【作者】 李海清；

【导师】 赵强； 陈安清； 夏利民；

【作者基本信息】 复旦大学 ， 心脏外科， 2009， 博士

【摘要】 心肌梗死是心力衰竭的主要原因。一些临床研究显示绝经前的心力衰竭女性预后比相应年龄的男性好。而且,多中心有关心力衰竭试验的回顾性分析表明,使用雌激素的绝经女性心力衰竭的预后比未使用雌激素的绝经女性预后好,这些都提示雌激素对心血管系统具有保护作用。但随机临床试验显示雌激素会增加发生不良心血管事件的风险,使在绝经妇女中普遍使用雌激素替代治疗的方案受到质疑。然而,动物实验研究和流行病学研究强烈提示雌激素对心血管系统有益,使得雌激素对心血管系统的有益作用研究开始转向新的可能机制和途径,即近十年来始终是研究的热点之一:内皮祖细胞(endothelial progenitor cells,EPCs)。EPCs修复梗死心脏主要是通过促进心肌梗死部位新生血管形成,增加血流灌注,促进组织再生,限制梗死部位疤痕发展,延缓心室重塑,从而改善心功能。在冠心病和有患冠心病危险因素的患者,EPCs的数量及其迁移、增殖、归巢及血管新生等功能明显降低。因此,在EPCs移植治疗心肌梗死过程中,如何提高EPCs的归巢及其血管新生功能成为当前研究的焦点。基质细胞衍生因子-1α(stromal cell-derived factor-1α,SDF-1α)与其唯一受体CXCR4构成的SDF-1α/CXCR4轴在EPCs归巢及新生血管形成过程中起着重要作用。有关雌激素是否可通过作用于SDF-1α/CXCR4轴调节EPCs的归巢和血管新生功能及其作用机制,目前研究尚少。由于全身应用雌激素副作用大,且作用的组织细胞广泛,难以阐述清楚雌激素的具体作用机制。因此,本实验首先通过建立卵巢切除模型,初步研究生理性雌激素对骨髓源性内皮祖细胞(bonemarrow-derived endothelial progenitor cells,BM-EPCs)在体外向SDF-1α迁移和血管新生功能的影响及作用机制;然后在体外进一步研究雌激素预处理对BM-EPCs迁移和血管新生功能的影响及其作用机制;最后研究雌激素预处理BM-EPCs对其在移植治疗心肌梗死中体内归巢的影响,并研究雌激素预处理BM-EPCs在移植治疗心肌梗死中的作用及其机制。第一部分生理性雌激素对骨髓源性内皮祖细胞迁移及血管新生功能的影响[目的]研究生理性雌激素对骨髓源性内皮祖细胞(BM-EPCs)迁移及血管新生功能的影响及其机制。[方法]6周龄雌性BALB/C小鼠随机分成卵巢切除组、假手术组和正常组。分别于1周和4周后,ELISA法检测各组小鼠血清雌激素浓度。4周后,取胫骨和股骨骨髓,培养、鉴定BM-EPCs。Transwell小室检测各组BM-EPCs经或未经CXCR4抑制剂AMD3100处理后向基质细胞衍生因子-1α(SDF-1α)的迁移功能。血管生成试验检测各组BM-EPCs经或未经CXCR4抑制剂AMD3100处理后形成管样物的长度。RT-PCR、流式细胞术和Western blot检测各组BM-EPCs CXCR4的表达。[结果]卵巢切除1周和4周后,卵巢切除组小鼠血清雌激素浓度(23.09±4.01pmol/L)(20.75±2.75 pmol/L)均明显低于假手术组(894.53±71.98 pmol/L)(910.18±58.77 pmol/L)(P＜0.01)和正常组(867.52±77.08 pmol/L)(901.66±78.57pmol/L)(P＜0.01),而假手术组和正常组无显著差异(P＞0.05)。卵巢切除组BM-EPCs向SDF-1α迁移的数量(102.67±7.02/400倍视野)明显低于假手术组(172.00±9.17/400倍视野)(P＜0.01)和正常组(174.67±10.41/400倍视野)(P＜0.01),而假手术组和正常组无明显差异(P＞0.05)。经CXCR4抑制剂AMD3100处理后,卵巢切除组、假手术组和正常组向SDF-1α迁移的数量均明显减少(55.33±5.51/400倍视野vs 102.67±7.02/400倍视野)(57.00±4.58/400倍视野vs172.00±9.17/400倍视野)(60.00±5.00/400倍视野vs 174.67±10.41/400倍视野)(P＜0.01),但组间比较没有显著差异(P＞0.05)。卵巢切除组BM-EPCs形成管样物的长度(5386±405μm/50倍视野)明显低于假手术组(7768±466μm/50倍视野)(P＜0.01)和正常组(7514±547μm/50倍视野)(P＜0.01),而假手术组和正常组无显著差异(P＞0.05)。正常组BM-EPCs经CXCR4抑制剂AMD3100处理后,形成管样物的长度明显降低(3292±310μm/50倍视野vs 7514±547μm/50倍视野)(P＜0.01)。卵巢切除组BM-EPCs CXCR4的表达明显低于假手术组和正常组(P＜0.01),而假手术组和正常组无显著差异(P＞0.05)。[结论]生理性雌激素通过调节BM-EPCs功能性CXCR4的表达而增强其迁移及血管新生功能。第二部分雌激素预处理提高骨髓源性内皮祖细胞迁移及血管新生功能的体外实验研究[目的]研究体外雌激素预处理对骨髓源性内皮祖细胞(BM-EPCs)迁移及血管新生功能的影响及其机制。[方法]6周龄雌性BALB/C小鼠卵巢切除4周后,取胫骨和股骨骨髓,培养、鉴定BM-EPCs。分别采用0nmol/L、1nmol/L、10nmol/L、100nmol/L 17β-雌二醇或相应浓度17β-雌二醇和雌激素受体拮抗剂ICI182 780与BM-EPCs共培养。48小时后,Transwell小室检测各组BM-EPCs经或未经CXCR4抑制剂AMD3100处理后向基质细胞衍生因子-1α(SDF-1α)的迁移功能。血管生成试验检测BM-EPCs经或未经AMD3100处理后形成管样物的长度。RT-PCR、流式细胞术和Western blot检测各组BM-EPCs CXCR4的表达。[结果]17β-雌二醇呈剂量依赖性增强BM-EPCs的迁移及CXCR4的表达(P＜0.05),但被雌激素受体拮抗剂ICl182 780完全阻断(P＞0.05)。同样,17β-雌二醇明显增强BM-EPCs的血管新生功能(P＜0.05),但被雌激素受体拮抗剂ICI182780完全阻断(P＞0.05)。经CXCR4抑制剂AMD3100处理后,17β-雌二醇预处理的BM-EPCs的迁移和血管新生功能均明显受损(P＜0.05)。[结论]雌激素通过雌激素受体途径上调BM-EPCs功能性CXCR4的表达而增强其迁移及血管新生功能。第三部分雌激素预处理骨髓源性内皮祖细胞提高细胞移植治疗急性心肌梗死疗效的体内实验研究[目的]研究雌激素预处理骨髓源性内皮祖细胞(BM-EPCs)对其体内归巢及治疗急性心肌梗死疗效的影响。[方法]6周龄雌性BALB/C小鼠切除卵巢4周后,结扎前降支建立心肌梗死模型,RT-PCR及免疫组化检测心肌梗死前和心肌梗死后1d、3d、7d、14d、21d心肌梗死区基质细胞衍生因子-1α(SDF-1α)的表达。心肌梗死后3d小鼠分成3组,分别经尾静脉注射经17β-雌二醇预处理的BM-EPCs、未经17β-雌二醇预处理的BM-EPCs和生理盐水作为空白对照。BM-EPCs移植后4d、11d,MRI实时示踪和普鲁士蓝染色分析心肌梗死区BM-EPCs的归巢量。BM-EPCs移植后25d,超声心动图检测3组小鼠的左室收缩末内径(LVDs)、左室舒张末内径(LVDd)、缩短分数(FS)和射血分数(EF);免疫组化检测3组小鼠心肌梗死区新生血管密度;Masson染色检测3组小鼠左室纤维化面积与左室总面积的百分比。