间充质干细胞通过隧道纳米管转运线粒体减轻内皮细胞缺血损伤并改善大鼠缺血性卒中预后的研究
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摘要
前言
缺血性脑血管疾病已成为严重威胁人类健康的重大疾病,并造成了严重的社会经济负担。尽管缺血性脑血管疾病的治疗研究取得了一定进展,但是现有的治疗手段仍十分有限,治疗效果并不理想。近年来,干细胞移植在缺血性脑血管疾病治疗中的应用研究,为解决这一难题提供了新的思路和方法。作为一种被广泛研究的多能干细胞,骨髓来源的间充质干细胞(mesenchymal stem cells, MSCs)可以自身取材,既不产生免疫排斥反应,也不存在伦理问题,是目前较为理想的种子细胞。
在缺血性脑血管疾病中,血管内皮的损伤是主要的起病始动因素；而血管的修复,尤其是缺血半暗区受损血管的修复,又是挽救缺血受损神经元的前提；同时脑微血管内皮细胞的损伤是也导致血脑屏障开放、脑水肿发生,从而加重神经元细胞损伤的重要因素。因此,保护和修复脑血管的内皮细胞才是防治缺血性脑血管疾病的关键。然而,目前在于细胞治疗脑梗死的研究中,多数研究聚焦于受损神经组织的修复,但在干细胞对缺血部位受损的脑血管内皮细胞影响的研究却亟待进一步深入。近期的研究提示,移植的间充质干细胞可以促进缺血局部区域的血管新生并减轻缺血组织的损伤；然而,其具体机制仍存在争议：部分学者认为干细胞通过转分化为缺血损伤的血管组织细胞促进了血管再生,另有学者认为移植的干细胞可以通过细胞融合发挥其保护作用,更有学者认为间充质干细胞通过其旁分泌的细胞因子促进了血管新生。因此,针对移植的间充质干细胞与缺血再灌注损伤的微血管及内皮细胞之间的相互作用需要进一步的研究。
在缺血再灌注损伤的复杂机制中,线粒体损伤是其中的关键环节。线粒体在有氧代谢、氧化磷酸化和细胞凋亡等多种病理生理过程中发挥着关键作用。有研究显示线粒体损伤的严重程度在一定程度上决定了内皮细胞的功能和命运,并影响到后续的血管新生及缺血组织的修复。然而,当前关于干细胞对损伤的内皮细胞线粒体功能影响的实验研究尚未见报道。
目前,一种新型通讯连接方式的发现正在引发人们对细胞通讯认识的革命：最近在动物细胞间发现了一种细长膜管状细胞连接一隧道纳米管((tunneling nanotubes, TNTs)。作为细胞间相互交流的通道,TNTs可以在相连接的细胞间形成复杂的通讯网络,并能够运输多种细胞组分以促进细胞间的相互交流,其中不仅包括细胞膜的成分、细胞质内的小分子,甚至线粒体等一些较大的细胞器也能通过TNTs进行转运。越来越多的研究提示TNTs是动物细胞间一种普遍存在的相互作用方式。由此我们提出以下假设：干细胞可以建立起与内皮细胞的直接连接方式——TNTs,并通过TNTs将自身的线粒体直接转运至内皮细胞中,修复受损内皮细胞的能量代谢,减少细胞凋亡,以保护内皮细胞、修复缺血受损的脑微血管系统,从而发挥其减轻缺血性卒中损伤并改善卒中预后的作用。
TNTs的发现揭示了一种全新的细胞间交流通道,引发了人们对细胞相互作用方式的重新思考,直到目前其研究仍处于初步阶段,许多问题还有待于进一步解决。我们在前期实验的基础上,结合干细胞和TNTs的最新研究进展,提出了干细胞通过TNTs直接转移线粒体以保护缺血受损内皮细胞的推测,以图揭示出干细胞修复缺血受损脑血管内皮细胞的机制,为干细胞在缺血性卒中治疗的临床应用提供了基础实验依据,同时也为干细胞治疗其他缺血性疾病的机制研究提供了借鉴。
第一部分间充质干细胞通过隧道纳米管转运线粒体保护类缺血n损伤的内皮细胞的研究
研究目的
1、探讨间充质干细胞与共培养的脐静脉内皮细胞间能否建立TNTs结构联系。
2、探究影响TNTs形成的因素并揭示TNTs的形成机制。
3、探讨线粒体能否通过TNTs在两种细胞间进行有效的转运。
4、探讨干细胞能否通过建立TNTs转运线粒体以保护类缺血损伤的内皮细胞。
研究方法
1、骨髓间充质干细胞与脐静脉内皮细胞的分离、培养、鉴定与标记。
密度梯度离心法分离骨髓间充质干细胞,经培养扩增后,流式细胞术鉴定间充质干细胞表型为CD29、CD44和CD105阳性, CD34和CD45阴性。脐静脉内皮细胞获赠于山东大学心血管蛋白质组重点实验室。采用慢病毒介导的增强型绿色荧光蛋白标记间充质干细胞,并通过流式细胞分选术将其纯化。另外,应用pAcGFP1-Mito vector和pDsRed2-Mito vector分别对脐静脉内皮细胞和骨髓间充质干细胞的线粒体进行标记。
2、对比分析正常条件下骨髓间充质干细胞与脐静脉内皮细胞的线粒体功能。
通过生物能量代谢监测仪分析细胞的耗氧率(oxygen consumption rate, OCR)和产酸率(extracellular acidification rate, ECAR),经细胞计数校正后的耗氧率和产酸率数值可直接反映出MSCs和HUVECs的线粒体功能差异。
3、建立干细胞与类缺血损伤后内皮细胞的共培养模型。
在缺氧培养箱中对HUVECs实施氧糖剥夺处理150分钟,再将处理的细胞进行复氧复糖处理,4小时后将等量的间充质干细胞与之直接混合培养,以建立干细胞与类缺血损伤后内皮细胞的共培养模型。
4、激光共聚焦显微镜观察隧道纳米管的形成及线粒体在其中的转运。
应用激光共聚焦显微镜观察共培养的MSCs和HUVECs之间隧道纳米管的形成以及线粒体在其中的转运情况,并对不同干预条件下的两种细胞间形成的TNT及膜突起(membrane protrusions, MPs)计数统计。
5、应用流式细胞术分选并定量分析混合培养后两种细胞间线粒体的相互转移率。
利用Hoechst33342标记的间充质干细胞细胞核,以及干细胞和内皮细胞两种细胞间的形态差异,采用流式细胞分选术将共培养48小时后的两种细胞分选开来。再应用流式细胞术定量分析线粒体在两种细胞间的相互转移率。
6、分析间充质干细胞对共培养脐静脉内皮细胞的有氧代谢、细胞活性及凋亡率的影响；并检测丝状肌动蛋白(filamentous actin, F-actin)解聚剂latrunculin-A (LatA)、磷脂酰丝氨酸位点阻断剂Annexin V和线粒体功能缺失的干细胞对其共培养效应的影响。
采用流式细胞分选术将不同的共培养组中的脐静脉内皮细胞分离出来。采用FITC Annexin V凋亡试剂盒分析内皮细胞凋亡率,采用Cell Counting Kit-8(CCK-8)分析内皮细胞活性,使用生物能量代谢监测仪分析内皮细胞的线粒体功能。
7、分析干细胞的旁分泌功能和细胞融合等因素对内皮细胞活性的影响。
收集浓缩不同处理条件下的间充质干细胞条件培养液,应用ELISA试剂盒检查其中的血管内皮细胞生长因子(vascular endothelial growth factor, VEGF)、血小板源性生长因子(platelet-derived growth factor BB, PDGF-BB)和成纤维细胞生长因子-2(fibroblast growth factor2,FGF-2)的含量,以明确干细胞的旁分泌功能对内皮细胞活性的影响。
采用直接测序法对比检测共培养前后的HUVECs和MSCs的线粒体DNA(mitochondrial DNA, mtDNA),明确共培养后被MSCs营救的HUVECs是否含有来源于MSCs的线粒体。应用短串联重复序列方法对比分析共培养前后的HUVECs和MSCs的核DNA(nuclear DNA, nDNA),明确共培养后被MSCs营救的HUVECs是否含有来源于MSCs的细胞核,也即是分析细胞融合的可能性。
将染色标记好的血小板和干细胞线粒体加入缺血受损的内皮细胞中直接共培养,激光共聚焦显微镜观察共培养48小时后的内皮细胞的生存状况,从而分析内皮细胞是否主动吞噬了细胞外游离的线粒体。
研究结果
1、生物能量代谢监测仪分析证实间充质干细胞具有显著优于脐静脉内皮细胞的线粒体功能(p<0.05)。
2、激光共聚焦显微镜证实共培养的间充质干细胞与脐静脉内皮细胞间可建立隧道纳米管结构联系,线粒体可在其中进行双向转运(两种细胞间的双向转移率大致均衡)。
3、激光共聚焦显微镜证实类缺血再灌注损伤能介导脐静脉内皮细胞与间充质干细胞间建立更为复杂和广泛的网络状TNT联系,并促使TNT中的线粒体双向交换变为单向转运(由干细胞向内皮细胞方向),且(流式细胞术证实)其转移率显著提高。
4、LatA和Annexin V可显著减少脐静脉内皮细胞与间充质干细胞间的TNTs数量,表明TNTs的形成是依赖F-actin的聚合和磷脂酰丝氨酸的外化的过程。
5、间充质干细胞通过隧道纳米管将自身线粒体转运至类缺血再灌注损伤的脐静脉内皮细胞内,以修复其有氧代谢功能并减少细胞凋亡。F-actin解聚剂LatA、磷脂酰丝氨酸位点阻断剂Annexin V均可显著抑制该保护效应(p<0.05)。另外,尽管线粒体功能缺失的MSCs完全丧失了对内皮细胞有氧代谢功能的修复,但仍可以部分减轻内皮细胞的凋亡(p<0.05),这提示TNTs还可能通过转运其他物质的方式发挥其对内皮细胞的保护效应。
6、LatA和Annexin V的处理对间充质干细胞的旁分泌促血管新生作用细胞因子(VEGF, PDGF-BB, FGF-2)的功能没有显著影响,因此间充质干细胞的旁分泌功能不是其保护内皮细胞的关键机制。另一方面,被拯救的脐静脉内皮细胞的nDNA来源母系的内皮细胞,而其：ntDNA则同时含有MSCs和母系内皮细胞两种组分,这一结果不仅支持线粒体在异种细胞间发生了转移,同时也排除了发生细胞融合的可能性。此外,将染色标记好的血小板和干细胞线粒体加入缺血损伤的内皮细胞中直接共培养48小时,内皮细胞的线粒体功能、细胞活性和凋亡率均没有明显改善,且细胞中未发现含有被染色标记的线粒体,表明缺失损伤后的脐静脉内皮细胞不具有主动吞噬线粒体的功能。
第二部分间充质干细胞保护大鼠脑微血管系统线粒体的功能并改善缺血性卒中预后的研究
研究目的
1、研究间充质干细胞移植对大脑中动脉闭塞后再灌注大鼠的脑梗死面积的影响。
2、探讨间充质干细胞移植对脑梗死大鼠运动神经功能恢复的影响。
3、研究间充质干细胞对脑梗死大鼠缺血脑组织血管新生的影响。
4、探讨间充质干细胞移植对缺血受损脑微血管的线粒体功能的影响。
5、进一步探讨隧道纳米管介导的线粒体转移机制在干细胞改善脑梗死大鼠预后中的作用。
研究方法
1、建立大鼠的大脑中动脉闭塞后再灌注模型。
线栓法制备大鼠的大脑中动脉闭塞(middle cerebral artery occlusion,MCAO)后再灌注模型。在MACO120分钟后撤出尼龙线恢复大脑中动脉的灌注,术中采用多普勒仪证实闭塞和再灌注的效果。术后对大鼠进行神经功能缺损评分,并根据评分结果选取大鼠进人下一步实验
2、建立经动脉途径将间充质干细胞植入缺血损伤的大鼠脑微血管系统的模型。
对大鼠的间充质干细胞进行分离、培养和鉴定。为示踪移植的干细胞及其线粒体,用Hoechst33342标记MSCs的细胞核,应用pDsRed2-Mito vector对干细胞的线粒体进行标记。在大脑中动脉闭塞再灌注术后24小时,使用显微注射针将5×105MSCs缓慢注入右侧颈内动脉系统并密切观察术中术后的大鼠病情变化。
3、2,3,5-氯化三苯基四氮唑染色法分析大鼠脑梗死体积。
间充质干细胞移植术7天后,采用2,3,5-氯化三苯基四氮唑(2,3,5-triphenyltetrazolium chloride,TTC)对不同处理组大鼠梗死后的脑组织进行染色,应用ImageJ软件进一步分析梗死体积。
4、测试脑梗死大鼠的运动神经功能。
应用转棒疲劳实验和跑轮实验对MCAO术前及MSCs移植术后的第1、7、14和28天的大鼠进行测试,以评估间充质干细胞移植及其它不同处理因素对脑梗死大鼠的运动神经功能恢复的影响。
5、探讨脑微血管的密度与接受移植干细胞线粒体的宿主细胞数量间的关系。
受试大鼠接受动脉注射的绿色荧光染料(fluorescein isothiocyanate-dextran amine, FD-2000S)以达到激光共聚焦显微镜下脑微血管充分显影的效果。将脑组织固定并制作冰冻切片。共聚焦显微镜下观察梗死核心区和缺血半暗区的脑微血管的密度与接受干细胞线粒体的宿主细胞(DsRed2+/Hoechst33342-cells)数量间的关系。
6、分离纯化脑微血管片段,并检测其线粒体功能。
取梗死侧的大脑半球,采用两次酶消化法和密度梯度离心法分离和纯化脑微血管片段。将纯化的脑微血管片段置于Ⅳ型胶原和纤连蛋白覆盖的XF24组织培养皿内,使用生物能量代谢监测仪分析微血管片段的线粒体功能。
研究结果
1、间充质干细胞移植显著减少大脑中动脉闭塞大鼠的脑梗死面积(p<0.05)。
2、间充质干细胞移植可促进脑梗死大鼠运动功能的恢复(p<0.05)。
3、间充质干细胞移植显著增加缺血半暗区的脑微血管密度(p<0.05)。
4、间充质干细胞可有效改善缺血损伤脑微血管片段的线粒体功能(p<0.05)。
5、LatA和Annexin V并不影响缺血半暗区内DsRed2+/Hoechst33342+细胞的数量(DsRed2+/Hoechst33342+细胞即为示踪出的植入术后全部的MSCs,包括了可能发生细胞融合和转分化的MSCs),证实LatA和Annexin V对干细胞的归巢、滞留和迁移没有造成显著影响。然而,TNTs的抑制剂LatA和Annexin V可显著减少缺血半暗区内DsRed2+/Hoechst33342-细胞的数量(DsRed2+/Hoechst33342-细胞为接受干细胞转移线粒体的宿主细胞),同时也显著抑制了干细胞的对脑梗死大鼠的保护效应(p<0.05),提示其保护效可能与隧道纳米管介导的线粒体转移密切相关；同时也进一步证实了旁分泌功能、细胞融合和转分化等因素不是干细胞保护效应的关键机制。
综上所述
1、骨髓间充质干细胞具有显著优于脐静脉内皮细胞的线粒体功能。间充质干细胞与内皮细胞间可建立隧道纳米管结构联系,两种细胞的线粒体可在其中进行双向转运。正常条件下两种细胞之间线粒体的转运频率大致均等。
2、类缺血再灌注损伤能介导内皮细胞与间充质干细胞间建立更为广泛和复杂的TNTs联系网络,并促使TNTs介导的线粒体双向均等交换转变为从干细胞向内皮细胞方向的单向转运,其转移率也显著提高。从而使间充质干细胞能有效修复受损内皮细胞的线粒体功能并减少细胞凋亡。
3、TNTs的形成可能反映了细胞在应激或损伤状态下表现出的一种自我防御和营救机制。TNTs的形成依赖于丝状肌动蛋白的聚合以及磷脂酰丝氨酸位点的外化与识别。
4、体内实验进一步证实：间充质干细胞通过隧道纳米管转运线粒体可能是其保护缺血受损脑微血管的线粒体功能、促进血管新生并改善卒中大鼠的预后的关键机制。而间充质干细胞的旁分泌细胞因子、细胞融合或转分化机制则并非其保护作用的主要因素。
Background
Ischemic stroke leads to high rates of disability and death, thereby imposing an enormous medical and social burden. Currently available therapies for injured cerebrovascular systems are insufficient. Meanwhile, stem cell transplantation can be used to repair ischemic vascular damage and thus offers new prospects for stroke therapy. As neither immune rejection, nor the ethical questions, the bone marrow mesenchymal stem cells (MSCs), which can be drawn from oneself, are the ideal seed cells.
