【作者】 刘鹏霞；

【导师】 韩忠朝；

【作者基本信息】 中国协和医科大学 ， 内科学， 2009， 博士

【摘要】 背景:血管内皮细胞不仅是血管腔与周围其它组织之间的生理屏障,而且参与多种血管生物学功能,包括血管舒张、血管收缩、血液凝固、动脉粥样硬化、血管生成和炎症。内皮功能失调是动脉粥样硬化和血管成形术后再狭窄发生的一个重要的病理生理学改变。干细胞具有修复损伤组织的潜能,干细胞技术的发展为修复损伤血管提供了新方向。间充质干细胞(Mesenchymal stem cell,MSC)属于成体干细胞,相对于胚胎干细胞和其它成体干细胞具有多方面的优点,因此近年来倍受研究者们的关注。研究证实,MSC能够分化为内皮细胞。MSC是否可以成为治疗内皮功能失调的种子细胞,值得我们去研究。目的:目前的研究中,我们通过球囊损伤术建立兔颈动脉内皮剥脱模型,旨在探讨MSC治疗对血管内皮细胞再生和血管功能恢复的作用。方法:异基因MSC由兔骨髓穿刺液培养获得。培养第三代的MSC经荧光染料CM-DiI标记后用于细胞治疗。通过在兔颈总动脉中穿过膨胀的球囊导管建立兔颈总动脉内皮剥脱模型。48只新西兰白兔随机分成三组:正常对照组4只,不接受任何处理;MSC注射组22只,血管球囊损伤术后立即接受细胞治疗;生理盐水注射组22只,血管球囊损伤术后立即接受生理盐水注射。MSC注射组动物在损伤的血管腔中给予10~5 MSC,生理盐水对照组动物给予等体积的生理盐水,夹闭动脉孵育30分钟,孵育完成后缝合手术切口。实验后第3天和第7天取MSC注射组兔(n=5)、生理盐水注射组兔(n=5)和正常对照组兔(n=4)的颈动脉,提取RNA,用实时定量PCR方法检测相关炎症因子的表达,包括白介素6、白介素10、转化生长因子β、干扰素γ和肿瘤坏死因子α。四周后处死剩余所有动物,分离颈动脉用于血管舒张功能检测和组织病理分析。结果:血管球囊损伤术后四周,激光共聚焦纤维镜下观察颈动脉,可见MSC注射组兔损伤的颈动脉血管壁中有红色荧光克隆存在,显示注射的MSC能够归巢到血管损伤部位。组织病理分析显示,MSC注射组兔与生理盐水注射组兔相比较,颈动脉再内皮化面积增加,内膜增生减少(p＜0.05)。MSC注射组兔与生理盐水注射组兔相比较,损伤部位颈动脉内皮依赖性舒张功能明显改善(p＜0.01)。实时定量PCR结果显示,与生理盐水注射组动物比较,损伤后第3天和第7天,MSC注射组兔颈动脉免疫抑制因子转化生长因子β(TGFβ)表达增加;损伤后第3天,MSC注射组兔颈动脉促炎因子白细胞介素6(IL-6)表达降低(p＜0.05)。结论:这些结果证明局部注射异基因MSC抑制了损伤局部的炎症反应,促进了球囊损伤术后颈动脉内皮再生及血管功能恢复,从而抑制了血管恶性重塑。这为我们了解间充质干细胞的生物学特性提供了新的证据、同时也为治疗血管功能失调以及阻止血管成形术后狭窄提供了新的策略。背景:动脉粥样硬化是严重危害人类健康的疾病,是心脑血管疾病的主要原因。动脉粥样硬化是选择性影响大、中动脉的慢性炎症疾病,斑块破裂和阻塞会引起急性临床并发症如心肌梗塞和脑卒中。到目前为止,引起动脉粥样硬化的直接病因尚未完全明确,导致动脉粥样硬化的相关因素统称危险因素,包括吸烟、肥胖、高血压、高血脂、高同型半胱氨酸血症以及家族遗传史等。间充质干细胞(Mesenchymalstem cell,MSC)是广泛存在于多种成体组织当中的一类多能干细胞,相对其它干细胞具有多方面的优点。研究证实,MSC能够分化为内皮细胞促进血管生成,改善缺血心肌。MSC用于急性心肌梗死的治疗,成为了近年来基础与临床研究的热点。但是,目前还没有MSC治疗对动脉粥样硬化的作用的报道。MSC治疗在动脉粥样硬化机体条件下是否安全,对动脉粥样硬化具有怎样的作用,值得我们去研究。目的:目前的研究中,我们通过建立兔动脉粥样硬化模型,探索静脉输注MSC在动脉粥样硬化条件下的安全性及其对动脉粥样硬化的影响。方法:体外培养兔骨髓穿刺液,获得MSC。30只新西兰白兔随机分为三组:正常饮食组6只,普通饲料饲养;高脂饮食组12只,高胆固醇高脂饲料喂养5周后经耳缘静脉注射生理盐水;MSC注射组12只,高胆固醇高脂饲料喂养5周后经耳缘静脉注射1.15×10~7/kg体重MSC。分别于实验前,实验第5周,第9周和第13周测体重和空腹血脂水平。实验第13周时处死所有动物,形态学分析检测动脉粥样硬化斑块形成和主动脉外膜滋养血管增生,结果:MSC注射组与高脂饮食组相比,主动脉窦斑块面积显著增大(斑块面积占主动脉窦面积的百分比分别为23.35±3.51%和11.39±3.08%,p＜0.05),整体主动脉斑块形成明显增多(斑块面积占主动脉面积的百分比分别为76.64±12.70%和57.61±9.00%,p＜0.05)。MSC注射组兔主动脉外膜滋养血管增生明显,毛细血管密度增加。结论:我们首次证明异基因MSC输注促进高脂血症兔动脉粥样硬化斑块进展,可能与促进外膜滋养血管增生有关。采用MSC或包含MSC的细胞进行细胞治疗时应采取策略减少MSC促进斑块形成的作用。更多还原

