【作者】 侯剑峰；

【导师】 胡盛寿；

【作者基本信息】 中国协和医科大学 ， 心血管外科， 2009， 博士

【摘要】 第一部分:低能激光照射对体外培养的骨髓间充质干细胞生物学性状影响实验研究目的:骨髓间充质干细胞(Bone-marrow mesenchymal stem cells,BMSCs)是目前细胞移植治疗和组织工程研究的热点种子细胞。低能激光照射(Low-level laser irradiation,LLLI)在以往的研究中已被证实能够影响多种体外培养细胞的细胞进程,但是其对体外培养的BMSCs的影响鲜有研究,本部分实验研究的主要目的是探讨不同能量密度的LLLI对体外培养的大鼠骨髓间充质干细胞增殖,分泌功能及肌样细胞分化的影响。方法:贴壁培养法自Sprague-Dawley大鼠新鲜骨髓中获取BMSCs,采用635nm半导体低能激光(输出功率60 mW)以0,0.5,1.0,2.0和5.0 J/cm~2能量密度体外照射,24小时后检测上清液乳酸脱氢酶含量;酶联免疫吸附法(ELISA)测定上清液中血管内皮生长因子(Vascular endothelial growth factor,VEGF)及神经生长因子(Nervegrowth factor,NGF)含量;MTT法及BrdU法绘制BMSCs增殖曲线;免疫细胞化学法检测5-氮胞苷(5-aza)诱导后肌系细胞早期标志物Sarcomericα-actin及Desmin表达。结果:635nm波长LLLI对体外培养的BMSCs没有显著的细胞毒性,并且能够促进体外培养的大鼠BMSCs的增殖,最佳能量密度为0.5 J/cm~2:大鼠BMSCs能够在体外培养条件下分泌VEGF、NGF,能量密度为5.0 J/cm~2的LLLI能够显著促进BMSCs分泌VEGF、NGF;能量密度为5.0 J/cm~2的LLLI能够显著促进5-aza诱导后BMSCsSarcomericα-actin及Desmin表达。结论:635nm波长LLLI能够显著促进体外培养的大鼠BMSCs增殖,分泌功能及肌样细胞分化,且与照射剂量有关。因此,LLLI可能作为一种有效的细胞预处理手段用于细胞移植治疗心肌梗死。第二部分:低能激光照射预处理梗死后心肌组织提高骨髓间充质干细胞早期移植存活实验研究目的:骨髓间充质干细胞(Bone-marrow mesenchymal stem cells,BMSCs)移植已成为心肌梗死治疗的新策略,但是移植后干细胞存活率低下极大限制了该治疗策略的疗效。在本部分研究中,我们设想低能激光照射(Low-level laser irradiation,LLLI)预处理能够改善梗死后心肌微环境,提高移植细胞早期存活率及细胞移植的疗效。方法:BMSCs取自雄性Sprague-Dawley(SD)大鼠,冠状动脉结扎法制作雌性SD大鼠心肌梗死(Myocardial infarction,MI)模型。3周后,经超声筛选合格心肌梗死大鼠随机分组评价LLLI预处理对梗死后心肌微环境影响及LLLI预处理后对移植后细胞存活影响。LLLI预处理采用635 nm半导体低能激光(输出功率:5 mW;照射时间:150秒;能量密度:0.96 J/cm~2)开胸单次照射。LLLI预处理后1小时、1天、1周,以免疫蛋白印迹法(Western blotting)和实时定量聚合酶链反应(Real-timePCR)法分别检测预LLLI处理区内心肌组织中血管内皮生长因子(Vascularendothelial growth factor,VEGF)和葡萄糖调节蛋白78(Glucose-regulated protein 78,GRP78)的表达,分别以黄嘌呤氧化酶法和硫代巴比妥酸法测定LLLI预处理区内心肌组织中超氧化物歧化酶(Superoxide dismutase,SOD)活性和丙二醛(Malondialdehyde,MDA)含量。LLLI预处理后即刻,于LLLI预处理的心肌梗死边带区单点注射DAPI标记的BMSCs 2×10~6个/50μl或等体积的无血清细胞培养液。BMSCs移植后1小时、1天、1周,Real-time PCR法检测移植BMSCs存活,TUNEL法检测移植BMSCs凋亡。免疫组化法检测梗死区及梗死边带区新生毛细血管密度,免疫荧光法检测移植BMSCs肌样细胞分化,超声心动图评价左心功能。结果:LLLI预处理显著提高了梗死后心肌组织中VEGF、GRP78的表达及SOD的活性,降低了MDA的含量。尽管在治疗后左心功能的改善上LLLI+BMSCs组与BMSCs组之间不存在统计学差异,但是LLLI+BMSCs组中早期移植BMSCs存活率,移植BMSCs的凋亡率,以及新生毛细血管数量都显著高于BMSCs组。结论:LLLI预处理梗死后心肌为提高移植后干细胞的早期存活率,增强细胞移植治疗心肌梗死的疗效提供了一种有效而且无创的干预措施。更多还原

