【作者】 张坡；

【导师】 黄岚；

【作者基本信息】 第三军医大学 ， 内科学， 2007， 博士

【摘要】 研究背景内皮细胞损伤和功能不全是血管损伤后有害修复、动脉粥样硬化的始动因素和根本原因,平滑肌细胞增生迁移是其继发因素和外在表现。修复损伤血管的关键在于抑制平滑肌细胞过度增生与促进损伤内皮重建。既往关于增殖性血管疾病治疗的研究热点主要集中于平滑肌细胞增生机制的探讨及如何有效抑制平滑肌细胞过度增生。雷帕霉素和紫杉醇药物洗脱支架是临床防治损伤血管过度的有效手段,但往往会干扰内皮修复,引起损伤血管段再内皮化不全或延迟及迟发性支架内血栓形成。因此,筛选选择性抑制新生内膜增生不影响甚至损伤血管再内皮化的洗脱支架包被药物是新一代洗脱支架的研制方向。既往认为,参与损伤血管修复的细胞成分是血管壁平滑肌细胞和内皮细胞。近来研究发现,骨髓中存在血管祖细胞——平滑肌祖细胞(smooth muscle progenitor cells, SPCs)和内皮祖细胞(endothelial progenitor cells, EPCs)。血管损伤后这些细胞可动员入外周血,归巢于损伤血管段,平滑肌祖细胞分化为平滑肌细胞,促进新生内膜生成,同时可导致管腔狭窄;内皮祖细胞分化为内皮细胞,重建管腔内皮细胞单层,抑制损伤血管有害重构。最近动物实验和临床研究均证实新生内膜中约一半以上平滑肌细胞是骨髓来源的,约25%内皮细胞是内皮祖细胞来源的。说明骨髓源平滑肌祖细胞和内皮祖细胞是参与损伤血管修复的重要细胞源。因此,抑制平滑肌祖细胞增殖可减少血管损伤后新生内膜过度增生,促进内皮祖细胞增殖,有助于损伤血管再内皮化,抑制新内膜发展。他汀类药物具有良好的调脂作用,能够降低低密度脂蛋白胆固醇和升高高密度脂蛋白胆固醇。同时,在胆固醇水平相当的情况下,他汀类药物能显著降低心血管事件的发生率。此外,他汀类药物还具有抑制血小板聚集和血管中膜平滑肌细胞增殖,改善内皮功能,抑制炎症反应及增加斑块稳定性作用。新近研究显示他汀类药物能增加内皮祖细胞数量、改善内皮祖细胞功能。他汀类药物的这些生物学效应提示其有可能成为理想的第二代洗脱支架药物。由于损伤位点有效药物浓度过低,临床口服他汀类药物并不能抑制支架内再狭窄。局部用药有靶区有效血药浓度高、作用时间长和全身毒性低优点。尚不清楚局部应用他汀类药物能否有效抑制新生内膜增生,同时加速损伤区再内皮化。研究目的:1.观察临床广泛应用的洗脱支架包被药物雷帕霉素对内皮祖细胞和平滑肌祖细胞分化、增殖、迁移和黏附的影响;2.观察辛伐他汀对血管内皮细胞、平滑肌细胞、内皮祖细胞和平滑肌祖细胞分化、增殖、迁移和黏附的影响。3.观察辛伐他汀对平滑肌细胞表达平滑肌祖细胞趋化因子SDF-1α的影响;4.建立大鼠颈动脉球囊损伤模型,观察局部应用辛伐他汀能否抑制损伤血管新生内膜过度增生并促进损伤血管再内皮化,为应用他汀类药物包被洗脱支架提供实验依据。实验方法1.观察雷帕霉素对内皮祖细胞和平滑肌祖细胞分化、增殖、迁移和黏附的影响:采用密度梯度离心法从大鼠骨髓获取单个核细胞,分别重悬于平滑肌祖细胞和内皮祖细胞培养基中,接种在纤维连接素包被培养板,加入不同浓度雷帕霉素(0,0.01,0.1,1,10,100 ng/ml),培养12天后,α-SMA和CD34免疫荧光染色鉴定骨髓源性平滑肌祖细胞,荧光显微镜鉴定FITC-UEA-Ⅰ和Di I-acLDL双染阳性细胞为正在分化的内皮祖细胞,并在倒置荧光显微镜下计数。收集原代培养8天贴壁细胞,分别加入不同浓度雷帕霉素(0,0.1,1,10,100,200 ng/ml)培养0、12、24、48、96 h。然后分别采用MTT比色法、改良的Boyden小室和黏附能力测定实验观察内皮祖细胞和平滑肌祖细胞的增殖、迁移和黏附能力。2.观察辛伐他汀对血管内皮细胞、平滑肌细胞、内皮祖细胞和平滑肌祖细胞分化、增殖、迁移和黏附的影响:分别采用酶消化法和组织块法分离培养大鼠血管内皮细胞和平滑肌细胞,加入不同浓度辛伐他汀(0,0. 01,0.1,1,10μmol/L)培养0、6、12、24、48 h,然后分别采用3H-TdR掺入法、改良的Boyden小室和黏附能力测定实验观察内皮细胞和平滑肌细胞的增殖、迁移能力。采用前述方法分离培养内皮祖细胞和平滑肌祖细胞,将其接种在纤维连接素包被培养板,加入不同浓度辛伐他汀(0,0. 01,0.1,1,10μmol/L),培养8 d,前述方法鉴定内皮祖细胞和平滑肌祖细胞,倒置荧光显微镜下计数。收集原代培养8 d的内皮祖细胞和平滑肌祖细胞,分别加入不同浓度辛伐他汀(0,0. 01,0.1,1,10μmol/L)培养0、6、12、24、48 h。然后分别采用3H-TdR掺入法、改良的Boyden小室和黏附能力测定实验观察内皮祖细胞和平滑肌祖细胞的增殖、迁移和黏附能力。Western blot检测不同浓度辛伐他汀(0,0. 01,0.1,1,10μmol/L)作用24小时对内皮细胞、平滑肌细胞、内皮祖细胞和平滑肌祖细胞周期抑制蛋白P27表达的影响。3.观察辛伐他汀对平滑肌细胞表达平滑肌祖细胞趋化因子SDF-1α的影响:不同浓度辛伐他汀(0,0. 01,0.1,1,10μmol/L)干预平滑肌细胞24 h,1μmol/L辛伐他汀干预平滑肌细胞6～48 h ,RT-PCR检测SDF-1αmRNA表达。4.局部应用辛伐他汀对损伤血管新生内膜过度增生和再内皮化影响:建立大鼠颈动脉球囊损伤模型,将400μg辛伐他汀溶于50μl F-127基质胶中,加在颈动脉外膜区域模拟洗脱支架。2周后,处死动物,取目标血管段,固定,石蜡包埋切片,HE染色观察新生内膜厚度,vWF染色观察损伤血管再内皮化程度。主要结果1.雷帕霉素对内皮祖细胞和平滑肌祖细胞分化、增殖、迁移和黏附的影响:成功分离培养骨髓源内皮祖细胞和平滑肌祖细胞。内皮祖细胞vWF染色阳性,UEA-Ⅰ和DiLDL双染色阳性,可见梭形细胞首尾相连形成线状结构。平滑肌祖细胞α-SMA、CD34染色阳性。雷帕霉素浓度依赖性抑制骨髓单个核细胞向内皮祖细胞和平滑肌祖细胞分化。0.1 ng/ml的雷帕霉素作用12天,分化EPC数量减少59.3±5.8%(n=5,P<0.01),平滑肌祖细胞数量减少70.5±34.5%(n=5,P<0.01)。雷帕霉素浓度和时间依赖性抑制内皮祖细胞和平滑肌祖细胞增殖。1 ng/ml作用24 h显著抑制EPC (1 ng/ml雷帕霉素组与对照组之比为0.418±0.018 vs 0.580±0.034,n=5,P<0.01)和SPC(1 ng/ml雷帕霉素组与对照组之比为0.476±0.016 vs 0.687±0.043,n=5,P<0.01)增殖。1 ng/ml雷帕霉素作用12 h对EPC和SPC增殖无明显影响(n=5,P>0.05)。0.1 ng/ml雷帕霉素对EPC和SPC增殖影响不明显(n=5,P>0.05)。雷帕霉素也浓度依赖性抑制EPC和SPC迁移和黏附能力。2.