【作者】 周宁；

【导师】 王海昌； 高峰； 边杰芳；

【作者基本信息】 第四军医大学 ， 内科学， 2009， 博士

【摘要】 研究背景近年来,随着社会经济发展、人民生活水平提高、就业压力增大、生活方式改变、人口急速老龄化和易患因素发病率的逐年上升,心血管疾病已成为危害人们健康的“第一杀手”。由于科技进步,针对心血管疾病的药物和介入治疗取得了长足进步,然而,冠状动脉旁路移植术(CABG)和周围动脉旁路移植术在复杂心血管病变的处理上仍有着不可替代的作用。自体静脉移植物,尤其是大隐静脉移植物(SVG),由于方便取材和管段较长,是动脉旁路移植术中最常用移植物。但是,较之乳内动脉移植物(IMAG),较低的通畅率严重降低了SVG的临床应用价值。因此,如何提高SVG通畅率,仍是众多学者研究的热点。越来越多的临床证据表明极化液(GIK)/胰岛素应用可明显改善CABG术后糖尿病和非糖尿病患者的预后,而且,减少CABG术后糖尿病患者缺血性心血管事件的发生率。我们的前期实验发现GIK不仅对缺血/再灌注(I/R)心肌细胞有保护作用,而且对I/R冠脉内皮细胞具有显著的抗凋亡作用,而其中胰岛素起着关键作用,它是通过PI3K/Akt-eNOS-NO发挥对上述细胞的保护作用。研究发现动脉旁路移植术后早期,移植静脉的内皮细胞(ECs)和血管平滑肌细胞(VSMCs)的凋亡与早中期SVG的失败关系密切:ECs凋亡是SVG附壁血栓形成、新生内膜增生的始动因素,而VSMCs大量凋亡又可促进别的VSMCs增殖和迁移,导致新生内膜过度增生。有学者发现在非胰岛素抵抗状态下,应用胰岛素可抑制大鼠球囊损伤后颈动脉新生内膜过度增生。然而,极化液/胰岛素对I/R移植静脉有何影响,尤其是对I/R移植静脉ECs和VSMCs是否有直接的保护作用及其机制尚不明确。另外有学者发现较持久的胰岛素刺激可促进ECs中eNOS表达的上调,我们推测由此带来的血管内皮功能改善、一氧化氮(NO)释放增加,对动脉血管损伤后新生内膜过度增生有着抑制作用。但胰岛素应用后对在体的SVG新生内膜过度增生有无抑制效应尚未见报道。研究目的1.明确给予胰岛素治疗是否可以减轻在体I/R后移植静脉内皮细胞和血管平滑肌细胞的凋亡程度;2.探讨胰岛素对抗移植静脉ECs和VSMCs凋亡的信号转导机制,尤其是胰岛素与移植静脉PI3K/Akt信号转导途径之间的关系;3.观察动脉旁路移植术后短期内应用胰岛素是否对移植静脉新生内膜过度增生有抑制作用,并探讨可能机制。材料和方法杂种犬40只,雌雄不拘,体重12 ~ 15 kg,随机分为5组,每组8只,以3%戊巴比妥钠静脉注射麻醉后,行颈部单侧倒置颈外静脉-颈总动脉端端吻合模拟动脉旁路移植术模型,分别于移植静脉再灌注前5分钟直到之后4小时,给予以下4种液体以2 mL/kg/h静脉输注:(1)假手术组(Sham:0.9% NaCL);(2)生理盐水(Vehicle: 0.9%氯化钠);(3) GK(葡萄糖-钾液:葡萄糖: 250g/L,氯化钾:80 mmol/L);(4) GIK(葡萄糖: 250g/L,胰岛素: 60U/L,氯化钾:80 mmol/L);(5) GIK+Wort (Wortmannin,2.0μg/kg/h,再灌注前5 min开始,持续1 h静脉输注)。之后取出颈外静脉移植物行TUNEL凋亡染色和激活的caspase-3免疫组化染色,用Western blot法检测颈外静脉移植物中caspase-3活性、Akt和eNOS磷酸化水平。在另一组实验中,杂种犬16只,随机分为2组,每组8只,建立同样模型,分别于移植静脉再灌注前5分钟直到之后4小时,给予以下2种液体以2 mL/kg/h静脉输注:(1)生理盐水;(2) GIK;之后分别给予生理盐水0.5 mL和胰岛素4 U (并按此分为两组),2次/日,皮下注射,连续1月,于术后29天,腹腔注射BrdU(50 mg/kg),24小时后处死犬,取出静脉移植物和正常颈外静脉,分别行BrdU掺入和eNOS免疫组化染色,用Western blot法检测其中α-平滑肌肌动蛋白表达状况,用Verhoeff-van Gieson染色法衡量新生内膜增生程度。实验结果第一部分实验:1.术后短期内移植静脉中存在ECs和VSMCs的大量凋亡;2. GK处理可加重ECs和VSMCs的凋亡(与生理盐水组比较,P < 0.05 );3. GIK可显著降低I/R后移植静脉ECs和VSMCs的凋亡指数(分别与生理盐水组和GK组比较,P < 0.001)和中/内膜caspase-3的激活程度(分别与生理盐水组和GK组比较,P < 0.01);4. GK处理后明显抑制移植静脉中Akt和eNOS的磷酸化,并刺激caspase-3的活化(与生理盐水组比较,P < 0.05),GIK处理后则明显增强了Akt和eNOS的磷酸化(分别与生理盐水组和GK组比较,P < 0.001),并明显抑制caspase-3的活化(P < 0.05);加用Wortmannin后,GIK的这种效应被明显消除;第二部分实验:1.在术后1月,应用胰岛素可明显促进犬移植静脉中α-平滑肌肌动蛋白的表达(分别与犬正常颈外静脉和生理盐水组比较,P < 0.001);2.应用胰岛素后明显抑制犬移植静脉中/内膜的细胞增殖(细胞增殖指数11.15%±0.93%与生理盐水组的25.55%±2.02%比较,P < 0.001);3.应用胰岛素后明显促进犬移植静脉内膜eNOS的表达(分别与正常静脉组和生理盐水组比较,P < 0.05);4.胰岛素应用可减轻移植静脉新生内膜增生程度(中/内膜厚度136.51±8.97μm,与生理盐水组的185.92±8.34μm比较, P < 0.05)。结论1.再灌注期间高血糖症损伤自体移植静脉,应用胰岛素可有效保护自体移植静脉;2.胰岛素保护自体移植静脉的效应是通过PI3K-Akt信号转导途径抑制I/R后ECs和VSMCs凋亡来实现的;3.术后短期内连续应用胰岛素治疗可有效抑制自体移植静脉新生内膜过度增生;4.胰岛素减轻犬自体移植静脉新生内膜过度增生的效应可能有赖于抑制VSMCs的增殖和血管内皮功能的改善。更多还原