Cerebrovascular injury induced by ischemia-reperfusion has a key function in determining the survival of nerve cells after recanalization in focal ischemia by clogging microcirculation and disrupting the blood-brain barrier integrity. Recent reports have shown that MSCs participate in neovascularization and attenuate ischemic injury after focal cerebral ischemia. However, whether MSCs transdifferentiate into and replace lost cerebrovascular cells or perform paracrine function by secreting proangiogenic factors are unclear. Therefore, the mechanism of action of MSCs especially in the interaction between brain microvessel and grafted MSCs need to be further investigated.
Among the many complex mechanisms underlying vascular endothelium ischemia-reperfusion injury, mitochondrial damage appears to contribute significantly to these pathological processes. Mitochondria are essential organelles that play prominent roles in biological processes such as aerobic metabolism, oxidative phosphorylation, and cell death pathways. Endothelial mitochondria have been recognized as playing critical roles in the signaling cellular responses to environmental cues, which may determine the endothelial function and fate, thereby influencing angiogenesis in ischemia-reperfusion injury. Limited experimental data are available on the effects of stem cells on injured mitochondria in endothelial cells.
Recent studies have discovered highly sensitive nanotubular structures named tunneling nanotubes (TNTs) that bridge adjacent animal cells, enabling them to form complex networks. As a novel mechanism of cell-cell communication, TNTs facilitate the exchange of cellular components and signaling molecules between connected cells such as plasma membrane components, calcium ions, pathogens, and organelles, including mitochondria. We hypothesized that stem cells and post-ischemic endothelial cells interact with each other through the formation of TNTs and that this novel mechanism might be responsible for the beneficial effects exerted by engrafted stem cells.
The discovery of TNT reveals a new style for intercellular communication, which has sparked the rethink the way of cell interaction. Until now the research on TNT is still in the preliminary stage, many problems has yet to be further investigated. Our findings challenge the classical view on stem cell transplantation for stroke therapy and may also have implications in the treatment of other ischemic diseases.
Part I:Mesenchymal stem cells rescue injured endothelial cells in an in vitro ischemia-reperfusion model via tunneling nanotube-mediated mitochondrial transfer
Objectives
1. To observe the TNT connections between the HUVECs and the MSCs.
2. To explore the formation mechanism of TNTs between the HUVECs and the MSCs.
3. To investigate the transfer of the mitochondria between the HUVECs and the MSCs through the TNT.
4. To explore the protective effect of the MSCs on the injured HUVECs via TNT-mediated mitochondrial transfer.
Methods
1. Cell culture, identification, label, and lentiviral transduction.
The human MSCs were isolated from the bone marrow of the subjects using a density gradient. The HUVECs were obtained from the Key Laboratory of Cardiovascular Proteomics of Shandong Province. The MSCs were analyzed by fluorescence-activated cell sorting (FACS) to evaluate the cell surface markers. The MSCs expressed the antigens CD105, CD29, and CD44; however, they were negative for CD45and CD34. The HUVECs and MSCs were labeled for distinction before the co-cultivation. The MSCs were incubated with lentiviral vector pWPT-enhanced green fluorescence protein (EGFP). To investigate the mitochondrial transfer, the MSC nuclei were stained with Hoechst33342before co-cultivation. The mitochondria of the MSCs and the HUVECs were labeled. The pDsRed2-Mito vector and pAcGFPl-Mito vector was used to label the mitochondria of the HUVECs and MSCs, respectively.
2. Functional Measurements of Mitochondrial Activity in the MSCs and the HUVECs.
The measurements of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), which are indicators of aerobic respiration and glycolysis, were conducted with an XF24Extracellular Flux Analyzer. The mitochondrial function of the MSCs and the HUVECs were analyzed, and the results were normalized to the cell number.
3.In Vitro Ischemia-Reperfusion Model and Co-culture Model
In vitro ischemia-reperfusion was simulated by conducting oxygen glucose deprivation (OGD) and reoxygenation (RO) on the HUVECs in an anoxia chamber. After150min of the OGD, the RO was performed by reinstating the cells under the normoxic conditions and the pre-OGD medium. At4h of RO, an equal number of MSCs were directly added to the damaged HUVECs.
4. The laser scanning confocal microscopy was conducted to investigate the TNT-like structures connections and the transport of mitochondria.
The laser scanning confocal microscopy was conducted during and after the in vitro ischemia to investigate the morphological changes and possible interactions among the co-cultured HUVECs and MSCs. The TNT-like structures connections between the HUVECs and the MSCs and the membrane protrusions (MPs) were counted after24h of co-culture.
5. The exchange rate of mitochondria between the HUVECs and the MSCs was analyzed using FACS analysis.
To investigate the mitochondrial transfer further, the exchange rate of mitochondria between the HUVECs and the MSCs was analyzed after co-culture for48h using FACS analysis. We could also distinguish the two different cells by their Hoechst33342label and different morphologies using FACS.
6. To investigate the protective effects of MSCs, the cell viability, apoptosis, and mitochondrial activity the of the HUVECs was analyzed respectively.
To provide direct evidence for the functional role of mitochondria transfer via TNT-like structures, we generated MSCs with mitochondrial dysfunction using mtDNA depletion by cell treatment with ethidium bromide, which do not affect the formation of TNTs and the transfer of other molecules and organelles. After co-culture for48h, the cell apoptosis of the HUVECs we analyzed using the FITC Annexin V Apoptosis Detection Kit I (BD PharmingenTM, San Diego, CA, USA). The cell viability of the HUVECs was estimated using the Cell Counting Kit-8(CCK-8; Dojindo, Japan). The mitochondrial function of the HUVECs was analyzed using the XF24Extracellular Flux Analyzer.
7. The possiblity of cell fusion, the paracrine function and the passive phagocytosis of mitochondria was respectively analyzed.