辛伐他汀对血管内皮细胞、平滑肌细胞、内皮祖细胞和平滑肌祖细胞分化、增殖、迁移和黏附的影响:辛伐他汀浓度和时间依赖性抑制平滑肌细胞增殖和迁移。0.01μmol/L辛伐他汀即可抑制平滑肌细胞增殖(0.01μmol/L辛伐他汀组与对照组之比为5647±268 vs 6038±218,n=5,P<0.05)和迁移(0.01μmol/L辛伐他汀组与对照组之比为41±3 vs 45±3,n=5,P<0.05)。0.01～10μmol/L的辛伐他汀不影响内皮细胞增殖(10μmol/L辛伐他汀组与对照组之比为4310±132 vs 4321±133,n=5,P>0.05)和迁移(10μmol/L辛伐他汀组与对照组之比为35±5 vs 37±5,n=5,P>0.05)。辛伐他汀浓度依赖性抑制骨髓单个核细胞向平滑肌祖细胞分化,促进其向内皮祖细胞分化。1μmol/L辛伐他汀作用24小时分化平滑肌祖细胞减少62.1±3.5%(1μmol/L辛伐他汀与对照组之比为32±5 vs 85±4,n=5,P<0.01),内皮祖细胞增加1.2±0.1倍(1μmol/L辛伐他汀与对照组之比为87±5 vs 39±4,n=5,P<0.01)。辛伐他汀浓度和时间依赖性促进内皮祖细胞增殖,最大作用剂量1.0μmol/ L,24 h达作用高峰。浓度和时间依赖性抑制平滑肌祖细胞增殖,1.0μmol/ L辛伐他汀作用24小时,内皮祖细胞数量增加2.2±0.1倍(1μmol/L辛伐他汀与对照组之比为3762±138 vs 1249±146,n=5,P<0.01),平滑肌祖细胞数量减少62.1±5.6%(1μmol/L辛伐他汀与对照组之比为1962±145 vs 4070±184,n=5,P<0.01)。辛伐他汀也抑制平滑肌祖细胞迁移和黏附,促进内皮祖细胞迁移和黏附。辛伐他汀浓度依赖性促进平滑肌细胞和平滑肌祖细胞周期抑制蛋白P27表达,抑制内皮祖细胞P27表达,不影响内皮细胞P27表达。3.辛伐他汀对平滑肌细胞表达平滑肌祖细胞趋化因子SDF-1α的影响:辛伐他汀浓度和时间依赖性抑制平滑肌细胞SDF-1αmRNA表达。4.局部应用辛伐他汀对大鼠损伤血管新生内膜过度增生和再内皮化影响:局部应用辛伐他汀抑制大鼠损伤颈动脉新生内皮过度增生,对照组与辛伐他汀组新生内膜面积之比为0.58±0.13 mm~2 vs 0.27±0.12 mm~2 (n=6,P<0.01),辛伐他汀组内膜/中膜比减少(对照组与辛伐他汀组之比为1.95±0.27 vs 0.97±0.24,n=6,P<0.01)。内皮细胞vWF免疫组化染色未观察到对照组和辛伐他汀组再内皮化差异。全文结论1.雷帕霉素时间和浓度依赖性抑制骨髓源平滑肌祖细胞分化、增殖、迁移和黏附;雷帕霉素也减少骨髓源内皮祖细胞数量和抑制其功能。寻找新的洗脱支架药物需考虑其对平滑肌祖细胞和内皮祖细胞的影响。2.辛伐他汀选择性促进骨髓单个核细胞向内皮祖细胞分化,促进内皮祖细胞增殖、迁移和黏附,抑制其向平滑肌祖细胞分化。抑制平滑肌细胞和平滑肌祖细胞增殖、迁移和黏附,不影响内皮细胞增殖和迁移。3.辛伐他汀浓度和时间依赖性抑制平滑肌细胞SDF-1αmRNA表达,间接抑制平滑肌祖细胞参与的新生内膜增生。4.局部应用辛伐他汀能有效抑制损伤动脉新内膜过度增生,不影响损伤血管段再内皮化。更多还原

【Abstract】 BACKGROUNDThe injury and dysfunction of endothelial cells play a key role in the starting events of atherosclerasis and harmful renovation and induce vascular injuries, proliferation and migration of smooth muscle cells(SMCs). To inhibit smooth SMCs proliferation and accelerate reendothelialization is the key for the repairment of injured vessel. In the past decades, intense efforts has been applied to discern the mechanisms that regulate SMCs proliferation after angioplasty and to develop therapies to inhibit SMCs overgrowth. Drug-eluting stents that employing cytostatic/immunomodulatory compound rapamycin and the chemotherapeutic paclitaxcel have been show to induce neointimal lesion formation. These therapies can also perturb endothelial recovery, and lead to incomplete, delayed reendothelialization or late stent thrommosis. Accordingly, identification of compounds that select to inhibit neointimal formation, as well as do not adversely affect endothelial regrowth could be of substantial clinical usefulness and is regarded as the main target for evolutionary concepts toward the development of next generation drug-eluting stents.In traditional opinions, it is thought that vascular SMCs and ECs were the predominant cells which take part in repairment of injured vessel. Recent studies found that vascular function not only depends on cells within the vessels, but also significantly modulated by circulating smooth muscle progenitor cells(SPCs) and endothelial progenitor cells(EPCs) derived from the bone marrow. Following vascular injuries, vascular