monkey Models

Objective: The applications of exosome and multimodal brain imaging technology in early diagnosis, prognosis prediction, and therapeutic targets receive more and more attractions. We aimed to explore the potential mechanisms of neural stem cell (NSCs) transplantation in treating ischemic stroke through exosomal miRNA sequencing and functional MRI scans.

Methods: In this study, plasma-derived exosomal miRNA sequencing was performed six weeks after NSCs transplantation, and the differentially expressed miRNAs were found in comparison with the control group. Then GO enrichment analysis and KEGG pathway enrichment analysis were performed. We selected bilateral motor system cortex (area F1 to F7) as regions of interest in the seed-based functional connectivity analysis. The strength of functional connectivity was compared between the NSCs treatment groups and the control group.

Results: We selected the top 20 differentially expressed miRNAs according to fold changes and did thorough literature reviews. The majority of exosomal miRNAs were not found to be associated with neuroprotection except the down-regulation of miR-182 in the stereotactic injection group, the down-regulation of miR-92a-3p in the trans-nasal injection group, and the down-regulation of miR-143-3p in the trans-arterial injection group. GO analysis revealed that target genes of differentially expressed miRNAs showed decreasing enrichment trends in cell killing, viral components, and electron carriers and increasing trends in cell aggregation, extracellular matrix, and collagen trimer. KEGG pathway analysis showed that the target genes of differentially expressed miRNAs were mainly involved in signaling pathways of endocytosis and regulation of actin cytoskeleton. The functional connectivity between the right area F5 and the right area 23b as well as left area F6 and left area 24c’ was enhanced in the trans-nasal injection group, and the functional connectivity between multiple brain areas in the trans-arterial injection group was enhanced.

Conclusion: The down-regulation of exosomal miR-182, miR-92a-3p and miR-143-3p expressions after NSCs transplantation may be important factors for neuroprotection. NSCs transplantation may promote the recovery of neurological function by enhancing the functional connectivity between the motor system cortex and other brain areas.