The MSC conditioned media were collected after48h of culture, and the concentration of the MSC conditioned media cytokines was measured using ELISA kits [vascular endothelial growth factor,(VEGF); platelet-derived growth factor BB,(PDGF-BB); fibroblast growth factor2,(FGF-2)].
To investigate the possiblity of cell fusion, the nuclear DNA and mitochondrial DNA (mtDNA) extracted from the HUVECs, the MSCs, and rescued HUVECs were assayed by direct sequencing of the PCR products and the GeneScan analysis based on the short tandem repeat sequences.
To investigate the possibility that the HUVECs phagocytose MSC debris containing mitochondria and thus acquire double-labeled fluorescent dyes, the mitochondria isolated from MSCs and human platelets were used as sources of membrane-bound functional mitochondria. These were then co-cultured with the injured HUVECs for48h.
Results
1. MSCs have superior mitochondrial function compared to HUVECs.
2. MSCs and endothelial cells can exchange mitochondria via the TNT-like structures (the basal level of bi-directional exchange of mitochondria occurs with equal frequencies).
3. OGD/RO stress-induced mitochondrial transfer through the TNT-like structures becomes frequent and almost unidirectional from the MSCs to the injured endothelial cells.
4. The TNT formations between the MSCs and HUVECs are substantially reduced after the LatA or Annexin V treatment. The formation of the TNT-like structures connecting the endothelial cells to the MSCs is dependent on the F-actin polymerization of and the exofacial PS domains.
5. Mesenchymal stem cells can rescue aerobic respiration and protect the endothelial cells from apoptosis via tunneling nanotube like structure-mediated mitochondrial transfer. The addition of LatA or Annexin V partially suppresses this effect. The TNT-mediated protection on aerobic respiration is completely abrogated by co-cultures with the MSCs having mitochondrial dysfunction,(no statistical differences comparing with the OGD/RO group), while that the protective effect on HUVECs viability is partially suppressed (significant differences comparing with the OGD/RO group, p<0.05), suggesting that the transfer of functional mitochondria from MSCs to HUVECs was required to rescue the endothelial cells from damage, but not unique.
6. The cell fusion, paracrine function, and passive phagocytosis of mitochondria can be excluded as the key mechanism for protective effects of MSCs. The potential side effects of LatA or Annexin V on the cultures of the MSCs or HUVECs were investigated. The addition of LatA or Annexin V caused negligible changes in the cell viability of MSCs or HUVECs based on the CCK-8assay and also affected the MSCs paracrine function of several primary proangiogenic factors (VEGF, PDGF-BB, FGF-2) insignificantly by ELISA analysis, suggesting that the TNT-like structures might be essential, whereas the soluble cytokines were less likely to act as the principal rescue factors.
The nuclear DNA of the rescued HUVECs from five independent samples was conformably from the HUVECs and did not contain the nuclear genome from the MSCs. Cell fusion can be excluded as the mechanism for the mitochondrial transfer.
The HUVECs became senescent, and double-labeled cellswere not observed. No significant changes in cell viabilityand mitochondrial activity were observed in the co-cultures compared with the injured HUVEC culture alone, which may exclude the possibility that the HUVECs recovered from injury through passive phagocytosis of the mitochondria.
Part Ⅱ:Mesenchymal stem cells protect mitochondrial function of cerebral microvasculature and promote recovery from ischemic stroke
Objectives
1. To investigate the effect of MSCs on the infarct cortex.
2. To explore the effect of MSCs on the neurobehavioral functions after stroke.
3. To explore the therapeutic effect of MSCs on the angiogenesis in ischemia-reperfusion mjury.
4. To investigate the effect of MSCs on mitochondrial activity of micro vascular fragments in ischemic cerebral hemisphere.
5. To explore the role of TNT-mediated mitochondrial transfer in MSC grafting for the treatment of ischemic stroke in vivo.
Methods
1. The middle cerebral artery occlusion (MCAO) and reperfusion model.
MCAO and reperfusion surgery were carried out as previously described. A nylon filament with a rounded tip was inserted into the right internal carotid artery at approximately18.5mm to20.0mm to occlude the origin of the right middle cerebral artery. After120min of MCAO, the filament was withdrawn to restore the blood flow (both occlusion and reperfusion were confirmed by laser Doppler). After the surgery, the rats were tested for neurological deficits according to Longa and Bederson's five score regulation. The rats with scores of2or3were kept for the subsequent experiments.
2. The culture, identification, label and transplantation of MSCs.
The rat MSCs were isolated and cultured as previously described. To investigate the mitochondria transfer, MSC nuclei were stained with Hoechst33342, and the mitochondria of the MSCs were also labeled using pDsRed2-Mito vector. The transduced cells were purified by FACS, and then expanded. At24h after brain ischemia and reperfusion, randomly assigned animals received IA injection of MSCs, as previously described. The right carotid artery was again exposed, whereas the external carotid artery and the pterygopalatine and superior thyroid arteries were ligated or coagulated. Up to5X105MSCs in10μL of PBS were injected into the common carotid artery using a10u L syringe with a33G microneedle. No difference in mortality and morbidity was found in the different experimental groups.
3. Analysis of Infarct Volume.
2,3,5-triphenyltetrazolium chloride (TTC) staining for determining the therapeutic effect of MSCs on the infarct cortex. Rats (n=5per group) were anesthetized with chloral hydrate (0.4g/kg body weight, intraperitoneally) and decapitated at7days after MSC transplantation. The brains were removed and sliced into2mm-thick coronal sections, and then immersed in2%2,3,5-triphenyltetrazolium chloride (TTC; Sigma, USA) in PBS for20min. The size of the infarct area was analyzed through the digital images of the brain slices using ImageJ software. The infarct volume percentage was calculated as a percentage of the contralateral hemisphere.
4. Behavioral tests.
Two behavioral tests were performed to evaluate the motor function before MCAO and at1,7,14, and28days after MSC transplantation (n=20per group). A rotarod test evaluated the coordinated movements of the limbs and the body through balance on a rotarod, which was slowly accelerated from4rpm to40rpm within4min. The length of time that the animals stayed on the rod was recorded. Data were calculated as the percentage of mean duration (10trials) on the rotor-rod compared with the baseline values. Another behavioral test was performed using the running wheel systems. Rats were transferred to individual cages containing an exercise wheel coupled to a bicycle computer. Total running distance, average speed, and maximum speed were recorded at different time points.
5. Analysis of Microvessel Density.
Microvessel density was measured as previously described. The rats were anesthetized and received IA injections fluorescein isothiocyanate-dextran amine (FD-2000S) to label the blood vessels at15days after MSC transplantation. The rat brains were rapidly fixed in4%paraformaldehyde and cryo-cut at30μm. Under a confocal microscope, we observed the microvessel densities in both the ischemic core and the peri-ischemic region. Microvessel density, the proportion of the area occupied by the labeled microvessels to the whole picture in pixels, was measured using ImageJ. The location of the DsRed2+/Hoechst33342+cells and DsRed2+/Hoechst33342-cells was also observed under the confocal microscope.
6. Brain Microvascular Fragment Isolation and the mitochondrial activity measurements.
The brain microvascular fragments of the right ischemic cerebral hemisphere of the rats were isolated using the methods described in our previous study. Fresh rat right cerebral cortices were cut into uniform1mm3sections. The sections were subsequently digested in a type II collagenase solution and collagenase/dispas solution. The microvascular fragments were purified twice in20%bovine serum albumin and33%continuous Percoll density gradient centrifugation. Purified microvascular fragments were plated on collagen type IV and fibronectin-coated XF24tissue culture plate. The mitochondrial function of the HUVECs was analyzed using the XF24Extracellular Flux Analyzer. OCR and ECAR were normalized to protein concentration for microvascular fragments. Protein measurements of the microvascular fragments were performed using a BCA Protein Assay Kit.
Results
1. MSCs can decrease the infarct area of stroke rats significantly (p<0.05).
2. MSCs can improve the motor function of stroke rats significantly (p<0.05).
3. MSCs can promote angiogenesis in ischemic penumbra of stroke rats significantly (p<0.05).
4. MSCs can protect the mitochondrial function of damaged cerebral microvasculature significantly (p<0.05).
5. The the addition of LatA or Annexin V does not affect the number of DsRed2+/Hoechst33342+cells (all of the transplanted MSCs, including the transdifferentiated MSCs and the MSCs occurring cell fusion with the host cells) in the peri-infarct area, suggesting that LatA or Annexin V had little impact on the homing, retention, or migration of MSCs to the peri-infarct area. However, The addition of LatA or Annexin V can significantly decrease the number of DsRed2+/Hoechst33342-cells, and the beneficial effect of MSC grafting for the treatment of ischemic stroke is also significantly suppressed by LatA or Annexin V (p<0.05), suggesting that the beneficial effect may be principally based on TNT-mediated mitochondrial transfer, not on the paracrine, cell fusion or transdifferentiation mechanism.
Conclusions
1. MSCs have superior mitochondrial function compared to HUVECs. MSCs and endothelial cells can exchange mitochondria via the TNT-like structures (the basal level of bi-directional exchange of mitochondria occurs with equal frequencies).
2. OGD/RO stress-induced mitochondrial transfer by TNT formation becomes frequent and almost unidirectional from MSCs to injured endothelial cells, thereby resulting in rescue of aerobic respiration and protection of endothelial cells from apoptosis.
3. TNT formation might represent a defense and rescue mechanism. The formation of TNTs connecting endothelial cells to MSCs is dependent on F-actin polymerization of and the exofacial PS domains.
4. MSC transplantation in vivo indirectly supports the function of TNT-mediated mitochondrial transfer in protecting the brain microvascular system from ischemic-reperfusion injury, enhancing angiogenesis, reducing infarct volume, and improving functional recovery. While the cell fusion, paracrine function, passive phagocytosis of mitochondria, and the transdifferentiation can be excluded as the key mechanism for protective effects of MSCs.