progenitor cells can be mobilized into the peripheral blood flow, home to the site of vascular injury, and SPCs can differentiate into smooth muscle cells and accelerate neointima formation and lead to luminal stenosis; and EPCs differentiate into endothelial cells and rebuild luminal endothelial cells layer and inhibit bad vascular remodeling. Recent animal and clinic studies both showed that the most of neointima SMCs were derived from bone marrow and about 25% ECs at the vascular injury site came from the differentiated EPCs. These data indicate that SPCs and EPCs derived from bone marrow are important sources of repairing cells after vascular injury. Therefore, inhibition of SPCs proliferation can induce neointima formation, and improvement of EPCs proliferation can accelerate reendothelialization of injured vessel and inhibit restenosis.Statins can effectively modulate the lipid profile of an individual patient. Moreover, there is compelling evidence that statins may also exhibit non cholesterol dependent pleiotropic effects, such as inhibiting SMCs poliferation, promoting EPCs proliferation, improving endothelial function, inhibiting platelet function, anti-inflammation and stabilizing atherosclerotic plaques. These biological properties of statins suggest that it can be a perfect candidate for coat compound of next generation drug-eluting stents. However, systemic statin therapy is not effective for the limitation of human coronary artery neointima formation because bioactive concentrations were neither achieved nor maintained at the site of the potential lesion. Local delivery of vasculature facilitates the achievement of high regional drug concentration, with prolonged retention at therapeutic doses less likely to product systemic toxicity. Nevertheless, it remains unknown whether local statin therapy can effectively inhibit restenosis and accelerate endothelial regrowth.OBJECTIVES1. To investigate effects of sirolimus on the differentiation, proliferation, adhension, and migration of EPCs and SPCs.2. To study effects of simvastin on EPCs and SPCs differentiation, in addition, on proliferation, adhension, and migration of ECs, SMCs, EPCs and SPCs.3. To explore effects of simvasation on SMCs SDF-1αm RNA expression.4. To study effects of locally delivered simvastatin on neointima formation and reendothelialization after vascular injury.METHODS1. Effects of sirolimus on EPCs and SPCs differentiation, proliferation, adhension, and migration: (1) The bone marrow mononuclear cells(MNCs) were isolated from the bone marrow of rats by density gradient centrifugation with Ficoll. MNCs were cultured in fibronectin-coated dishes in endothelial progenitor cells growth medium or smooth muscle progenitor cells growth supplements with or without sirolimus(final concentrations: 0.01, 0.1, 1, 10, 100 ng/ml) for 12 days. (2) After 8 days primarily cultured, attached cells were treated with sirolimus(final