[结果](1)心肌梗死后1d,SDF-1α表达即明显增高;在心肌梗死后3d达高峰;心肌梗死后7d仍维持在较高水平(P＜0.01)。但在心肌梗死后14d、21d,SDF-1α表达明显降低,与正常心肌中SDF-1α表达水平无明显差异(P＞0.05)。(2)BM-EPCs移植后4d、11d,17β-雌二醇预处理组心肌梗死区中归巢的BM-EPCs数量明显高于未经17β-雌二醇预处理组(P＜0.05)。(3)BM-EPCs移植后25d,17β-雌二醇预处理组小鼠心室扩张程度明显轻于空白对照组(LVDs:3.09±0.05 vs3.27±0.10 mm,P＜0.05:LVDd:4.18±0.07 vs 4.3±0.05 mm,P＜0.05),未经17β-雌二醇预处理组小鼠心室扩张程度与空白对照组无明显差异(LVDs:3.18±0.07 vs 3.27±0.10 mm,P＞0.05:LVDd:4.24±0.06 vs 4.31±0.05 mm,P＞0.05)。17β-雌二醇预处理组小鼠的心功能也好于空白对照组(FS,%:33±3.8 vs26±3.2,P＜0.05),未经17β-雌二醇预处理组小鼠的心功能与空白对照组无明显差异(FS,%:28±4.7 vs 26±3.2,P＞0.05)。虽然17β-雌二醇预处理组和未经17β-雌二醇预处理组小鼠EF值分别比对照组增加了4.72%和3.29%,但3组小鼠的EF值无统计学差异(P＞0.05)。(4)17β-雌二醇预处理BM-EPCs移植组心肌梗死区域新生血管密度明显高于对照组(1428±214/mm~2 vs 1070±168/mm~2,P＜0.05),未经17β-雌二醇预处理BM-EPCs移植组心肌梗死区域新生血管密度与对照组无显著差异(1214±157/mm~2 vs 1070±168/mm~2,P＞0.05)。(5)经17β-雌二醇预处理BM-EPCs移植组左室纤维化面积占整个左室面积的百分比明显低于对照组(8.8±4.9%vs 49.0±4.6%,P＜0.05),而未经17β-雌二醇预处理组与对照组无显著差异(41.6±5.2%vs 49.0±4.6%,P＞0.05)。[结论](1)急性心肌梗死后1周内,心肌梗死区中SDF-1α表达水平处于较高水平,为BM-EPCs移植最佳时期。(2)雌激素预处理可增强BM-EPCs归巢,促进心肌梗死区新生血管形成,减轻心室重塑,从而改善心功能。更多还原

【Abstract】 Myocardial infarction has been a major cause of heart failure.Several studies demonstrated that premenopausal women with heart failure have a better prognosis than age-matched men.Whether endogenous sex hormones contribute to these differences in prognosis remains unknown.However,observational studies have demonstrated that postmenopausal women taking estrogen after a myocardial infarction have a lower incidence of heart failure.Furthermore,retrospective analysis of multicenter heart failure trials have shown that postmenopausal women taking estrogen have a better prognosis than women not on estrogen,supporting that estrogen may have beneficial effects on cardiovascular system.Hormone replacement therapy(HRT) in postmenopausal women was the accepted standard of care for many years until a randomized clinical trial showed that HRT actually increased the risk of cardiac events.Nevertheless,extensive experimental and epidemiological evidence indicates that estrogen has a protective role against cardiovascular system,which connects current studies about effects of estrogen on cardiovascular system with endothelial progenitor cells(EPCs) being a research focus for ten years.Experimental studies from several laboratories have reported that EPCs repair infarcted heart primarily through inducing neovascularization,resulting in enhancing perfusion,contributing to regeneration,confining extension of scar,inhibiting left ventricular remodeling and improving cardiac function.However,EPCs from patients with coronary artery disease and with risk factors for coronary artery disease show a lower number and impaired functions including migration,proliferation,homing, angiogenic activity and so on,compared with healthy volunteers.Accordingly,in an effort to enhance EPCs-mediated cardiovascular reparative benefits,current studies are focused on how to improve homing and neovascularization of EPCs.Stromal cell-derived factor-1αexclusively binds to CXCR4 and has CXCR4 as its only receptor.The SDF-1α/CXCR4 axis plays a pivotal role in homing and neovascularization of EPCs.Now,it is still not clear that whether estrogen mediates