【Abstract】 BackgroundWith the change of life-style, enhancement of social stress, increase of risk factors such as hypertension, dyslipidemia, diabetes mellitus and smoking, and aging of population, recent decades have seen that cardiovascular disease has been becoming the first killer of people in China. Regardless of a great progress of medicinal and intervational therapies, peripheral and coronary artery bypass grafing (CABG) is still in an indispensible position of treating complex and multiple vascular lesions because of demand of complete revascularization. However, compared with the arterial conduit, especially, internal mammary artery graft, saphenous graft (SVG) is more prone to neointimal hyperplasia and atherosclerosis, subsequently, easy to restenosis. Thus, a lower long-term patency of SVG than that of the arterial graft greatly limits its therapeutic property in arterial bypass grafting.Growing evidence from clinical and experimental studies indicates that endothelial cell (EC) injury resulting in endothelial dysfuction due to ischemia/repefusion (I/R) induced injury, increased stretch stress under arterialization initiates the thrombosis and neointimal hyperplasia in autologous vein grafts. Additionally, the death of vascular smooth muscle cells (VSMCs) can provoke migration of another VSMCs into tunica intima and infiltration of inflammatory cells into the vein graft wall, and cytokines and growth factors released from the inflammatory cells, in turn, inducing the migration and proliferation of VSMCs, resultly, enhancing neointimal hyperplasia.Considerable studies have demonstrated that glucose-insulin-potassium (GIK) /insulin remarkably improves the prognosis of pateints undergoing CABG; furthermore, a recent clinical trial indicated that insulin (GIK) infusion significantly reduces reccurent ischemic episodes in post-CABG patients with diabetes. Our previous studies have demonstrated that insulin, as the key compound of GIK, plays a pivotal role in inhibiting the apoptosis of both I/R cardiac myocytes and coronary arterial endothelial cells via PI3K/Akt-eNOS -NO cell survival signaling pathway. Importantly, recent studies indicated that insulin markedly inhibits neointimal hyperplasia induced by balloon injury in the rat carotid artery.Nevertheless, it is still to be evaluated whether GIK/insulin plays a protective role in vein grafts and inhibits neointimal hyperplasia in autologous vein grafts.AimsThe purposes of this study were (1) to verify whether apoptotic phenomenon of ECs and VSMCs exists in vein grafts at the earlier stage of postoperation; (2) to assess whether the administration of GIK/ insulin plays a protective role in I/R vein grafts; and if so, (3) to elucidate the invovled mechanisms; (4) to investigate influence of a short-term continuous insulin administration on neointimal hyperplasia in autologous vein grafts.Materials and methodsThirty-two mongrel dogs were subjected to external jugular-carotid interposition bypass grafting, and underwent infusions of vehicle (saline), GIK (glucose, 250 g/L; insulin, 60 U/L; potassium, 80 mmol/L), GK or GIK plus wortmannin (i.v., 2.0μg/kg/min for 1 hour immediately after reperfusion) 5 minutes before and 4 hours after reperfusion, respectively (n = 8, in each group). Other 8 sham I/R dogs underwent the same operation except the excision of the external jugular vein. All dogs were euthanized at 4 