concentrations: 0.1, 1, 10, 100, 200 ng/ml) or vehicle for various time points(0h, 12h, 24h, 48h, 96h). SPCs were identified as adherent cells positive forα-SMA by indirect immunofluorescent staining. And EPCs were characterized as adherent cells double positive stained for DiI-acLDL-uptake and FITC-UEA-Ⅰ(lectin) binding under immunofluence microscope by direct fluorescent staining. EPCs and SPCs proliferation, migration were assayed with MTT assay and modified Boyden chamber assay respectively. Adhension assay of EPCs and SPCs were performed by replating them on fibronectin-coated dishes, and the adhension cells were then courted.2. To study effects of simvastin on EPCs and SPCs differentiation, in addition, on proliferation, adhension, and migration of ECs, SMCs, EPCs and SPCs: Rat aorta SMCs were cultured in high-glucose DMEM medium and Rat aorta ECs were cultured in M199 medium. EPCs and SPCs were cultured based on above meathod. Treatment with simvastatin was performed in fully supplemented media for the indicated time(0h, 6h, 12h, 24h, 48 h) and concentration ranges(0,0. 01,0.1,1,10μmol/L). The proliferation of SMCs, ECs, EPCs and SPCs were assayed with 3H-TdR incorporation assay. Migration and adhension assay of SMCs, ECs, EPCs and SPCs were performed as same as above procedure. In addition, western blot was performed for the assessment of effects of a series concentration of simvastatin(0,0. 01,0.1,1,10μmol/L) on cyclin-dependent kinase inhibitor p27 protein expression of ECs, SMCs, EPCs and SPCs for 24 hours.3. To explore effects of simvasation on SMCs SDF-1αm RNA expression: SMCs were treated with a range concentration of simvastatin(0,0. 01,0.1,1,10μmol/L) for 24h or with 1μmol/L simvastatin for 0h, 6h, 12h, 24h, 48 h. SDF-1αmRNA expression was detected by RT-PCR.4. To study effects of locally delivered simvastatin on neointima formation and reendothelialization after vascular injury: the model of vascular restenosis established by balloon injury of rat carotid arteries was used. 400μg of simvastatin suspended in 100μL of Pluronic F-127 gel was administrated to the perivascular area of injured vessel, and the same volume of Pluronic gel without simvastatin was used in control group. Two weeks after surgery, rats were sacrificed. The common carotid arteries were embedded in paraffin, and cross sections were made. Sections were stained with hematoxylin-eosin for assessement of Neointima thickness and with vWF antibody for observation of endothelial coverage.RESULTS1. Effects of sirolimus on EPCs and SPCs differentiation, proliferation, adhension, and migration: EPCs and SPCs were successfully cultured in vitro. EPCs were doubly stained by DiI-acLDL and FITC-UEA-Ⅰwith“line”structure. SPCs were positive forα-SMA and CD34 and mRNA forα-SMA was detected in adherent SPCs. The number ofα-SMA-positive SPCs and DiI-acLDL and FITC-UEA-Ⅰ-double-positve EPCs differentiated from MNCs at 12 days was