【作者】 陈鑫；

【导师】 沈振亚；

【作者基本信息】 苏州大学 ， 胸心血管外科， 2007， 博士

【摘要】 第一部分:MI大鼠心肌组织中SDF-1与MCP-1的动态变化及意义目的:有研究提示移植后的骨髓干细胞可以植入到心肌梗死周围区域中,而细胞因子单核细胞趋化蛋白-1(monocyte chemotactic protein 1, MCP-1)及基质细胞衍生因子-1(stromal cell-derived factor 1, SDF-1)可能对干细胞具有趋化作用。移植干细胞植入到心肌梗死和/或周围区域的现象是否与心肌梗死后细胞因子水平和分布存在联系尚不十分明确。本研究结扎大鼠冠状动脉左前降支建立心肌梗死(myocardial infarction, MI)模型,探讨MI后心肌组织中不同部位和不同时段细胞因子MCP-1及SDF-1表达的动态变化及其意义。方法:以原位杂交及免疫组化方法检测大鼠心肌梗死前及梗死后1、2、4、7、14、28、56天梗死中心区域、梗死周围区域及无梗死正常区域SDF-1、MCP-1 mRNA及其蛋白的表达情况;同时建立单纯开胸假手术对照组,于术后1、2、4、7、14、28、56d以同样方法检测SDF-1、MCP-1 mRNA及蛋白的表达情况。结果:心肌梗死大鼠中:梗死中心区域MCP-1于心肌梗死后第1d升高,第2d达峰值,至第14d渐降至正常水平;梗死周围区域MCP-1心肌梗死后第1天升高,7天达峰值,至第28天恢复正常水平;梗死中心区域及周围区域SDF-1均于心肌梗死后第1天升高并达峰值,至第14天渐恢复至正常水平;无梗死正常区域中MCP-1、SDF-1的表达在正常水平,且不随时间改变而改变。假手术非梗死组大鼠心肌中MCP-1、SDF-1的表达在正常水平,且不随时间改变而改变。MCP-1、SDF-1 mRNA与蛋白的表达变化趋势相一致。结论:心肌梗死后早期梗死中心及周围区域SDF-1、MCP-1表达水平升高;根据梗死中心区和周围区域细胞因子SDF-1、MCP-1表达的动态变化情况选择治疗时间窗,可能有助于趋化更多的干细胞到达损伤部位,提高治疗效果,可能对心肌梗死后组织修复产生影响。第二部分:SDF-1与MCP-1对骨髓间质干细胞归巢到急性梗死心肌中作用的研究目的:心肌梗死后早期,SDF-1、MCP-1表达迅速升高,并对MSCs的归巢起到了重要的促进作用。但内源性的SDF-1、MCP-1表达时段短、表达水平低,与心肌梗死后超急性期炎症反应的时间窗重叠,可使趋化作用受到限制。本研究通过外源性的SDF-1、MCP-1进行干预,探讨外源性SDF-1与MCP-1在骨髓间质干细胞归巢到急性梗死心肌中的作用。方法:细胞培养采用健康F344大鼠麻醉后取双下肢股骨后处死,股骨移入超净台内,咬骨钳咬碎股骨,以IMDM培养基冲洗髓腔获取骨髓干细胞。采用全骨髓培养法培养扩增,利用MSCs贴附于塑料培养皿的特点,在传代增殖过程中通过换液逐渐去除造血系细胞等杂质细胞。经21~28天培养,约传代3~4代的细胞即可用于移植。细胞移植前予以含10μmol/L BrdU的培养基孵育24小时进行细胞标记。近交系F344大鼠随机分为5组,其中4组为心肌梗死大鼠,1组为正常大鼠。大鼠心肌梗死模型建立前行超声心动图检查心功能(left ventricular ejection fraction, LVEF; left ventricular fractional shortening, LVFS)。心肌梗死大鼠于梗死4天后行超声心动图检查心功能,然后3组梗死大鼠在心肌梗死区及其周围注射外源性MCP-1、SDF-1、MCP-1+SDF-1进行干预,剩余1组梗死大鼠于相应部位注射等量的生理盐水进行对照,正常大鼠也于相应部位注射等量的生理盐水进行对照。随后所有大鼠经尾静脉注射BrdU标记的MSCs(5×106),MSCs移植3天后处死一半大鼠检测心肌梗死区MSCs的归巢量,28天后剩余一半大鼠检测大鼠心功能状况后处死,取出心脏检测心肌梗死区及其周围血管内皮生长因子(vascular endothelial growth factor,VEGF)的表达和新生血管密度。结果:心肌梗死组MSCs归巢量大于正常大鼠对照组。MCP-1、SDF-1、MCP-1+SDF-1处理组大鼠心肌梗死区MSCs归巢量、新生毛细血管密度明显优于对照组。MCP-1+SDF-1处理组大鼠心肌梗死区MSCs归巢量及新生毛细血管密度大于MCP-1、SDF-1处理组,但三组间MSCs移植后心功能水平无明显统计学差异。结论:SDF-1、MCP-1能够促进MSCs归巢并改善心功能,促进毛细血管增生可能为其改善心功能的机制之一;SDF-1、MCP-1联合使用较单用更有效,但尚未达到两者的相加作用。其原因可能与炎症损伤干细胞、受体重叠、内陷等有关,尚需进一步研究明确其具体原因;以获得更佳的治疗效果。第三部分:SDF-1、MCP-1对骨髓间质干细胞归巢剂量-效应关系的研究目的:外源性细胞因子SDF-1和MCP-1可以促进MSCs归巢,但归巢量与细胞因子的水平是否存在联系还有待探讨。本研究探讨SDF-1、MCP-1在骨髓间质干细胞归巢到梗死心肌中的剂量-效应关系及二者之间是否存在协同效应。方法:大鼠于心肌梗死模型建立前、MSCs移植前及移植后28天行超声心动图检查心功能(LVEF; LVFS)。大鼠共分为12组,5组大鼠心肌梗死56天后于心肌梗死区及其周围分别均匀注射不同浓度(50ng/ml、100ng/ml、200ng/ml、400ng/ml、800ng/ml)的MCP-1 50μl,5组大鼠心肌梗死56天后于心肌梗死区及其周围分别均匀注射不同浓度(50ng/ml、100ng/ml、200ng/ml、400ng/ml、800ng/ml)的SDF-1 50μl,1组大鼠心肌梗死56天后于心肌梗死区及其周围均匀注射200ng/ml MCP-1+200ng/ml SDF-1各50μl,1组大鼠心肌梗死56天后于心肌梗死区及其周围均匀注射等量的生理盐水进行对照。