hours postoperationally to harvest vein grafts. Active caspase-3 immunohistochemistry and TUNEL stainings were used to detect apoptosis of ECs and VSMCs in vein grafts, and Western blotting was used to determine Akt/phosphorylated Akt (pAkt), eNOS/phosphorylated eNOS (peNOS) and active caspase-3 levels in vein grafts. In a separate study, 16 dogs undergoing the same operation were randomly administered with subcutaneous injection of 0.5-mL saline or 4-U insulin two times per day for 30 days postoperatively(n = 8, in each group).At 1 month postoperationally, dogs were sacrificed for sampling vein grafts. BrdU incorporation immunohistochemistry and Verhoeff-Van Gieson stainings were used for cell proliferation and intimal/medial thickness assays. Western blotting of alpha-smooth muscle actin was used to assess the phenotype of VSMCs, and immunohistochemistry staining of eNOS was utilized to evaluate endothelial function of vein grafts.Results 1. Much apoptosis of ECs and VSMCs existed in canine vein grafts at the early stage after reperfusion in vivo;2. Treatment with GK aggravated apoptosis of both ECs and VSMCs (P < 0.05 vs vehicle ); however, treatment with GIK significantly reduced the apoptosis of both ECs and VSMCs, evidenced by decrease of apoptotic index (P < 0.001 vs vehicle and GK) and inhibition of caspase-3 activation in canine vein grafts (P < 0.01 vs vehicle and GK);3. Treatment with GK attenuated the phosphorylation of Akt and eNOS in canine vein grafts (P < 0.05, respectively vs vehicle); meanwhile, treatment with GIK enhanced the phosphorylation of Akt and eNOS nearly by 2 and 2.5 times, respectively (P < 0.001, respectively vs vehicle). However, wortmannin largely abolished the GIK-elicited effects;4. At 1 month postoperatively, administration of insulin greatly increased alpha-smooth muscle actin expression in canine vein grafts (P < 0.001 vs normal control and vehicle groups, respectively );5. Administration of insulin inhibited the cell proliferation in canine vein grafts (11.15%±0.93% vs 25.55%±2.02%, P < 0.001 vs vehicle);6. Administration of insulin increased the eNOS expression in intima of canine vein grafts (P < 0.05, respectively vs vehicle);7. Administration of insulin inhibited significantly neointimal hyperplasia of canine vein grafts (intimal/medial thickness 136.51±8.97μm vs 185.92±8.34μm, P < 0.05 vs vehicle).Conclusions1. Hyperglycemia aggravates the injury to ischemic/reperfused canine autologous vein grafts through provoking the apoptosis of ECs and VSMCs.2. Insulin exerts its anti-apoptotic effect on ECs and VSMCs via PI3K/Akt pathway in canine autologous vein grafts.3. Administration of insulin at the early stage of postoperation plays an active role in preventing vein grafts from early degeneration.4. Postoperational insulin administration exerts an preventive effect on neointimal hyperplasia in autologous vein grafts probably through improvement of endothelial function and inhibition of the proliferation of VSMCs.更多还原