significantly lower in sirolimus treated cells than that of vehicle-treated cells in dose-depended manner. At a concentration as low as 0.1 ng/mL, sirolimus dramatically reduced the number of SPCs to 59.3±5.8% of control(0.1 ng/mL sirolimus versus control: 23±3 versus 78±5, n=5,P<0.01) and reduced the number of EPCs to 70.5±34.5%(0.1 ng/mL sirolimus versus control: 37±5 versus 90±7, n=5,P<0.01). Sirolimus significantly inhibited the proliferative capacity of EPCs and SPCs in a time and dose dependent manner. There was no significant effects on EPCs and SPCs proliferation by sirolimus at 0.1 ng,mL, whereas at higher concentration(≥1 ng/mL), sirolimus inhibited the proliferation of EPCs(1 ng/mL sirolimus versus control: 0.414±0.019 versus 0.580±0.034, n=5,P<0.01) and SPCs(1 ng/mL sirolimus versus control: 0.476±0.016 versus 0.687±0.043, n=5,P<0.01). Moreover, sirolimus also significantly dose-dependently inhibited the migratory and adhensive capacity of EPCs and SPCs.1ng/mL sirolimus significantly inhibited EPCs adhension(1 ng/mL sirolimus versus control: 34±5 versus 52±6, n=5,P<0.01) and SPCs adhension(1 ng/mL sirolimus versus control: 27±2 versus 51±3, n=5,P<0.01).2. To study effects of simvastin on EPCs and SPCs differentiation, in addition, on proliferation, adhension, and migration of ECs, SMCs, EPCs and SPCs: Rat SMCs proliferation was inhibited by simvastatin at concentration as low 0.01μmol/L(0.01μmol/L simvastatin versus control: 5647±268 versus 6038±218 , n=5,P<0.05), the same concentration had no significant effect on rat ECs proliferation. Treatment with simvastatin at dosages of 1μmol/L or higher led to very prominent growth arrest in SMCs but not ECs. Remarkably, 3H-TdR incorporation was constantly maintained at significantly higher levels in ECs with increasing relative differences at higher simvastatin concentrations. (10μmol/L simvastatin versus control: 4310±132 versus 4321±133, n=5,P>0.05)In addition, simvastatin led to dose dependent inhibition of SMCs migration(0.01μmol/L simvastatin versus control: 41±3 vs 45±3, n=5,P<0.05). In contrast to SMCs, simvastatin had no detectable effect on migration of ECs at concentrations of 0.01 to 10μmol/L in vitro(10μmol/L simvastatin versus control: 35±5 versus 37±5, n=5,P>0.05). EPCs and SPCs are also important tools for assessment of stent-coated compounds. Incubation of isolated rat MNCs with simvastatin dose-dependently increased the number of EPCs differentiated from MNCs and decreased the number of SPCs. The number of differentiating EPCs significantly increased 1.2±0.1 fold(1μmol/L simvastatin versus control: 87±5 versus 39±4, n=5,P<0.01) at concentration of 1μmol/L simvastatin and the number of differentiating SPCs dramatically reduced 62.1±3.5% of control(1μmol/L simvastatin versus control: 32±5 versus 85±4, n=5,P<0.01) at the same concentration. In addition, simvastatin time- and dose-dependently promoted EPCs proliferation, while reached