然后各组大鼠经尾静脉注射BrdU标记的MSCs(5×106),MSCs移植3天后各组处死一半大鼠检测梗死区及其周围MSCs的归巢量,28天后检测剩余一半大鼠心功能状况、以及梗死区及其周围VEGF的表达及新生血管密度。结果:MCP-1、SDF-1注射组MSCs归巢量大于生理盐水对照组;且随着MCP-1、SDF-1剂量的增加,MSCs归巢量亦相应增加,呈线性关系。VEGF的表达及新生血管密度与MSCs归巢量相一致。SDF-1与MCP-1联合注射组MSCs归巢量、新生毛细血管数高于同剂量的单纯SDF-1或MCP-1注射组。各组大鼠心功能较MSCs移植前改善,但各组之间无显著差异。结论:心肌梗死56天后无明显MSCs自发归巢。SDF-1、MCP-1能够促进MSCs归巢,归巢的MSCs可能通过分泌VEGF而促进血管新生;而且MSCs的归巢量与SDF-1、MCP-1的剂量呈线性剂量-效应关系。但对改善心功能无明显作用,可能与大鼠梗死后干预时间过晚有关。SDF-1、MCP-1联合使用较单用更有效,但尚未达到两者的相加作用。第四部分:MCP-1与SDF-1在G-CSF外周动员自身骨髓干细胞归巢中作用的研究目的:G-CSF能够动员骨髓干细胞进入外周血液循环,但外源性SDF-1、MCP-1对G-CSF动员释放的骨髓干细胞的归巢是否有促进作用还不十分清楚。本部分研究探讨细胞因子MCP-1与SDF-1在G-CSF动员自身骨髓干细胞归巢治疗缺血性心脏病中的作用。方法:心肌梗死模型制作成功8周后,所有大鼠给予皮下注射瑞白(重组人粒细胞集落刺激因子,rhG-CSF)20μg·kg-1·d-1,共5天。随后予以超声心动图检查心功能(EF、FS),再将大鼠随机分为3组,每组15只,一组为SDF-1注射组,一组为MCP-1注射组,一组为生理盐水对照组。SDF-1及MCP-1注射组大鼠于心肌梗死区及其周围5个区域均匀注射SDF-1及MCP-1 400ng/ml各10μl,对照组于相应区域注射生理盐水各10μl。接着每只大鼠腹腔注射BrdU 50mg·kg-1·d-1,共15天。细胞因子注射4周(心肌梗死后12周+5天)以后超声心动图检查心功能后戊巴比妥钠(50mg/kg)腹腔注射麻醉大鼠后,经主动脉注入质量分数为10%的氯化钾2ml,使心脏停搏于舒张期。随即开胸取出大鼠心脏,进行CD45、CD90、BrdU、VEGF及VIII因子免疫组化染色。结果:MCP-1、SDF-1注射组与生理盐水对照组相比,梗死区域BrdU、CD45、CD90阳性细胞明显增多,VEGF表达及毛细血管数明显增加。MCP-1、SDF-1注射组中,归巢的CD45+细胞多于CD90+细胞。但各组心功能无明显统计学差异。结论:MCP-1、SDF-1能有效促进G-CSF外周动员的骨髓干细胞归巢到心肌梗死区域,归巢的骨髓干细胞可能促进血管新生。更多还原

【Abstract】 Part I: Expression and significance of SDF-1 and MCP-1 after myocardial infarctionObjective:To investigate the dynamic changes of the expression of SDF-1 and MCP-1 in and around myocardial infarct site.Methods:Protein and mRNA of MCP-1 and SDF-1 expression were measured by immunohistochemistry and in situ hybridization in mid-infarct zone, peri-infarct zone and non-MI normal zone in infarcted hearts or sham operated hearts at 1, 2, 4, 7, 14, 28 and 56 days post operation.Results:MCP-1 expression increased at the first day, peaked at 2nd day and decreased thereafter post MI in the center of myocardial infarct site, peaked at 7th and decresed thereafter post MI in peri-myocardial infarct site. SDF-1 expression increased and peaked at the first day and decreased thereafter post MI in the center of myocardial infarct site and peri-myocardial infarct site. However, both MCP-1 and SDF-1 remained unchanged away from myocardial site and in sham operated hearts.Conclusions:Myocardial MCP-1 and SDF-1 expression were increased in mid- and