缺血性脑血管疾病已成为严重威胁人类健康的重大疾病,并造成了严重的社会经济负担。尽管缺血性脑血管疾病的治疗研究取得了一定进展,但是现有的治疗手段仍十分有限,治疗效果并不理想。近年来,干细胞移植在缺血性脑血管疾病治疗中的应用研究,为解决这一难题提供了新的思路和方法。作为一种被广泛研究的多能干细胞,骨髓来源的间充质干细胞(mesenchymal stem cells, MSCs)可以自身取材,既不产生免疫排斥反应,也不存在伦理问题,是目前较为理想的种子细胞。
在缺血性脑血管疾病中,血管内皮的损伤是主要的起病始动因素；而血管的修复,尤其是缺血半暗区受损血管的修复,又是挽救缺血受损神经元的前提；同时脑微血管内皮细胞的损伤是也导致血脑屏障开放、脑水肿发生,从而加重神经元细胞损伤的重要因素。因此,保护和修复脑血管的内皮细胞才是防治缺血性脑血管疾病的关键。然而,目前在于细胞治疗脑梗死的研究中,多数研究聚焦于受损神经组织的修复,但在干细胞对缺血部位受损的脑血管内皮细胞影响的研究却亟待进一步深入。近期的研究提示,移植的间充质干细胞可以促进缺血局部区域的血管新生并减轻缺血组织的损伤；然而,其具体机制仍存在争议：部分学者认为干细胞通过转分化为缺血损伤的血管组织细胞促进了血管再生,另有学者认为移植的干细胞可以通过细胞融合发挥其保护作用,更有学者认为间充质干细胞通过其旁分泌的细胞因子促进了血管新生。因此,针对移植的间充质干细胞与缺血再灌注损伤的微血管及内皮细胞之间的相互作用需要进一步的研究。
在缺血再灌注损伤的复杂机制中,线粒体损伤是其中的关键环节。线粒体在有氧代谢、氧化磷酸化和细胞凋亡等多种病理生理过程中发挥着关键作用。有研究显示线粒体损伤的严重程度在一定程度上决定了内皮细胞的功能和命运,并影响到后续的血管新生及缺血组织的修复。然而,当前关于干细胞对损伤的内皮细胞线粒体功能影响的实验研究尚未见报道。
目前,一种新型通讯连接方式的发现正在引发人们对细胞通讯认识的革命：最近在动物细胞间发现了一种细长膜管状细胞连接一隧道纳米管((tunneling nanotubes, TNTs)。作为细胞间相互交流的通道,TNTs可以在相连接的细胞间形成复杂的通讯网络,并能够运输多种细胞组分以促进细胞间的相互交流,其中不仅包括细胞膜的成分、细胞质内的小分子,甚至线粒体等一些较大的细胞器也能通过TNTs进行转运。越来越多的研究提示TNTs是动物细胞间一种普遍存在的相互作用方式。由此我们提出以下假设：干细胞可以建立起与内皮细胞的直接连接方式——TNTs,并通过TNTs将自身的线粒体直接转运至内皮细胞中,修复受损内皮细胞的能量代谢,减少细胞凋亡,以保护内皮细胞、修复缺血受损的脑微血管系统,从而发挥其减轻缺血性卒中损伤并改善卒中预后的作用。
TNTs的发现揭示了一种全新的细胞间交流通道,引发了人们对细胞相互作用方式的重新思考,直到目前其研究仍处于初步阶段,许多问题还有待于进一步解决。我们在前期实验的基础上,结合干细胞和TNTs的最新研究进展,提出了干细胞通过TNTs直接转移线粒体以保护缺血受损内皮细胞的推测,以图揭示出干细胞修复缺血受损脑血管内皮细胞的机制,为干细胞在缺血性卒中治疗的临床应用提供了基础实验依据,同时也为干细胞治疗其他缺血性疾病的机制研究提供了借鉴。
第一部分间充质干细胞通过隧道纳米管转运线粒体保护类缺血n损伤的内皮细胞的研究
研究目的
1、探讨间充质干细胞与共培养的脐静脉内皮细胞间能否建立TNTs结构联系。
2、探究影响TNTs形成的因素并揭示TNTs的形成机制。
3、探讨线粒体能否通过TNTs在两种细胞间进行有效的转运。
4、探讨干细胞能否通过建立TNTs转运线粒体以保护类缺血损伤的内皮细胞。
研究方法
1、骨髓间充质干细胞与脐静脉内皮细胞的分离、培养、鉴定与标记。
密度梯度离心法分离骨髓间充质干细胞,经培养扩增后,流式细胞术鉴定间充质干细胞表型为CD29、CD44和CD105阳性, CD34和CD45阴性。脐静脉内皮细胞获赠于山东大学心血管蛋白质组重点实验室。采用慢病毒介导的增强型绿色荧光蛋白标记间充质干细胞,并通过流式细胞分选术将其纯化。另外,应用pAcGFP1-Mito vector和pDsRed2-Mito vector分别对脐静脉内皮细胞和骨髓间充质干细胞的线粒体进行标记。
2、对比分析正常条件下骨髓间充质干细胞与脐静脉内皮细胞的线粒体功能。
通过生物能量代谢监测仪分析细胞的耗氧率(oxygen consumption rate, OCR)和产酸率(extracellular acidification rate, ECAR),经细胞计数校正后的耗氧率和产酸率数值可直接反映出MSCs和HUVECs的线粒体功能差异。
3、建立干细胞与类缺血损伤后内皮细胞的共培养模型。
在缺氧培养箱中对HUVECs实施氧糖剥夺处理150分钟,再将处理的细胞进行复氧复糖处理,4小时后将等量的间充质干细胞与之直接混合培养,以建立干细胞与类缺血损伤后内皮细胞的共培养模型。
4、激光共聚焦显微镜观察隧道纳米管的形成及线粒体在其中的转运。
应用激光共聚焦显微镜观察共培养的MSCs和HUVECs之间隧道纳米管的形成以及线粒体在其中的转运情况,并对不同干预条件下的两种细胞间形成的TNT及膜突起(membrane protrusions, MPs)计数统计。
5、应用流式细胞术分选并定量分析混合培养后两种细胞间线粒体的相互转移率。
利用Hoechst33342标记的间充质干细胞细胞核,以及干细胞和内皮细胞两种细胞间的形态差异,采用流式细胞分选术将共培养48小时后的两种细胞分选开来。再应用流式细胞术定量分析线粒体在两种细胞间的相互转移率。
6、分析间充质干细胞对共培养脐静脉内皮细胞的有氧代谢、细胞活性及凋亡率的影响；并检测丝状肌动蛋白(filamentous actin, F-actin)解聚剂latrunculin-A (LatA)、磷脂酰丝氨酸位点阻断剂Annexin V和线粒体功能缺失的干细胞对其共培养效应的影响。
采用流式细胞分选术将不同的共培养组中的脐静脉内皮细胞分离出来。采用FITC Annexin V凋亡试剂盒分析内皮细胞凋亡率,采用Cell Counting Kit-8(CCK-8)分析内皮细胞活性,使用生物能量代谢监测仪分析内皮细胞的线粒体功能。
7、分析干细胞的旁分泌功能和细胞融合等因素对内皮细胞活性的影响。
收集浓缩不同处理条件下的间充质干细胞条件培养液,应用ELISA试剂盒检查其中的血管内皮细胞生长因子(vascular endothelial growth factor, VEGF)、血小板源性生长因子(platelet-derived growth factor BB, PDGF-BB)和成纤维细胞生长因子-2(fibroblast growth factor2,FGF-2)的含量,以明确干细胞的旁分泌功能对内皮细胞活性的影响。
采用直接测序法对比检测共培养前后的HUVECs和MSCs的线粒体DNA(mitochondrial DNA, mtDNA),明确共培养后被MSCs营救的HUVECs是否含有来源于MSCs的线粒体。应用短串联重复序列方法对比分析共培养前后的HUVECs和MSCs的核DNA(nuclear DNA, nDNA),明确共培养后被MSCs营救的HUVECs是否含有来源于MSCs的细胞核,也即是分析细胞融合的可能性。
将染色标记好的血小板和干细胞线粒体加入缺血受损的内皮细胞中直接共培养,激光共聚焦显微镜观察共培养48小时后的内皮细胞的生存状况,从而分析内皮细胞是否主动吞噬了细胞外游离的线粒体。
研究结果
1、生物能量代谢监测仪分析证实间充质干细胞具有显著优于脐静脉内皮细胞的线粒体功能(p<0.05)。
2、激光共聚焦显微镜证实共培养的间充质干细胞与脐静脉内皮细胞间可建立隧道纳米管结构联系,线粒体可在其中进行双向转运(两种细胞间的双向转移率大致均衡)。
3、激光共聚焦显微镜证实类缺血再灌注损伤能介导脐静脉内皮细胞与间充质干细胞间建立更为复杂和广泛的网络状TNT联系,并促使TNT中的线粒体双向交换变为单向转运(由干细胞向内皮细胞方向),且(流式细胞术证实)其转移率显著提高。
4、LatA和Annexin V可显著减少脐静脉内皮细胞与间充质干细胞间的TNTs数量,表明TNTs的形成是依赖F-actin的聚合和磷脂酰丝氨酸的外化的过程。
5、间充质干细胞通过隧道纳米管将自身线粒体转运至类缺血再灌注损伤的脐静脉内皮细胞内,以修复其有氧代谢功能并减少细胞凋亡。F-actin解聚剂LatA、磷脂酰丝氨酸位点阻断剂Annexin V均可显著抑制该保护效应(p<0.05)。另外,尽管线粒体功能缺失的MSCs完全丧失了对内皮细胞有氧代谢功能的修复,但仍可以部分减轻内皮细胞的凋亡(p<0.05),这提示TNTs还可能通过转运其他物质的方式发挥其对内皮细胞的保护效应。
6、LatA和Annexin V的处理对间充质干细胞的旁分泌促血管新生作用细胞因子(VEGF, PDGF-BB, FGF-2)的功能没有显著影响,因此间充质干细胞的旁分泌功能不是其保护内皮细胞的关键机制。另一方面,被拯救的脐静脉内皮细胞的nDNA来源母系的内皮细胞,而其：ntDNA则同时含有MSCs和母系内皮细胞两种组分,这一结果不仅支持线粒体在异种细胞间发生了转移,同时也排除了发生细胞融合的可能性。此外,将染色标记好的血小板和干细胞线粒体加入缺血损伤的内皮细胞中直接共培养48小时,内皮细胞的线粒体功能、细胞活性和凋亡率均没有明显改善,且细胞中未发现含有被染色标记的线粒体,表明缺失损伤后的脐静脉内皮细胞不具有主动吞噬线粒体的功能。
第二部分间充质干细胞保护大鼠脑微血管系统线粒体的功能并改善缺血性卒中预后的研究
研究目的
1、研究间充质干细胞移植对大脑中动脉闭塞后再灌注大鼠的脑梗死面积的影响。
2、探讨间充质干细胞移植对脑梗死大鼠运动神经功能恢复的影响。
3、研究间充质干细胞对脑梗死大鼠缺血脑组织血管新生的影响。
4、探讨间充质干细胞移植对缺血受损脑微血管的线粒体功能的影响。
5、进一步探讨隧道纳米管介导的线粒体转移机制在干细胞改善脑梗死大鼠预后中的作用。
研究方法
1、建立大鼠的大脑中动脉闭塞后再灌注模型。
线栓法制备大鼠的大脑中动脉闭塞(middle cerebral artery occlusion,MCAO)后再灌注模型。在MACO120分钟后撤出尼龙线恢复大脑中动脉的灌注,术中采用多普勒仪证实闭塞和再灌注的效果。术后对大鼠进行神经功能缺损评分,并根据评分结果选取大鼠进人下一步实验
2、建立经动脉途径将间充质干细胞植入缺血损伤的大鼠脑微血管系统的模型。
对大鼠的间充质干细胞进行分离、培养和鉴定。为示踪移植的干细胞及其线粒体,用Hoechst33342标记MSCs的细胞核,应用pDsRed2-Mito vector对干细胞的线粒体进行标记。在大脑中动脉闭塞再灌注术后24小时,使用显微注射针将5×105MSCs缓慢注入右侧颈内动脉系统并密切观察术中术后的大鼠病情变化。
3、2,3,5-氯化三苯基四氮唑染色法分析大鼠脑梗死体积。
间充质干细胞移植术7天后,采用2,3,5-氯化三苯基四氮唑(2,3,5-triphenyltetrazolium chloride,TTC)对不同处理组大鼠梗死后的脑组织进行染色,应用ImageJ软件进一步分析梗死体积。
4、测试脑梗死大鼠的运动神经功能。
应用转棒疲劳实验和跑轮实验对MCAO术前及MSCs移植术后的第1、7、14和28天的大鼠进行测试,以评估间充质干细胞移植及其它不同处理因素对脑梗死大鼠的运动神经功能恢复的影响。
5、探讨脑微血管的密度与接受移植干细胞线粒体的宿主细胞数量间的关系。
受试大鼠接受动脉注射的绿色荧光染料(fluorescein isothiocyanate-dextran amine, FD-2000S)以达到激光共聚焦显微镜下脑微血管充分显影的效果。将脑组织固定并制作冰冻切片。共聚焦显微镜下观察梗死核心区和缺血半暗区的脑微血管的密度与接受干细胞线粒体的宿主细胞(DsRed2+/Hoechst33342-cells)数量间的关系。
6、分离纯化脑微血管片段,并检测其线粒体功能。
取梗死侧的大脑半球,采用两次酶消化法和密度梯度离心法分离和纯化脑微血管片段。将纯化的脑微血管片段置于Ⅳ型胶原和纤连蛋白覆盖的XF24组织培养皿内,使用生物能量代谢监测仪分析微血管片段的线粒体功能。
研究结果
1、间充质干细胞移植显著减少大脑中动脉闭塞大鼠的脑梗死面积(p<0.05)。
2、间充质干细胞移植可促进脑梗死大鼠运动功能的恢复(p<0.05)。
3、间充质干细胞移植显著增加缺血半暗区的脑微血管密度(p<0.05)。
4、间充质干细胞可有效改善缺血损伤脑微血管片段的线粒体功能(p<0.05)。
5、LatA和Annexin V并不影响缺血半暗区内DsRed2+/Hoechst33342+细胞的数量(DsRed2+/Hoechst33342+细胞即为示踪出的植入术后全部的MSCs,包括了可能发生细胞融合和转分化的MSCs),证实LatA和Annexin V对干细胞的归巢、滞留和迁移没有造成显著影响。然而,TNTs的抑制剂LatA和Annexin V可显著减少缺血半暗区内DsRed2+/Hoechst33342-细胞的数量(DsRed2+/Hoechst33342-细胞为接受干细胞转移线粒体的宿主细胞),同时也显著抑制了干细胞的对脑梗死大鼠的保护效应(p<0.05),提示其保护效可能与隧道纳米管介导的线粒体转移密切相关；同时也进一步证实了旁分泌功能、细胞融合和转分化等因素不是干细胞保护效应的关键机制。
综上所述
1、骨髓间充质干细胞具有显著优于脐静脉内皮细胞的线粒体功能。间充质干细胞与内皮细胞间可建立隧道纳米管结构联系,两种细胞的线粒体可在其中进行双向转运。正常条件下两种细胞之间线粒体的转运频率大致均等。
2、类缺血再灌注损伤能介导内皮细胞与间充质干细胞间建立更为广泛和复杂的TNTs联系网络,并促使TNTs介导的线粒体双向均等交换转变为从干细胞向内皮细胞方向的单向转运,其转移率也显著提高。从而使间充质干细胞能有效修复受损内皮细胞的线粒体功能并减少细胞凋亡。
3、TNTs的形成可能反映了细胞在应激或损伤状态下表现出的一种自我防御和营救机制。TNTs的形成依赖于丝状肌动蛋白的聚合以及磷脂酰丝氨酸位点的外化与识别。
4、体内实验进一步证实：间充质干细胞通过隧道纳米管转运线粒体可能是其保护缺血受损脑微血管的线粒体功能、促进血管新生并改善卒中大鼠的预后的关键机制。而间充质干细胞的旁分泌细胞因子、细胞融合或转分化机制则并非其保护作用的主要因素。
Background
Ischemic stroke leads to high rates of disability and death, thereby imposing an enormous medical and social burden. Currently available therapies for injured cerebrovascular systems are insufficient. Meanwhile, stem cell transplantation can be used to repair ischemic vascular damage and thus offers new prospects for stroke therapy. As neither immune rejection, nor the ethical questions, the bone marrow mesenchymal stem cells (MSCs), which can be drawn from oneself, are the ideal seed cells.
Cerebrovascular injury induced by ischemia-reperfusion has a key function in determining the survival of nerve cells after recanalization in focal ischemia by clogging microcirculation and disrupting the blood-brain barrier integrity. Recent reports have shown that MSCs participate in neovascularization and attenuate ischemic injury after focal cerebral ischemia. However, whether MSCs transdifferentiate into and replace lost cerebrovascular cells or perform paracrine function by secreting proangiogenic factors are unclear. Therefore, the mechanism of action of MSCs especially in the interaction between brain microvessel and grafted MSCs need to be further investigated.
Among the many complex mechanisms underlying vascular endothelium ischemia-reperfusion injury, mitochondrial damage appears to contribute significantly to these pathological processes. Mitochondria are essential organelles that play prominent roles in biological processes such as aerobic metabolism, oxidative phosphorylation, and cell death pathways. Endothelial mitochondria have been recognized as playing critical roles in the signaling cellular responses to environmental cues, which may determine the endothelial function and fate, thereby influencing angiogenesis in ischemia-reperfusion injury. Limited experimental data are available on the effects of stem cells on injured mitochondria in endothelial cells.