the homing and angiogenesis of EPCs by regulating SDF-1α/CXCR4 axis.Because of adverse reaction due to systemically applying estrogen and wide tissues and cells being targeted by estrogen,the effects and molecular mechanisms of estrogen on EPCs is hard to be clarified.Therefore,first,in this experiment,we investigated the effects and mechanisms of physiological estrogen on the migratory and angiogenic activity of bone marrow-derived endothelial progenitor cells(BM-EPCs) through establishing ovariectomized mouse models.Second,the effects and specific mechanisms of 17β-estradiol preconditioning in vitro on the migratory and angiogenic capacity of BM-EPCs were further illuminated.Finally,we studied the effects of 17β-estradiol preconditioning on homing of BM-EPCs in vivo and deciphered the effects and mechanisms of 17β-estradiol preconditioned BM-EPCs-mediated cardiac repair after acute myocardial infarction.PartⅠEffects of physiological estrogen on the migratory and angiogenic capacity of bone marrow-derived endothelial progenitor cellsObjective To investigate the effects and mechanisms of physiological estrogen on the migratory and angiogenic capacity of bone marrow-derived endothelial progenitor cells(BM-EPCs).Methods Six weeks female BALB/C mice were randomly divided into ovariectomized group,sham operative group and normal group.ELISA was taken to measure serum levels of estrogen in each group 1 and 4 weeks after observation.4 weeks later,we cultured and identified BM-EPCs,assessed migratory capacity of BM-EPCs of each group toward stromal cell-derived factor-1α(SDF-1α) with or without treatment of CXCR4 inhibitor AMD3100 by Transwell chamber,measured tube length of BM-EPCs of each group with or without AMD3100,and detected expression of CXCR4 by RT-PCR,fluorescence-activated cell sorting(FACS),and Western blotting.Results First,serum levels of estrogen in the ovariectomized group 1 and 4 weeks after ovariectomy were both significantly lower than sham operative group and normal group(23.09±4.01 pmol/L vs 894.53±71.98 pmol/L,23.09±4.01 pmol/L vs 867.52±77.08 pmol/L,respectively,after 1 week,P＜0.01;20.75±2.75 pmol/L vs 910.18±58.77 pmol/L,20.75±2.75 pmol/L vs 901.66±78.57 pmol/L,respectively, after 4 weeks,P＜0.01),but there were no difference between sham and normal group (P＞0.05).Second,the number of BM-EPCs migrated toward SDF-1αin ovariectomized group(102.67±7.02 per high power field) was significantly decreased than in sham operative group(172.00±9.17 per high power field;P＜0.01) and in normal group(174.67±10.41 per high power field;P＜0.01),respectively.However, there were no difference between sham and normal group(P＞0.05).After administration of AMD3100,the number of BM-EPCs migrated to SDF-1αin each group was significantly reduced(ovariectomized group,55.33±5.51 vs 102.67±7.02 per high power field,P＜0.01;sham group,57.00±4.58 vs 172.00±9.17 per high power field,P＜0.01;normal group,60.00±5.00 vs 174.67±10.41 per high power field,P＜0.01),but there was no difference between them(P＞0.05).Third,tube length of BM-EPCs in ovariectomized group(5386±405μm per power field) significantly decreased than in sham operative group(7768±466μm per power field; P＜0.01) and