硫化氢对内皮细胞迁移和缺血过程中血管新生作用的研究

【作者】 王铭洁；

【导师】 朱依纯； 姚泰； 陆利民；

【作者基本信息】 复旦大学 ， 生理学， 2008， 博士

【摘要】 硫化氢（H₂S）是一种无色有臭鸡蛋味的气体,大量吸入会导致中毒。但是很多种哺乳动物细胞自身能产生H₂S,如实验大鼠血液中H₂S浓度达到46μmol/L。体内的H₂S主要通过含硫氨基酸代谢产生,胱硫醚-β-合酶（cystathionineβ-synthase,CBS）和胱硫醚-γ-裂解酶（cystathionineγ-lyase,CSE）是两个能分解L-半胱氨酸产生硫化氢的酶。CBS主要存在于神经系统,对于心血管组织,内源性H₂S则主要通过CSE的催化产生。研究发现在心血管系统中,H₂S能开放ATP敏感的钾离子(K_ATP)通道,使血管平滑肌舒张,降低血压;体内H₂S生成减少与自发性高血压的形成有密切关系;外源性给予H₂S能有效保护心肌的缺血性损伤。由于H₂S是一种小分子量的气体分子,能穿透细胞膜直接起效,在体内产生并受内源性关键酶的调节,生理浓度时有明确的功能并能被外源性气体模拟,H₂S成为继一氧化氮（NO）和一氧化碳（CO）后的第三个气体信号分子。血管新生是指在原有血管床基础上长出新的血管,存在于组织生长发育和修复过程中,肿瘤生长也与其关系密切,因而调节血管新生具有重要的临床应用价值。我们实验室近期研究表明,内皮细胞上存在CSE的表达,生理浓度H₂S不同程度地促进了细胞的粘附、迁移和管腔形成能力,使整合素α2β1的表达增强,Akt磷酸化呈时间和浓度依赖性增加。这是国际上第一篇关于H₂S促进血管新生作用的研究论文。本文作者在该论文中分工研究H₂S对迁移的影响。本文包括了该论文中已报道的结果和进一步研究的内容。已知H₂S能促进C57小鼠Matrigelplug模型以及鸡胚绒毛膜尿囊（CAM）模型中的血管新生,但是在体内缺血情况下,H₂S是否能促进血管新生,并改善缺血区血供,目前尚未见报道。因此通过我们对整体缺血模型中H₂S作用的研究,将更深入地认识这个新的气体信号分子,对其促血管新生作用的应用前景也将逐步明朗。首先,建立了多种内皮细胞迁移模型,从不同的角度论证H₂S对内皮细胞迁移过程的影响。我们先后采用划痕损伤修复、跨膜迁移和单细胞实时动态3种方法,观察到10和20μmol/L的H₂S供体硫氢化钠（NaHS）促进单层内皮细胞划痕损伤修复、促进内皮细胞跨膜迁移和促进单个内皮细胞运动的作用。接着采用内皮细胞肌动蛋白骨架荧光染色法,观察到1和10μmol/L的NaHS能促进内皮细胞骨架重排,肌动蛋白多聚化后形成明显的横贯细胞的应力纤维。进一步地,我们使用10μmol/L的格列苯脲（glibenclamide）阻断K_ATP通道或10mmol/L的炔丙基甘氨酸（DL-propargylglycine,PAG）抑制CSE活性（从而阻断内源性H₂S的产生）,观察到H₂S促进内皮细胞跨膜迁移的作用被阻断,肌动蛋白多聚化倾向也削弱了。说明H₂S促进内皮细胞迁移和骨架重排的作用同细胞膜上K_ATP通道的开放有关,内源性H₂S参与对内皮细胞迁移的调控。然后,建立了大鼠一侧下肢缺血模型,并成功利用该模型研究H₂S在体内缺血情况下是否能促进血管新生,并改善缺血区血供。不同浓度NaHS（10,50以及200μmol/kg/d）和CSE质粒肌肉注射的治疗作用在造模28天后通过以下四种指标进行评价:1)缺血区域血流量;2)缺血下肢肌肉形态学研究;3)毛细血管密度定量以及4)髂动脉血流量和最大血管舒张储备。运用荧光微球法比较患侧和健侧肌肉的区域血流量,结果显示50μmol/kg NaHS腹腔注射以及肌肉注射CSE质粒均能显著改善缺血区血供。HE染色也表明NaHS腹腔注射组和CSE质粒肌肉注射组的肌肉形态基本正常,肌纤维间可见较多血管。用CD31免疫组化染色法观察腓肠肌横截面毛细血管密度,结果显示50μmol/kg NaHS腹腔注射以及肌肉注射CSE质粒均能增加缺血下肢肌肉毛细血管密度。采用超声多普勒血流仪,比较患侧和健侧的髂动脉血流量以及最大血管舒张储备,结果显示50μmol/kg NaHS腹腔注射以及肌肉注射CSE质粒能部分恢复缺血下肢的最大血管舒张储备。以上结果说明H₂S通过促进缺血情况下的血管新生,发挥其改善缺血区血供的作用。最后,我们初步探讨了内皮细胞和心脏组织块的立体培养方法,提供了一系列更好地模拟体内情况的体外模型,为今后进一步研究H₂S的作用机制打下基础。总之,我们在实验中发现H₂S能促进内皮细胞迁移和骨架重排,通过促进整体缺血情况下的血管新生,发挥其改善缺血区血供的作用。更多还原