【Abstract】 Objectives:Bone marrow derived mesenchymal stem cells(BMSCs) have shown to be an appealing source for cell therapy and tissue engineering.Previous studies have confirmed that the application of low-level laser irradiation(LLLI) could affect the cellular process. However,little is known about the effects of LLLI on BMSCs.The aim of this study was designed to investigate the influence of LLLI at different energy densities on BMSCs proliferation,secretion and myogenic differentiation.Materials and Methods:BMSCs were harvested from rat fresh bone marrow and exposed to a 635nm diode laser(60mW;0,0.5,1.0,2.0,or 5.0 J/cm~2).The lactate dehydrogenase(LDH) release was used to assess the cytotoxicity of LLLI at different energy densities.Cell proliferation was evaluated by using 3-(4,5-dimethylithiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT) and 5-bromo-20-deoxyuridine(BrdU) assay.Production of vascular endothelial growth factor(VEGF) and nerve growth factor(NGF) were measured by enzyme-linked immunosorbent assay(ELISA).Myogenic differentiation, induced by 5-azacytidine(5-aza),was assessed by using immunocytochemical staining for the expression of sarcomericα-actin and desmin.Results:Cytotoxicity assay showed no significant difference between the non-irradiated group and irradiated groups.LLLI significantly stimulated BMSCs proliferation and 0.5 J/cm~2 was found to be an optimal energy density.VEGF and NGF were identified and LLLI at 5.0 J/cm~2 significantly stimulated the secretion.After 5-aza induction,myogenic differentiation was observed in all groups and LLLI at 5.0 J/cm~2 dramatically facilitated the differentiation.Conclusions:LLLI stimulates proliferation,increases growth factors secretion and facilitates myogenic differentiation of BMSCs.Therefore,LLLI may provide a novel approach for the preconditioning of BMSCs in vitro prior to transplantation. Objectives:Recent years have witnessed a growing interest and enthusiasm in the application of bone-marrow derived stem cell-based therapies to repair or regenerate damaged myocardium.However,progress in stem cell therapy is hampered by the poor survival of implanted cells.We hypothesized that low-level laser irradiation(LLLI) preconditioning prior to bone-marrow mesenchymal stem cells(BMSCs) transplantation might remodel the hostile milieu of infarcted myocardium and subsequently enhance early survival and therapeutic potential of implanted cells.Materials and Methods:BMSCs were isolated from male donor rat.Myocardial infarction was induced by left anterior descending artery ligation in female rats.Three weeks later,female rats were randomly divided for LLLI preconditioning study and the following LLLI preconditioning and cell survival study.After chest-opened,a 635 nm,5 mW diode laser was performed with energy density of 0.96 J/cm~2 for 150 seconds for the purpose of myocardial preconditioning.In LLLI preconditioning study,vascular endothelial growth factor(VEGF),glucose-regulated protein 78(GRP78),superoxide dismutase(SOD) and malondialdehyde(MDA) in the infarcted myocardium were evaluated at 1 hour,1 day and 1 week after laser irradiation.In cell survival study,2 millions male BMSCs or no-serum culture media was injected into infarcted myocardium with or without LLLI preconditioning.Cell survival was assayed with quantitative real-time polymerase chain reaction to identify Y chromosome gene and apoptosis was assayed with TUNEL staining. Capillary density,myogenic differentiation and left ventricular function were tested at 1 week.Results:After LLLI preconditioning,increased VEGF and GRP78 expression,as well as the enhanced SOD activity and inhibited MDA production,was observed.Compared with BMSCs transplantation and culture media injection group,although there was no difference in the improved heart function and myogenic differentiation,LLLI preconditioning significantly enhanced early cell survival rate by 2-fold,decreased the apoptotic percentage of implanted BMSCs in infarcted myocardium and thus increased the number of newly formed capillaries.Conclusions:LLLI preconditioning could be a novel non-invasive approach for intraoperative cell transplantation to enhance cell early survival and therapeutic potential.更多还原