in normal group(7514±547μm per power field;P＜0.01),but there were no difference between sham group and normal group(P＞0.05).After treatment with AMD3100,the tube length of BM-EPCs in normal group was significantly impaired(3292±310μm with AMD3100 vs 7514±547μm per power field without AMD3100;P＜0.01).Finally,CXCR4 expression of BM-EPCs from ovariectomized group significantly decreased than sham operative group and normal group, respectively(P＜0.01).However,the difference was not shown between sham group and normal group(P＞0.05).Conclusion Physiological estrogen improves migratory and angiogenic capacity of BM-EPCs by up-regulating functional CXCR4 expression.PartⅡIn vitro experimental study of estrogen preconditioning for enhancing the migratory and angiogenic activity of bone marrow-derived endothelial progenitor cellsObjective To investigate the effects and mechanisms of estrogen preconditioning in vitro on the migratory and angiogenic capacity of bone marrow-derived endothelial progenitor cells(BM-EPCs).Methods 6 weeks aged BALB/C mice were ovariectomized and 4 weeks later, BM-EPCs were cultured and identified from ovariectomized BALB/C mice tibia and femur.After 48 hours coculture with 0 nmol/L,1 nmol/L,10 nmol/L,100 nmol/L 17β-estradiol,or,corresponding concentration of 17β-estradiol and estrogen receptors antagonist ICI182 780,BM-EPCs migratory activity toward stromal cell-derived factor-1αwere assessed by transwell chamber with or without treatment of CXCR4 inhibitor AMD3100,angiogenic capacity of BM-EPCs was evaluated by measuring tube length also with or without administration of AMD3100,and CXCR4 expression of BM-EPCs were detected by RT-PCR,FACS,and Western blotting.Results Migratory activity and CXCR4 expression of BM-EPCs were increased by 17β-estradiol in a dose-dependent manner(P＜0.05),however,these effects were completely blocked by ICI182 780(P＞0.05).17β-estradiol prominently enhances angiogenic capacity of BM-EPCs which was fully blocked by ICI182 780 as well. After administration of AMD3100,both migratory and angiogenic activity of BM-EPCs preconditioned with 17β-estradiol were significantly impaired(P＜0.05).Conclusion Estrogen enhances migratory and angiogenic activity of BM-EPCs by up-regulating functional CXCR4 expression via estrogen receptors pathway.PartⅢIn vivo experimental study of estrogen preconditioned bone marrow-derived endothelial progenitor cells transplanted for enhancing recovery after acute myocardial infarctionObjective To investigate the effects of estrogen preconditioning on homing and treatment of bone marrow-derived endothelial progenitor cells(BM-EPCs) transplanted for infarcted myocardium.Methods Female BALB/C mice aged six weeks were ovariectomized.Four weeks later,acute myocardial infarction models were established by ligating the anterior descending(LAD) branch of the left coronary artery.RT-PCR and immunohistochemistry were taken to detect the expression of stromal cell-derived factor-1αin myocardial infarcted area just before myocardial infarction and 1,3,14, 21days after myocardial infaction.3 Days after myocardial infarction,mice were randomly divided into three groups,and each group received 17β-estradiol preconditioned