peri-infarct site only in the early phase post MI. It may affect the repair of infarct myocardium. Part II: The effects of SDF-1 & MCP-1 on mesenchymal stem cells homing to myocardial infarct siteObjective:In the first part, we found that the expression of SDF-1 and MCP-1 increase in the early phase post MI, and the expression is not the same in different site of the myocardium.In this part, we are to investigate the effects of SDF-1 and MCP-1 on MSCs homing to myocardial infarct site.Methods:MSCs from donor rat were cultured and labeled with BrdU. MCP-1, SDF-1, MCP-1+SDF-1 or saline was injected into mid- and peri-infarct myocardium 4 days after MI. Then, a total of 5×106 cells in 2.5 mL of PBS or equal volume PBS alone were injected through the tail vein. The number of the labeled MSCs in the infarcted hearts was counted 3 days post injection. Cardiac function, expression of vascular endothelial growth factor (VEGF) and blood vessel density were assessed 28 days post injection. MI and non-MI saline injection groups were established as control.Results:The MSCs enrichment in the host hearts were more abundant in the MI groups than that in the non-MI group; MCP-1, SDF-1, MCP-1+SDF-1 injected group than control groups. Cardiac function improvement and blood vessel density in MCP-1, SDF-1, MCP-1+SDF-1 injected groups were more than control groups. MSCs enrichment and blood vessel density in MCP-1+SDF-1 injected group were more than MCP-1, SDF-1 injected group; but cardiac function had no difference among the three groups.Conclusion:SDF-1 and MCP-1 may enhance MSCs homing to injured heart and improved cardiac function by promoting neovascularization. The homing enhancement effect of SDF-1 plus MCP-1 is better than SDF-1 or MCP-1 respectively. Part III: The dose-effect relation of SDF-1 & MCP-1 on MSCs homing to myocardial infarct siteObjective:To investigate the dose-effect relation of SDF-1 and MCP-1 on MSCs homing to myocardial infarct site.Methods:MSCs from donor rat were cultured and labeled with BrdU. Fifty-six days after MI, different dose of MCP-1, SDF-1 or MCP-1+SDF-1 was injected into mid- and peri-infarcted zone evenly. Then, a total of 5×106 BrdU labeled MSCs in 2.5 mL of PBS were injected through the tail vein. The number of the labeled MSCs in the infarcted hearts was counted 3 days post injection. Cardiac function, expression of VEGF and blood vessel