Recent studies have discovered highly sensitive nanotubular structures named tunneling nanotubes (TNTs) that bridge adjacent animal cells, enabling them to form complex networks. As a novel mechanism of cell-cell communication, TNTs facilitate the exchange of cellular components and signaling molecules between connected cells such as plasma membrane components, calcium ions, pathogens, and organelles, including mitochondria. We hypothesized that stem cells and post-ischemic endothelial cells interact with each other through the formation of TNTs and that this novel mechanism might be responsible for the beneficial effects exerted by engrafted stem cells.
The discovery of TNT reveals a new style for intercellular communication, which has sparked the rethink the way of cell interaction. Until now the research on TNT is still in the preliminary stage, many problems has yet to be further investigated. Our findings challenge the classical view on stem cell transplantation for stroke therapy and may also have implications in the treatment of other ischemic diseases.
Part I:Mesenchymal stem cells rescue injured endothelial cells in an in vitro ischemia-reperfusion model via tunneling nanotube-mediated mitochondrial transfer
Objectives
1. To observe the TNT connections between the HUVECs and the MSCs.
2. To explore the formation mechanism of TNTs between the HUVECs and the MSCs.
3. To investigate the transfer of the mitochondria between the HUVECs and the MSCs through the TNT.
4. To explore the protective effect of the MSCs on the injured HUVECs via TNT-mediated mitochondrial transfer.
Methods
1. Cell culture, identification, label, and lentiviral transduction.
The human MSCs were isolated from the bone marrow of the subjects using a density gradient. The HUVECs were obtained from the Key Laboratory of Cardiovascular Proteomics of Shandong Province. The MSCs were analyzed by fluorescence-activated cell sorting (FACS) to evaluate the cell surface markers. The MSCs expressed the antigens CD105, CD29, and CD44; however, they were negative for CD45and CD34. The HUVECs and MSCs were labeled for distinction before the co-cultivation. The MSCs were incubated with lentiviral vector pWPT-enhanced green fluorescence protein (EGFP). To investigate the mitochondrial transfer, the MSC nuclei were stained with Hoechst33342before co-cultivation. The mitochondria of the MSCs and the HUVECs were labeled. The pDsRed2-Mito vector and pAcGFPl-Mito vector was used to label the mitochondria of the HUVECs and MSCs, respectively.
2. Functional Measurements of Mitochondrial Activity in the MSCs and the HUVECs.
The measurements of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), which are indicators of aerobic respiration and glycolysis, were conducted with an XF24Extracellular Flux Analyzer. The mitochondrial function of the MSCs and the HUVECs were analyzed, and the results were normalized to the cell number.
3.In Vitro Ischemia-Reperfusion Model and Co-culture Model
In vitro ischemia-reperfusion was simulated by conducting oxygen glucose deprivation (OGD) and reoxygenation (RO) on the HUVECs in an anoxia chamber. After150min of the OGD, the RO was performed by reinstating the cells under the normoxic conditions and the pre-OGD medium. At4h of RO, an equal number of MSCs were directly added to the damaged HUVECs.
4. The laser scanning confocal microscopy was conducted to investigate the TNT-like structures connections and the transport of mitochondria.
The laser scanning confocal microscopy was conducted during and after the in vitro ischemia to investigate the morphological changes and possible interactions among the co-cultured HUVECs and MSCs. The TNT-like structures connections between the HUVECs and the MSCs and the membrane protrusions (MPs) were counted after24h of co-culture.
5. The exchange rate of mitochondria between the HUVECs and the MSCs was analyzed using FACS analysis.
To investigate the mitochondrial transfer further, the exchange rate of mitochondria between the HUVECs and the MSCs was analyzed after co-culture for48h using FACS analysis. We could also distinguish the two different cells by their Hoechst33342label and different morphologies using FACS.
6. To investigate the protective effects of MSCs, the cell viability, apoptosis, and mitochondrial activity the of the HUVECs was analyzed respectively.
To provide direct evidence for the functional role of mitochondria transfer via TNT-like structures, we generated MSCs with mitochondrial dysfunction using mtDNA depletion by cell treatment with ethidium bromide, which do not affect the formation of TNTs and the transfer of other molecules and organelles. After co-culture for48h, the cell apoptosis of the HUVECs we analyzed using the FITC Annexin V Apoptosis Detection Kit I (BD PharmingenTM, San Diego, CA, USA). The cell viability of the HUVECs was estimated using the Cell Counting Kit-8(CCK-8; Dojindo, Japan). The mitochondrial function of the HUVECs was analyzed using the XF24Extracellular Flux Analyzer.
7. The possiblity of cell fusion, the paracrine function and the passive phagocytosis of mitochondria was respectively analyzed.
The MSC conditioned media were collected after48h of culture, and the concentration of the MSC conditioned media cytokines was measured using ELISA kits [vascular endothelial growth factor,(VEGF); platelet-derived growth factor BB,(PDGF-BB); fibroblast growth factor2,(FGF-2)].
To investigate the possiblity of cell fusion, the nuclear DNA and mitochondrial DNA (mtDNA) extracted from the HUVECs, the MSCs, and rescued HUVECs were assayed by direct sequencing of the PCR products and the GeneScan analysis based on the short tandem repeat sequences.
To investigate the possibility that the HUVECs phagocytose MSC debris containing mitochondria and thus acquire double-labeled fluorescent dyes, the mitochondria isolated from MSCs and human platelets were used as sources of membrane-bound functional mitochondria. These were then co-cultured with the injured HUVECs for48h.
Results
1. MSCs have superior mitochondrial function compared to HUVECs.
2. MSCs and endothelial cells can exchange mitochondria via the TNT-like structures (the basal level of bi-directional exchange of mitochondria occurs with equal frequencies).
3. OGD/RO stress-induced mitochondrial transfer through the TNT-like structures becomes frequent and almost unidirectional from the MSCs to the injured endothelial cells.
4. The TNT formations between the MSCs and HUVECs are substantially reduced after the LatA or Annexin V treatment. The formation of the TNT-like structures connecting the endothelial cells to the MSCs is dependent on the F-actin polymerization of and the exofacial PS domains.
5. Mesenchymal stem cells can rescue aerobic respiration and protect the endothelial cells from apoptosis via tunneling nanotube like structure-mediated mitochondrial transfer. The addition of LatA or Annexin V partially suppresses this effect. The TNT-mediated protection on aerobic respiration is completely abrogated by co-cultures with the MSCs having mitochondrial dysfunction,(no statistical differences comparing with the OGD/RO group), while that the protective effect on HUVECs viability is partially suppressed (significant differences comparing with the OGD/RO group, p<0.05), suggesting that the transfer of functional mitochondria from MSCs to HUVECs was required to rescue the endothelial cells from damage, but not unique.