the maximum 24 hours after the simulation at 1μmol/L. In contrast with EPCs, SPCs proliferation was significantly inhibited by simvastatin in a time and dose dependent manner. The number of EPCs increased 2.2±0.1 fold at concentration of 1μmol/L simvastatin for 24 hours(1μmol/L simvastatin versus control: 3762±138 versus 1249±146, n=5,P<0.01), and that of SPCs decreased 62.1±5.6% of control at the same concentration for the same time(1μmol/L simvastatin versus control: 1962±145 versus 4070±184, n=5,P<0.01). Moreover, simvastatin also dose-dependently promoted EPCs adhension and migration, while dose-dependently inhibited SPCs adhension and migration. Consistent with above results, simvastatin dose-dependently promoted the p27 expression of SMCs and SPCs, and inhibited the p27 expression of EPCs, and had no effect on the p27 expression of ECs.3. To explore effects of simvasation on SMCs SDF-1αm RNA expression: simvastatin significantly reduced the SDF-1αm RNA expression of SMCs at a time and dose dependent manner.4. To study effects of locally delivered simvastatin on neointima formation and reendothelialization after vascular injury: local delivery of simvastatin significantly reduced neointimal formation(neointimal area 0.58±0.13 mm~2 versus 0.27±0.12 mm~2; intima/media ratio 1.95±0.27 versus 0.97±0.24,n=6,P<0.01) . Immunohistochemical assessment of injured segments with an endothelial cell marker(von Willebrand factor) revealed no appreciable difference between animals receiving simvastatin administration and control.CONCLUSIONS1. Inhibition of bone marrow-derived endothelial progenitor cells maybe reslt in delayed reendothelialization, which may lead to fatal late thrombosis. The clinical efficacy of sirolimus-eluting stent against restenosis might be achieved, at least in part, through its inhibitory effect on smooth muscle progenitor cells derived from bone marrow. It is necessary that think about the effects of new generation drug-eluting stent-coated compounds on the bone marrow-derived EPCs and SPCs.2. Simvastatin of equal dosage displayed a differential effect on SMCs and SPCs as compared to ECs and EPCs with regard to differentiation, proliferation, migration and adhension, inhibiting SPCs differentiation from the bone marrow MNCs, promoting EPCs differentiation from the bone marrow MNCs, limiting proliferation, migration and adhension of SMCs and SPCs, promoting proliferation, migration and adhension of EPCs, but having no effect on proliferation and migration of ECs.3. Simvastatin significantly reduced the SDF-1αm RNA expression of SMCs at a time and dose dependent manner, which might indirectly inhibiting neointimal hyperplasia by reducing mobilization and recruitment of the bone marrow smooth muscle progenitor cells.4. Local delivery of simvastatin can significantly reduce neointima hyperproliferation in injured artery, together without adverse interfere of reendothelialization.更多还原