BM-EPCs,non-17β-estradiol preconditioned BM-EPCs,and saline as control through tail vein,respectively.4 and 11 days after BM-EPCs transplantation, MRI trafficking and Prussian staining were used to quantify BM-EPCs homed to infarcted myocardium.25 Days after BM-EPCs transplantation,transthoracic echocardiography was performed to measure left ventricular systolic(LVDs) and diastolic(LVDd) dimensions,fractional shortening(FS) and ejection fraction(EF).At the same time,immunohistochemistry and Masson’s trichrome staining were used to assess the capillary density and the average ratio of fibrosis area to total left ventricular area,respectively.Results(1) SDF-1αexpression in infarcted myocardium area showed a higher level just 1 day after myocardial infarction,reached the highest level 3 days after myocardial infarction,and still maintained at a higher level 7 days after myocardial infarction(P＜0.01).However,14 and 21 days after myocardial infarction,expression of SDF-1αsignificantly decreased and had no statistic significance compared with in normal myocardium(P＞0.05).(2) Quantity of homed BM-EPCs in infarcted myocardium of 17β-estradiol preconditioned group is larger than of non-17β-estradiol preconditioned group(P＜0.01).(3) 25 Days after transplantation,echocardiography revealed less ventricular dilation in 17β-estradiol preconditioned group versus controlled group(LVDs:3.09±0.05 vs 3.27±0.10 mm,P＜0.05;LVDd,4.18±0.07 vs 4.3±0.05 mm,P＜0.05),but there were no significance between non-17β-estradiol preconditioned group versus controlled group(LVDs:3.18±0.07 vs 3.27±0.10 mm,P＞0.05;LVDd:4.24±0.06 vs 4.31±0.05 mm,P＞0.05).LV function was also significantly better in 17β-estradiol preconditioned group versus controlled group(FS, %:33±3.8 vs 26±3.2,P＜0.05),however,LV function of non-17β-estradiol preconditioned group was not significantly improved compared with of control(FS, %:28±4.7 vs 26±3.2,P＞0.05).Although EF value of 17β-estradiol and non-17β-estradiol preconditioned group increased 4.72%and 3.29%than of controlled group,respectively,there were no significance between them(P＞0.05).(4) Capillary density 25days after BM-EPCs transplantation in infarcted myocardium of 17β-estradiol preconditioned group was significantly greater than of controlled group (1428±214/mm~2 vs 1070±168/mm~2,P＜0.05).However,capillary density was similar between non-17β-estradiol preconditioned group and controlled group (1214±157/mm~2 vs 1070±168/mm~2,P＞0.05).(5) 25 Days after BM-EPCs transplantation,the area of left ventricular fibrosis was significantly less in 17β-estradiol preconditioned group than in controlled group(8.8±4.9%vs 49.0±4.6%, P＜0.05).Nevertheless,the area of left ventricular fibrosis of non-17β-estradiol preconditioned group had no significance compared with control(41.6±5.2%vs 49.0±4.6%,P＞0.05).Conclusions(1) During 1 week after acute myocardial infarction is optimal period for BM-EPCs transplantation for treating ischemic heart disease for SDF-1αexpression in infarcted myocardium showing a higher level.(2) estrogen preconditioning enhances homing of BM-EPCs which contributes to neovascularization,attenuating left ventricular remodeling,and improving cardiac function.更多还原