6. The cell fusion, paracrine function, and passive phagocytosis of mitochondria can be excluded as the key mechanism for protective effects of MSCs. The potential side effects of LatA or Annexin V on the cultures of the MSCs or HUVECs were investigated. The addition of LatA or Annexin V caused negligible changes in the cell viability of MSCs or HUVECs based on the CCK-8assay and also affected the MSCs paracrine function of several primary proangiogenic factors (VEGF, PDGF-BB, FGF-2) insignificantly by ELISA analysis, suggesting that the TNT-like structures might be essential, whereas the soluble cytokines were less likely to act as the principal rescue factors.
The nuclear DNA of the rescued HUVECs from five independent samples was conformably from the HUVECs and did not contain the nuclear genome from the MSCs. Cell fusion can be excluded as the mechanism for the mitochondrial transfer.
The HUVECs became senescent, and double-labeled cellswere not observed. No significant changes in cell viabilityand mitochondrial activity were observed in the co-cultures compared with the injured HUVEC culture alone, which may exclude the possibility that the HUVECs recovered from injury through passive phagocytosis of the mitochondria.
Part Ⅱ:Mesenchymal stem cells protect mitochondrial function of cerebral microvasculature and promote recovery from ischemic stroke
Objectives
1. To investigate the effect of MSCs on the infarct cortex.
2. To explore the effect of MSCs on the neurobehavioral functions after stroke.
3. To explore the therapeutic effect of MSCs on the angiogenesis in ischemia-reperfusion mjury.
4. To investigate the effect of MSCs on mitochondrial activity of micro vascular fragments in ischemic cerebral hemisphere.
5. To explore the role of TNT-mediated mitochondrial transfer in MSC grafting for the treatment of ischemic stroke in vivo.
Methods
1. The middle cerebral artery occlusion (MCAO) and reperfusion model.
MCAO and reperfusion surgery were carried out as previously described. A nylon filament with a rounded tip was inserted into the right internal carotid artery at approximately18.5mm to20.0mm to occlude the origin of the right middle cerebral artery. After120min of MCAO, the filament was withdrawn to restore the blood flow (both occlusion and reperfusion were confirmed by laser Doppler). After the surgery, the rats were tested for neurological deficits according to Longa and Bederson's five score regulation. The rats with scores of2or3were kept for the subsequent experiments.
2. The culture, identification, label and transplantation of MSCs.
The rat MSCs were isolated and cultured as previously described. To investigate the mitochondria transfer, MSC nuclei were stained with Hoechst33342, and the mitochondria of the MSCs were also labeled using pDsRed2-Mito vector. The transduced cells were purified by FACS, and then expanded. At24h after brain ischemia and reperfusion, randomly assigned animals received IA injection of MSCs, as previously described. The right carotid artery was again exposed, whereas the external carotid artery and the pterygopalatine and superior thyroid arteries were ligated or coagulated. Up to5X105MSCs in10μL of PBS were injected into the common carotid artery using a10u L syringe with a33G microneedle. No difference in mortality and morbidity was found in the different experimental groups.
3. Analysis of Infarct Volume.
2,3,5-triphenyltetrazolium chloride (TTC) staining for determining the therapeutic effect of MSCs on the infarct cortex. Rats (n=5per group) were anesthetized with chloral hydrate (0.4g/kg body weight, intraperitoneally) and decapitated at7days after MSC transplantation. The brains were removed and sliced into2mm-thick coronal sections, and then immersed in2%2,3,5-triphenyltetrazolium chloride (TTC; Sigma, USA) in PBS for20min. The size of the infarct area was analyzed through the digital images of the brain slices using ImageJ software. The infarct volume percentage was calculated as a percentage of the contralateral hemisphere.
4. Behavioral tests.
Two behavioral tests were performed to evaluate the motor function before MCAO and at1,7,14, and28days after MSC transplantation (n=20per group). A rotarod test evaluated the coordinated movements of the limbs and the body through balance on a rotarod, which was slowly accelerated from4rpm to40rpm within4min. The length of time that the animals stayed on the rod was recorded. Data were calculated as the percentage of mean duration (10trials) on the rotor-rod compared with the baseline values. Another behavioral test was performed using the running wheel systems. Rats were transferred to individual cages containing an exercise wheel coupled to a bicycle computer. Total running distance, average speed, and maximum speed were recorded at different time points.
5. Analysis of Microvessel Density.
Microvessel density was measured as previously described. The rats were anesthetized and received IA injections fluorescein isothiocyanate-dextran amine (FD-2000S) to label the blood vessels at15days after MSC transplantation. The rat brains were rapidly fixed in4%paraformaldehyde and cryo-cut at30μm. Under a confocal microscope, we observed the microvessel densities in both the ischemic core and the peri-ischemic region. Microvessel density, the proportion of the area occupied by the labeled microvessels to the whole picture in pixels, was measured using ImageJ. The location of the DsRed2+/Hoechst33342+cells and DsRed2+/Hoechst33342-cells was also observed under the confocal microscope.
6. Brain Microvascular Fragment Isolation and the mitochondrial activity measurements.
The brain microvascular fragments of the right ischemic cerebral hemisphere of the rats were isolated using the methods described in our previous study. Fresh rat right cerebral cortices were cut into uniform1mm3sections. The sections were subsequently digested in a type II collagenase solution and collagenase/dispas solution. The microvascular fragments were purified twice in20%bovine serum albumin and33%continuous Percoll density gradient centrifugation. Purified microvascular fragments were plated on collagen type IV and fibronectin-coated XF24tissue culture plate. The mitochondrial function of the HUVECs was analyzed using the XF24Extracellular Flux Analyzer. OCR and ECAR were normalized to protein concentration for microvascular fragments. Protein measurements of the microvascular fragments were performed using a BCA Protein Assay Kit.
Results
1. MSCs can decrease the infarct area of stroke rats significantly (p<0.05).
2. MSCs can improve the motor function of stroke rats significantly (p<0.05).
3. MSCs can promote angiogenesis in ischemic penumbra of stroke rats significantly (p<0.05).
4. MSCs can protect the mitochondrial function of damaged cerebral microvasculature significantly (p<0.05).
5. The the addition of LatA or Annexin V does not affect the number of DsRed2+/Hoechst33342+cells (all of the transplanted MSCs, including the transdifferentiated MSCs and the MSCs occurring cell fusion with the host cells) in the peri-infarct area, suggesting that LatA or Annexin V had little impact on the homing, retention, or migration of MSCs to the peri-infarct area. However, The addition of LatA or Annexin V can significantly decrease the number of DsRed2+/Hoechst33342-cells, and the beneficial effect of MSC grafting for the treatment of ischemic stroke is also significantly suppressed by LatA or Annexin V (p<0.05), suggesting that the beneficial effect may be principally based on TNT-mediated mitochondrial transfer, not on the paracrine, cell fusion or transdifferentiation mechanism.
Conclusions
1. MSCs have superior mitochondrial function compared to HUVECs. MSCs and endothelial cells can exchange mitochondria via the TNT-like structures (the basal level of bi-directional exchange of mitochondria occurs with equal frequencies).
2. OGD/RO stress-induced mitochondrial transfer by TNT formation becomes frequent and almost unidirectional from MSCs to injured endothelial cells, thereby resulting in rescue of aerobic respiration and protection of endothelial cells from apoptosis.
3. TNT formation might represent a defense and rescue mechanism. The formation of TNTs connecting endothelial cells to MSCs is dependent on F-actin polymerization of and the exofacial PS domains.
4. MSC transplantation in vivo indirectly supports the function of TNT-mediated mitochondrial transfer in protecting the brain microvascular system from ischemic-reperfusion injury, enhancing angiogenesis, reducing infarct volume, and improving functional recovery. While the cell fusion, paracrine function, passive phagocytosis of mitochondria, and the transdifferentiation can be excluded as the key mechanism for protective effects of MSCs.
引文
1. Liu, K., et al., The interactions between brain microvascular endothelial cells and mesenchymal stem cells under hypoxic conditions. Microvascular research,2008.75(1):p.59-67.
2. Lin, Y.C., et al., Human umbilical mesenchymal stem cells promote recovery after ischemic stroke. Stroke,2011.42(7):p.2045-53.
3. Nakano-Doi, A., et al., Bone marrow mononuclear cells promote proliferation of endogenous neural stem cells through vascular niches after cerebral infarction. Stem Cells,2010.28(7):p.1292-302.
4. Soleimani, M. and S. Nadri, A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow. Nat Protoc,2009.4(1):p.102-6.
5. Dar, A., et al., Multipotent vasculogenic pericytes from human pluripotent stem cells promote recovery of murine ischemic limb. Circulation,2012.125(1):p.87-99.
6. Kinnaird, T., et al., Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms. Circ Res,2004.94(5):p.678-85.
7. Chan, P.H., Mitochondrial dysfunction and oxidative stress as determinants of cell death/survival in stroke. Annals of the New York Academy of Sciences,2005.1042:p.203-9.
8. Li, J., et al., Reperfusion promotes mitochondrial dysfunction following focal cerebral ischemia in rats. PLoS One,2012.7(9):p. e46498.
9. Zhang, W.H., et al., Nortriptyline protects mitochondria and reduces cerebral ischemia/hypoxia injury. Stroke,2008.39(2):p.455-62.
10. Dyall, S.D., M.T. Brown, and P.J. Johnson, Ancient invasions:from endosymbionts to organelles. Science,2004.304(5668):p.253-7.
11. Galluzzi, L., et al., Mitochondrial control of cellular life, stress, and death. Circ Res,2012.111(9):p. 1198-207.
12. Spees, J.L., et al., Mitochondrial transfer between cells can rescue aerobic respiration. Proc Natl Acad Sci U S A,2006.103(5):p.1283-8.
13. Spees, J.L., et al., Differentiation, cell fusion, and nuclear fusion during ex vivo repair of epithelium by human adult stem cells from bone marrow stroma. Proc Natl Acad Sci U S A,2003.100(5):p. 2397-402.
14. Wen, YD., et al., Hydrogen sulfide protects HUVECs against hydrogen peroxide induced mitochondrial dysfunction and oxidative stress. PLoS One,2013.8(2):p. e53147.
15. Rustom, A., et al., Nanotubular highways for intercellular organelle transport. Science,2004. 303(5660):p.1007-10.
16. He, K., et al., Long-distance intercellular connectivity between cardiomyocytes and cardiofibroblasts mediated by membrane nanotubes. Cardiovasc Res,2011.92(1):p.39-47.
17. Onfelt, B., et al., Structurally distinct membrane nanotubes between human macrophages support long-distance vesicular traffic or surfing of bacteria. J Immunol,2006.177(12):p.8476-83.
18. Pasquier, J., et al., Preferential transfer of mitochondria from endothelial to cancer cells through tunneling nanotubes modulates chemoresistance. J Transl Med,2013.11:p.94.
19. Vallabhaneni, K.C., H. Haller, and I. Dumler, Vascular smooth muscle cells initiate proliferation of mesenchymal stem cells by mitochondrial transfer via tunneling nanotubes. Stem Cells Dev,2012. 21(17):p.3104-13.