density were assessed 28 days post injection. Saline injection group was established as control.Results:The MSCs enrichment in the host hearts were more abundant in the MCP-1, SDF-1 injected groups than that in saline injected group, and the MSCs enrichment increased dose-dependently with the increase of the dose of MCP-1, SDF-1. Expression of VEGF and blood vessel density accorded with the MSCs enrichment. The MSCs enrichment and blood vessel density in MCP-1+SDF-1 injected group were more than those in MCP-1, SDF-1 injected groups. Cardiac function was improved more than 28 days before, but no difference was found among all the groups.Conclusions:There is no significantly autologous MSCs homing 56d after MI. SDF-1 and MCP-1 may enhance MSCs homing to injured heart dose-dependently. Homed MSCs may secrete VEGF and promote neovascularization. But homed MSCs fail to improve cardiac function, it may due to late therapy. The homing enhancement effect of SDF-1 plus MCP-1 is better than SDF-1 or MCP-1 respectively. Part IV: The effects of SDF-1 & MCP-1 on autologous bone marrow stem cells mobilized by G-CSF homing to myocardial infarct siteObjective:To investigate the effects of SDF-1 and MCP-1 on autologous bone marrow stem cells mobilized by G-CSF homing to myocardial infarct site.Methods:Fifty-six days after MI, 20μg·kg-1·d-1 recombinant human G-CSF was injected hypodermically in the rats for 5 days. Then 50μl MCP-1, SDF-1 or saline was injected into peri-infarct myocardium evenly. BrdU was injected peritoneally 50mg/Kg per day for 15days. 13 days later, the rats were sacrificed and the hearts were harvested for BrdU、CD45、CD90、VEGF、VIII detection. Cardiac function was assessed by Ultrasonic Cardiography before MCP-1, SDF-1 injection and rats sacrificed.Results:BrdU+, CD45+, CD90+ cell enrichment in the host hearts, expression of VEGF and blood vessel density were more in MCP-1, SDF-1 injected groups than saline injected group. CD45+ cell enrichment was more than that of CD90+ cell in MCP-1, SDF-1 injected groups. Cardiac function had no difference among all the three groups.Conclusions:SDF-1 and MCP-1 may enhance autologous bone marrow stem cells mobilized by G-CSF homing to injured heart. Homed bone marrow stem cells may secrete VEGF and promote neovascularization. But homed bone marrow stem cells fail to improve cardiac function, it may due to late therapy.更多还原