【Abstract】 Background:The endothelium not only forms a selective barrier between the vessel lumen and surrounding tissue but also takes part in many aspects of vascular biology. including vasoconstriction,vasodilation,blood clotting,atherosclerosis,angiogenesis and inflammation.Endothelial dysfunction is one important pathophysiological step of atherosclerosis and restenosis after angioplasty.Stem cells hold a great potential for the regeneration of damaged tissues in cardiovascular diseases.The advancement of stem cell technology brings about prospectives in the treatment of endothelium dysfunction. Mesenchymal stem cells(MSCs) are one of stem cell types undergoing extensive investigation for cell-based therapeutic strategies.Aims:In the present study,we investigated the effect of MSC delivery on the regeneration of endothelium and recovery of endothelial function in denudated carotids.Methods:Allogeneic MSCs were obtained from rabbit bone marrow(BM) aspirates. MSCs of passage 3 were labeled with CM-DiI and used in the experiment.A rabbit model of common carotid artery denudation by passage of an inflated balloon catheter was used.New Zealand White rabbits were randomly divided into three groups:rabbits receiving administration of either 10~5 MSCs(n=22) or saline(n=22) into the lumen of injured artery immediately after balloon angioplasty and additional rabbits(n=4) were served as controls.We extracted total RNA from injured and contralateral uninjured carotids harvested at 3 and 7 days after balloon angioplasty from MSC-(n=5 for each time point) or saline-treated rabbits(n=5 for each time point).Real-time reverse transcription-polymerase chain reaction(RT-PCR) was performed to validate the expression of inflammation-related gene,including interleukin-6(IL-6),interleukin-10 (IL-10),transforming growth factor beta(TGF-β),interferon-gamma(IFN-γ),and tumor necrosis factor-alpha(TNF-α).Four weeks after balloon angioplasty.carotid arteries were harvested for vasodilatory function analysis and histological analysis.Results:Four weeks after transplant,fluorescence-labeled colonies of MSCs were found in the injured vessel wall of MSC-treated carotids.Local transfer of allogeneic MSCs as compared with saline administration caused enhanced reendothelialization and reduced neointima formation when assessed 4 weeks after denudation(p＜0.05).MSC delivery improved endothelium-dependent vasorelaxation at 4 weeks compared with saline(p＜0.01).Moreover,MSC treatment affected the expression level of inflammation-related genes,inducing an increase of TGF-βin injured carotids at 3 and 7 days after angioplasty and a decrease of IL-6 in injured carotids at 3 days after angioplasty(p＜0.05).Conclusions:Taken together,these data indicate that local delivery of allogeneic MSCs played a local immunomodulatory action,caused an increased reendothelialization and endothelium-dependent vasorelaxation as well as a markedly reduced neointima formation in rabbit model of common carotid artery denudation.These results bring new insights into MSC biology and the treatment of vascular dysfunction and prevention of restenosis after angioplasty. Background:Atherosclerosis,the primary cause of heart disease and stroke,is a multifactorial process with complicated aetiology.It is considered as a chronic inflammatory condition that can be converted into an acute clinical event by plaque rupture and thrombosis.Thrombo-occlusive complications of atherosclerosis,including stroke and myocardial infarction,are becoming major causes of morbidity and mortality in the industrialized world.Epidemiological studies have revealed several important environmental and genetic risk factors associated with atherosclerosis,such as elevated levels of homocysteine,hyperlipidemia,hypertension,diabetes,smoking,lack of exercises,family history etc.Mesenchymal stem cells(MSC) are present in adult tissues,including bone marrow and adipose,from which they can be easily isolated and cultured ex vivo.Recent experimental observations indicate that MSCs are capable of differentiating into endothelial cells,facilitate both myocardial repair and neovascularization in models of cardiac injury.But the safety of MSC transplantation in the setting of atherosclerosis has not yet been demonstrated.Aims:The present study was designed to evaluate the safety and the effects of MSC-based therapy in the setting of atherosclerosis.Methods:Allogeneic MSCs were obtained from rabbit bone marrow aspirates and expanded in vitro.New Zealand White rabbits were divided into three groups:24 rabbits with hypercholesterolemia receiving intravenous injection of either 1.15×10~7/kg MSCs (n=12) or saline(n=12) after 5 weeks on a high lipid diet and additional rabbits(n=6) fed with standard rabbit diet were served as controls.Body weight and blood lipids were measured at weeks 0.5,9 and 13 during the study.All rabbits were euthanized at week 13.Atherosclerotic plaque size,atherosclerotic plaque composition and vasa vasorum were evaluated using pathological analysis and immunocytochemical technique.Results:Aortic sinus lesion size was significantly increased in rabbits receiving MSCs compared with saline controls(23.35±3.51%and 11.39±3.08%respectively).The lesion size in whole aortas of MSC-treated rabbits was 76.64±12.70%versus 57.61±9.00%in saline-treated animals(p＜0.05).Moreover.Vasa vasorum networks in MSC-treated aortas are more numerous and have increased capillary density.Conclusions:We demonstrated for the first time that allogeneic MSC transfer may result in an increase in atherosclerotic lesion size.Cell therapy with MSCs or cell populations containing MSCs requires a strategy to attenuate the high potential of MSCs to contribute to atherogenesis in the context of atherosclerosis.更多还原