20. Wang, X. and H.H. Gerdes, Long-distance electrical coupling via tunneling nanotubes. Biochim Biophys Acta,2012.1818(8):p.2082-6.
21. Wang, Y, et al., Tunneling-nanotube development in astrocytes depends on p53 activation. Cell Death Differ,2011.18(4):p.732-42.
22. Yasuda, K.., et al., Adriamycin nephropathy:a failure of endothelial progenitor cell-induced repair. The American journal of pathology,2010.176(4):p.1685-95.
23. Koyanagi, M., et al., Cell-to-cell connection of endothelial progenitor cells with cardiac myocytes by nanotubes:a novel mechanism for cell fate changes? Circ Res,2005.96(10):p.1039-41.
24. Cui, X., et al., Grape seed proanthocyanidin extracts enhance endothelial nitric oxide synthase expression through 5'-AMP activated protein kinase/Surtuin 1-Krupple like factor 2 pathway and modulate blood pressure in ouabain induced hypertensive rats. Biol Pharm Bull,2012.35(12):p. 2192-7.
25. Zou, Y., et al., Characterization of nuclear localization signal in the N terminus of CUL4B and its essential role in cyclin E degradation and cell cycle progression. J Biol Chem,2009.284(48):p. 33320-32.
26. Rizzuto, R., et al., Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells. Curr Biol,1995.5(6):p.635-42.
27. Lidell, M.E., et al., The adipocyte-expressed forkhead transcription factor Foxc2 regulates metabolism through altered mitochondrial function. Diabetes,2011.60(2):p.427-35.
28. Cselenyak, A., et al., Mesenchymal stem cells rescue cardiomyoblasts from cell death in an in vitro ischemia model via direct cell-to-cell connections. BMC Cell Biol,2010.11:p.29.
29. Konishi, H., et al., Latrunculin a has a strong anticancer effect in a peritoneal dissemination model of human gastric cancer in mice. Anticancer Res,2009.29(6):p.2091-7.
30. Lou, E., et al., Tunneling nanotubes provide a unique conduit for intercellular transfer of cellular contents in human malignant pleural mesothelioma. PLoS One,2012.7(3):p. e33093.
31. Yasuda, K., et al., Tunneling nanotubes mediate rescue of prematurely senescent endothelial cells by endothelial progenitors:exchange of lysosomal pool. Aging (Albany NY),2011.3(6):p.597-608.
32. Lis, R., et al., Mesenchymal cell interaction with ovarian cancer cells induces a background dependent pro-metastatic transcriptomic profile. J Transl Med,2014.12:p.59.
33. Touboul, C., et al., Mesenchymal stem cells enhance ovarian cancer cell infiltration through IL6 secretion in an amniochorionic membrane based 3D model. J Transl Med,2013.11:p.28.
34. Gnecchi, M. and L.G. Melo, Bone marrow-derived mesenchymal stem cells:isolation, expansion, characterization, viral transduction, and production of conditioned medium. Methods Mol Biol, 2009.482:p.281-94.
35. Boomsma, R.A. and D.L. Geenen, Mesenchymal stem cells secrete multiple cytokines that promote angiogenesis and have contrasting effects on chemotaxis and apoptosis. PLoS One,2012.7(4):p. e35685.
36. Ling, B., et al., Effect of conditioned medium of mesenchymal stem cells on the in vitro maturation and subsequent development of mouse oocyte. Braz J Med Biol Res,2008.41(11):p.978-85.
37. Rogers, G.W., et al., High throughput microplate respiratory measurements using minimal quantities of isolated mitochondria. PLoS One,2011.6(7):p. e21746.
38. Sharma, P., et al., Positive association of tyrosine hydroxylase microsatellite marker to essential hypertension. Hypertension,1998.32(4):p.676-82.
39. Andres, A.M., et al., Comparative genetics of functional trinucleotide tandem repeats in humans and apes. J Mol Evol,2004.59(3):p.329-39.
40. Zhou, X., et al., Trimetazidine protects against smoking-induced left ventricular remodeling via attenuating oxidative stress, apoptosis, and inflammation. PLoS One,2012.7(7):p. e40424.
41. Invernizzi, F., et al., Microscale oxygraphy reveals OXPHOS impairment in MRC mutant cells. Mitochondrion,2012.12(2):p.328-35.
42. Hase, K., et al., M-Sec promotes membrane nanotube formation by interacting with Ral and the exocyst complex. Nat Cell Biol,2009.11(12):p.1427-32.
43. Chauveau, A., et al., Membrane nanotubes facilitate long-distance interactions between natural killer cells and target cells. Proc Natl Acad Sci U S A,2010.107(12):p.5545-50.
44. Depraetere, V, "Eat me" signals of apoptotic bodies. Nat Cell Biol,2000.2(6):p. E104.
45. Qu, X., et al., Autophagy gene-dependent clearance of apoptotic cells during embryonic development. Cell,2007.128(5):p.931-46.
46. Frisoni, L., et al., Nuclear autoantigen translocation and autoantibody opsonization lead to increased dendritic cell phagocytosis and presentation of nuclear antigens:a novel pathogenic pathway for autoimmunity? J Immunol,2005.175(4):p.2692-701.
47. Ravichandran, K.S., Find-me and eat-me signals in apoptotic cell clearance:progress and conundrums. J Exp Med,2010.207(9):p.1807-17.
48. Murphy, M.P., How mitochondria produce reactive oxygen species. Biochem J,2009.417(1):p. 1-13.
49. Logan, A., et al., Using exomarkers to assess mitochondrial reactive species in vivo. Biochim Biophys Acta,2014.1840(2):p.923-30.
50. Echtay, K.S., et al., Superoxide activates mitochondrial uncoupling protein 2 from the matrix side. Studies using targeted antioxidants. J Biol Chem,2002.277(49):p.47129-35.
51. Lee, J., S. Giordano, and J. Zhang, Autophagy, mitochondria and oxidative stress:cross-talk and redox signalling. Biochem J,2012.441(2):p.523-40.
52. Hughes, A.L. and D.E. Gottschling, An early age increase in vacuolar pH limits mitochondrial function and lifespan in yeast. Nature,2012.492(7428):p.261-5.
53. Markaki, I., et al, Long-Term Survival of Ischemic Cerebrovascular Disease in the Acute Inflammatory Stroke Study, a Hospital-Based Cohort Described by TOAST and ASCO. Cerebrovasc Dis,2013.35(3):p.213-219.
54. Gursoy-Ozdemir, Y., M. Yemisci, and T. Dalkara, Microvascular protection is essential for successful neuroprotection in stroke. Journal of neurochemistry,2012.123 Suppl 2:p.2-11.
55. Kim, D., et al., In vivo tracking of human mesenchymal stem cells in experimental stroke. Cell Transplant,2008.16(10):p.1007-12.
56. Chen, J., et al., Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats. Stroke,2001.32(4):p.1005-11.
57. Wei, N., et al., Delayed intranasal delivery of hypoxic-preconditioned bone marrow mesenchymal stem cells enhanced cell homing and therapeutic benefits after ischemic stroke in mice. Cell Transplant,2013.22(6):p.977-91.
58. Zhao, Y.H., Y. Guan, and W.K. Wu, Potential advantages of a combination of chinese medicine and bone marrow mesenchymal stem cell transplantation for removing blood stasis and stimulating neogenesis during ischemic stroke treatment. J Tradit Chin Med,2012.32(2):p.289-92.
59. Lee, J.S., et al., A long-term follow-up study of intravenous autologous mesenchymal stem cell transplantation in patients with ischemic stroke. Stem Cells,2010.28(6):p.1099-106.
60. Sokolova, I.B., et al., Effect of mesenchymal stem cell transplantation on cognitive functions in rats with ischemic stroke. Bull Exp Biol Med,2006.142(4):p.511-4.
61. De Keyser, J., Autologous mesenchymal stem cell transplantation in stroke patients. Ann Neurol, 2005.58(4):p.653-4; author reply 654-5.
62. Bang, O.Y., et al., Autologous mesenchymal stem cell transplantation in stroke patients. Ann Neurol, 2005.57(6):p.874-82.
63. Tsai, L.K., et al., Mesenchymal stem cells primed with valproate and lithium robustly migrate to infarcted regions and facilitate recovery in a stroke model. Stroke,2011.42(10):p.2932-9.
64. Croft, A.P. and S.A. Przyborski, Formation of neurons by non-neural adult stem cells:potential mechanism implicates an artifact of growth in culture. Stem Cells,2006.24(8):p.1841-51.
65. Terada, N., et al., Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature,2002.416(6880):p.542-5.
66. Davidson, S.M. and M.R. Duchen, Endothelial mitochondria:contributing to vascular function and disease. Cire Res,2007.100(8):p.1128-41.
67. Liu, K., et al., Mesenchymal stem cells rescue injured endothelial cells in an in vitro ischemia-reperfusion model via tunneling nanotube like structure-mediated mitochondrial transfer. Microvascular research,2014.92:p.10-8.
68. Longa, E.Z., et al., Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke, 1989.20(1):p.84-91.
69. Li, Y., et al., Exendin-4 ameliorates motor neuron degeneration in cellular and animal models of amyotrophic lateral sclerosis. PLoS One,2012.7(2):p. e32008.
70. Pendharkar, A.V., et al., Biodistribution of neural stem cells after intravascular therapy for hypoxic-ischemia. Stroke,2010.41(9):p.2064-70.
71. Wang, L.Q., et al., Timing and dose regimens of marrow mesenchymal stem cell transplantation affect the outcomes and neuroinflammatory response after ischemic stroke. CNS Neurosci Ther, 2014.20(4):p.317-26.
72. 孙明,赵育梅,徐超,局灶性脑缺血再灌注损伤核心区及半暗带的组织学定位.中华神经外科杂志,2003.19(4)：259-62.
2. Lin, Y.C., et al., Human umbilical mesenchymal stem cells promote recovery after ischemic stroke. Stroke,2011.42(7):p.2045-53.
3. Nakano-Doi, A., et al., Bone marrow mononuclear cells promote proliferation of endogenous neural stem cells through vascular niches after cerebral infarction. Stem Cells,2010.28(7):p.1292-302.
4. Soleimani, M. and S. Nadri, A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow. Nat Protoc,2009.4(1):p.102-6.
5. Dar, A., et al., Multipotent vasculogenic pericytes from human pluripotent stem cells promote recovery of murine ischemic limb. Circulation,2012.125(1):p.87-99.
6. Kinnaird, T., et al., Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms. Circ Res,2004.94(5):p.678-85.
7. Chan, P.H., Mitochondrial dysfunction and oxidative stress as determinants of cell death/survival in stroke. Annals of the New York Academy of Sciences,2005.1042:p.203-9.
8. Li, J., et al., Reperfusion promotes mitochondrial dysfunction following focal cerebral ischemia in rats. PLoS One,2012.7(9):p. e46498.
9. Zhang, W.H., et al., Nortriptyline protects mitochondria and reduces cerebral ischemia/hypoxia injury. Stroke,2008.39(2):p.455-62.
10. Dyall, S.D., M.T. Brown, and P.J. Johnson, Ancient invasions:from endosymbionts to organelles. Science,2004.304(5668):p.253-7.