【Abstract】 Hydrogen sulfide（H₂S） is well known as a toxic gas with the characteristic smell of rotten eggs.But it is becoming increasingly clear that mammalian cells also produce H₂S.The H₂S concentration in rat serum is about 46μmol/L.Endogenous H₂S is synthesized naturally in the body from L-cysteine mainly by the activity of two enzymes,cystathionineβ-synthase（CBS） and cystathionineγ-1yase（CSE）.CBS seems to be main HES-forming enzyme in the brain and nervous system,whereas CSE is the main H₂S-synthesizing enzyme in the cardiovascular system.It is reported that in cardiovascular system,HES can relax smooth muscle cells by activating ATP-dependent K⁺ channels（KATP） and decrease the blood pressure.The deficit of HES/CSE system may be responsible for the development of spontaneous hypertension.The administration of exogenous H₂S can effectively prevent myocardial ischemia injury.Since H₂S is a small molecule of gas,freely permeable to membrane,endogenously and enzymatically generated,has physiological functions,it is regarded as third gasotransmitters together with nitric oxide（NO） and carbon monoxide（CO）.Angiogenesis,the formation of new blood vessels from pre-existing vessels, occurs during embryonic development and reparative processes.Persistent and uncontrolled angiogenesis is observed in tumour development.So regulation of angiogenesis is important for clinical appliance.Our laboratory has recently demonstrated that endothelial cells express CSE,physiologically relevant dosages of HES promote a highly robust angiogenic response that is largely dependent on activation of Akt.This paper reported the angiogenic effect of HES for the first time, and my role in this paper is focus on endothelial cell migraion.So we will discuss those findings already puhlished and some new obserbvations.Recent investigations also implied that HES can promote angiogenesis in mice matdgel plug model and chicken chorioallantoid membranes（CAM）.That brings about the hypothesis of the present study,i.e.HES may regulate the process of angiogenesis in ischemic animal muscles and therefore improve blood supply to these regions.The purpose of the present study is to assess the role of HES in regulation of angiogenesis in ischemic conditions.First,various methods of endothelial cell migration were established and the influence of HES on endothelial cell migration was investigated from different aspects. Three kinds of models:scratch wound healing,transmembrane migration and time-lapse single cell migration were successively used and we observed that 10 and 20μmol/L NariS（H₂S donor） can promote scratch wound healing,transmembrane migration and time-lapse single cell migration ability.We also found that relatively low concentrations of NariS（1 and 10μmol/L） enhanced endothelial cell skeleton reorganazion and stress fiber formation using F-actin fluorescence staining method. Furthermore,we observed that KATP channel blocker Glibenclamide（10μmol/L） and CSE inhibitor DL-propargylglycine（PAG,10 mmol/L） both prevented H₂S-induced endothelial cell transmembrane migration and actin polymerization.These data suggested that H₂S stimulates endothelial cell migration by opening KATP channel. Endogenous H₂S participates in the regulation of endothelial cell migration process.Then we used rat unilateral hindlimb ischemia model to investigate the effect of H₂S on in vivo angiogenesis and blood supply recovery.The therapeutic effect of NaHS（10,50 and 200μmol/kg/d） and CSE plasmid injection was assessed 28 days after induction of ischemia by 1) regional blood flow analysis,2)histological study of hindlimb muscles,3) quantification of capillary density,and 4) iliac blood flow and maximal vasodilatory reserve.Both 50μmol/kg/d NariS and CSE plasmid injection improved residual muscle blood flow（1.073±0.153 and 1.089±0.178,respectively） compared with the control group（0.558±0.0524,p＜0.05） by a fluorescent microsphere assay.These results were confirmed by HE staining,showing relatively normal muscle fiber and capillary arrangement in rats treated with NariS and CSE plasmid.Capillary density count increased from 0.847±0.0365 CD31 positive cells/myofiber in untreated ischemic controls to 1.567±0.119 with 50μmol/kg/d Naris and 1.546±0.0953 with CSE plasmid injection（p＜0.01）.Maximal vasodilatory reserve tended to increase from 0.313±0.0134 ml/min in untreated ischemic controls to 0.824±0.128 ml/min with 50μmol/kg/d Naris and 0.657±0.105 ml/min with C SE plasmid injection（p＜0.01 and p＜0.05,respectively）.These data suggested that H₂S promotes in vivo angiogenesis in ischemic conditions and improve blood supply recovery.Furthermore,we studied three dimentional culture method for endothelial cells and cardiac explants,which provides series of in vitro models to mimic the in vivo conditions for further investigation.In summary,the present study provides the first evidence of H₂S on endothelial cell actin polymerization.H₂S can promote in vivo angiogenesis in ischemic conditions,which leads to blood supply recovery.更多还原