11. Galluzzi, L., et al., Mitochondrial control of cellular life, stress, and death. Circ Res,2012.111(9):p. 1198-207.
12. Spees, J.L., et al., Mitochondrial transfer between cells can rescue aerobic respiration. Proc Natl Acad Sci U S A,2006.103(5):p.1283-8.
13. Spees, J.L., et al., Differentiation, cell fusion, and nuclear fusion during ex vivo repair of epithelium by human adult stem cells from bone marrow stroma. Proc Natl Acad Sci U S A,2003.100(5):p. 2397-402.
14. Wen, YD., et al., Hydrogen sulfide protects HUVECs against hydrogen peroxide induced mitochondrial dysfunction and oxidative stress. PLoS One,2013.8(2):p. e53147.
15. Rustom, A., et al., Nanotubular highways for intercellular organelle transport. Science,2004. 303(5660):p.1007-10.
16. He, K., et al., Long-distance intercellular connectivity between cardiomyocytes and cardiofibroblasts mediated by membrane nanotubes. Cardiovasc Res,2011.92(1):p.39-47.
17. Onfelt, B., et al., Structurally distinct membrane nanotubes between human macrophages support long-distance vesicular traffic or surfing of bacteria. J Immunol,2006.177(12):p.8476-83.
18. Pasquier, J., et al., Preferential transfer of mitochondria from endothelial to cancer cells through tunneling nanotubes modulates chemoresistance. J Transl Med,2013.11:p.94.
19. Vallabhaneni, K.C., H. Haller, and I. Dumler, Vascular smooth muscle cells initiate proliferation of mesenchymal stem cells by mitochondrial transfer via tunneling nanotubes. Stem Cells Dev,2012. 21(17):p.3104-13.
20. Wang, X. and H.H. Gerdes, Long-distance electrical coupling via tunneling nanotubes. Biochim Biophys Acta,2012.1818(8):p.2082-6.
21. Wang, Y, et al., Tunneling-nanotube development in astrocytes depends on p53 activation. Cell Death Differ,2011.18(4):p.732-42.
22. Yasuda, K.., et al., Adriamycin nephropathy:a failure of endothelial progenitor cell-induced repair. The American journal of pathology,2010.176(4):p.1685-95.
23. Koyanagi, M., et al., Cell-to-cell connection of endothelial progenitor cells with cardiac myocytes by nanotubes:a novel mechanism for cell fate changes? Circ Res,2005.96(10):p.1039-41.
24. Cui, X., et al., Grape seed proanthocyanidin extracts enhance endothelial nitric oxide synthase expression through 5'-AMP activated protein kinase/Surtuin 1-Krupple like factor 2 pathway and modulate blood pressure in ouabain induced hypertensive rats. Biol Pharm Bull,2012.35(12):p. 2192-7.
25. Zou, Y., et al., Characterization of nuclear localization signal in the N terminus of CUL4B and its essential role in cyclin E degradation and cell cycle progression. J Biol Chem,2009.284(48):p. 33320-32.
26. Rizzuto, R., et al., Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells. Curr Biol,1995.5(6):p.635-42.
27. Lidell, M.E., et al., The adipocyte-expressed forkhead transcription factor Foxc2 regulates metabolism through altered mitochondrial function. Diabetes,2011.60(2):p.427-35.
28. Cselenyak, A., et al., Mesenchymal stem cells rescue cardiomyoblasts from cell death in an in vitro ischemia model via direct cell-to-cell connections. BMC Cell Biol,2010.11:p.29.
29. Konishi, H., et al., Latrunculin a has a strong anticancer effect in a peritoneal dissemination model of human gastric cancer in mice. Anticancer Res,2009.29(6):p.2091-7.
30. Lou, E., et al., Tunneling nanotubes provide a unique conduit for intercellular transfer of cellular contents in human malignant pleural mesothelioma. PLoS One,2012.7(3):p. e33093.
31. Yasuda, K., et al., Tunneling nanotubes mediate rescue of prematurely senescent endothelial cells by endothelial progenitors:exchange of lysosomal pool. Aging (Albany NY),2011.3(6):p.597-608.
32. Lis, R., et al., Mesenchymal cell interaction with ovarian cancer cells induces a background dependent pro-metastatic transcriptomic profile. J Transl Med,2014.12:p.59.
33. Touboul, C., et al., Mesenchymal stem cells enhance ovarian cancer cell infiltration through IL6 secretion in an amniochorionic membrane based 3D model. J Transl Med,2013.11:p.28.
34. Gnecchi, M. and L.G. Melo, Bone marrow-derived mesenchymal stem cells:isolation, expansion, characterization, viral transduction, and production of conditioned medium. Methods Mol Biol, 2009.482:p.281-94.
35. Boomsma, R.A. and D.L. Geenen, Mesenchymal stem cells secrete multiple cytokines that promote angiogenesis and have contrasting effects on chemotaxis and apoptosis. PLoS One,2012.7(4):p. e35685.
36. Ling, B., et al., Effect of conditioned medium of mesenchymal stem cells on the in vitro maturation and subsequent development of mouse oocyte. Braz J Med Biol Res,2008.41(11):p.978-85.
37. Rogers, G.W., et al., High throughput microplate respiratory measurements using minimal quantities of isolated mitochondria. PLoS One,2011.6(7):p. e21746.
38. Sharma, P., et al., Positive association of tyrosine hydroxylase microsatellite marker to essential hypertension. Hypertension,1998.32(4):p.676-82.
39. Andres, A.M., et al., Comparative genetics of functional trinucleotide tandem repeats in humans and apes. J Mol Evol,2004.59(3):p.329-39.
40. Zhou, X., et al., Trimetazidine protects against smoking-induced left ventricular remodeling via attenuating oxidative stress, apoptosis, and inflammation. PLoS One,2012.7(7):p. e40424.
41. Invernizzi, F., et al., Microscale oxygraphy reveals OXPHOS impairment in MRC mutant cells. Mitochondrion,2012.12(2):p.328-35.
42. Hase, K., et al., M-Sec promotes membrane nanotube formation by interacting with Ral and the exocyst complex. Nat Cell Biol,2009.11(12):p.1427-32.
43. Chauveau, A., et al., Membrane nanotubes facilitate long-distance interactions between natural killer cells and target cells. Proc Natl Acad Sci U S A,2010.107(12):p.5545-50.
44. Depraetere, V, "Eat me" signals of apoptotic bodies. Nat Cell Biol,2000.2(6):p. E104.
45. Qu, X., et al., Autophagy gene-dependent clearance of apoptotic cells during embryonic development. Cell,2007.128(5):p.931-46.
46. Frisoni, L., et al., Nuclear autoantigen translocation and autoantibody opsonization lead to increased dendritic cell phagocytosis and presentation of nuclear antigens:a novel pathogenic pathway for autoimmunity? J Immunol,2005.175(4):p.2692-701.
47. Ravichandran, K.S., Find-me and eat-me signals in apoptotic cell clearance:progress and conundrums. J Exp Med,2010.207(9):p.1807-17.
48. Murphy, M.P., How mitochondria produce reactive oxygen species. Biochem J,2009.417(1):p. 1-13.
49. Logan, A., et al., Using exomarkers to assess mitochondrial reactive species in vivo. Biochim Biophys Acta,2014.1840(2):p.923-30.
50. Echtay, K.S., et al., Superoxide activates mitochondrial uncoupling protein 2 from the matrix side. Studies using targeted antioxidants. J Biol Chem,2002.277(49):p.47129-35.
51. Lee, J., S. Giordano, and J. Zhang, Autophagy, mitochondria and oxidative stress:cross-talk and redox signalling. Biochem J,2012.441(2):p.523-40.
52. Hughes, A.L. and D.E. Gottschling, An early age increase in vacuolar pH limits mitochondrial function and lifespan in yeast. Nature,2012.492(7428):p.261-5.
53. Markaki, I., et al, Long-Term Survival of Ischemic Cerebrovascular Disease in the Acute Inflammatory Stroke Study, a Hospital-Based Cohort Described by TOAST and ASCO. Cerebrovasc Dis,2013.35(3):p.213-219.
54. Gursoy-Ozdemir, Y., M. Yemisci, and T. Dalkara, Microvascular protection is essential for successful neuroprotection in stroke. Journal of neurochemistry,2012.123 Suppl 2:p.2-11.
55. Kim, D., et al., In vivo tracking of human mesenchymal stem cells in experimental stroke. Cell Transplant,2008.16(10):p.1007-12.
56. Chen, J., et al., Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats. Stroke,2001.32(4):p.1005-11.
57. Wei, N., et al., Delayed intranasal delivery of hypoxic-preconditioned bone marrow mesenchymal stem cells enhanced cell homing and therapeutic benefits after ischemic stroke in mice. Cell Transplant,2013.22(6):p.977-91.
58. Zhao, Y.H., Y. Guan, and W.K. Wu, Potential advantages of a combination of chinese medicine and bone marrow mesenchymal stem cell transplantation for removing blood stasis and stimulating neogenesis during ischemic stroke treatment. J Tradit Chin Med,2012.32(2):p.289-92.
59. Lee, J.S., et al., A long-term follow-up study of intravenous autologous mesenchymal stem cell transplantation in patients with ischemic stroke. Stem Cells,2010.28(6):p.1099-106.
60. Sokolova, I.B., et al., Effect of mesenchymal stem cell transplantation on cognitive functions in rats with ischemic stroke. Bull Exp Biol Med,2006.142(4):p.511-4.
61. De Keyser, J., Autologous mesenchymal stem cell transplantation in stroke patients. Ann Neurol, 2005.58(4):p.653-4; author reply 654-5.
62. Bang, O.Y., et al., Autologous mesenchymal stem cell transplantation in stroke patients. Ann Neurol, 2005.57(6):p.874-82.
63. Tsai, L.K., et al., Mesenchymal stem cells primed with valproate and lithium robustly migrate to infarcted regions and facilitate recovery in a stroke model. Stroke,2011.42(10):p.2932-9.
64. Croft, A.P. and S.A. Przyborski, Formation of neurons by non-neural adult stem cells:potential mechanism implicates an artifact of growth in culture. Stem Cells,2006.24(8):p.1841-51.
65. Terada, N., et al., Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature,2002.416(6880):p.542-5.
66. Davidson, S.M. and M.R. Duchen, Endothelial mitochondria:contributing to vascular function and disease. Cire Res,2007.100(8):p.1128-41.
67. Liu, K., et al., Mesenchymal stem cells rescue injured endothelial cells in an in vitro ischemia-reperfusion model via tunneling nanotube like structure-mediated mitochondrial transfer. Microvascular research,2014.92:p.10-8.
68. Longa, E.Z., et al., Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke, 1989.20(1):p.84-91.
69. Li, Y., et al., Exendin-4 ameliorates motor neuron degeneration in cellular and animal models of amyotrophic lateral sclerosis. PLoS One,2012.7(2):p. e32008.
70. Pendharkar, A.V., et al., Biodistribution of neural stem cells after intravascular therapy for hypoxic-ischemia. Stroke,2010.41(9):p.2064-70.
71. Wang, L.Q., et al., Timing and dose regimens of marrow mesenchymal stem cell transplantation affect the outcomes and neuroinflammatory response after ischemic stroke. CNS Neurosci Ther, 2014.20(4):p.317-26.
72. 孙明,赵育梅,徐超,局灶性脑缺血再灌注损伤核心区及半暗带的组织学定位.中华神经外科杂志,2003.19(4)：259-62.
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