【作者】 刘华东；

【导师】 廖玉华； 董少红；

【作者基本信息】 华中科技大学 ， 心血管内科， 2008， 博士

【摘要】 目的(1)探讨简便、快捷建立大鼠颈动脉球囊损伤模型的方法;(2)探讨低分子肝素与普通肝素在建立大鼠颈动脉球囊损伤模型中的效果及差异;(3)探讨G-CSF对大鼠颈总动脉球囊损伤后内膜增生及再内皮化的影响;(4)评价G-CSF预处理及后处理两种不同处理方式对损伤血管的影响;(5)探讨大鼠Thy-1.1+干细胞局部移植对大鼠颈总动脉球囊损伤后内膜增生的影响,评价干细胞移植对再狭窄的作用;(6)探讨G-CSF与大鼠Thy-1.1+干细胞局部移植联合应用对损伤后血管内皮的修复是否具有协同作用。方法实验分三阶段进行。第一阶段:取60只雌性SD大鼠随机分为2组,每组30只,低分子肝素组及普通肝素组,对比低分子肝素与普通肝素在建立大鼠颈动脉球囊损伤模型中的效果。第二阶段:取90只雌性SD大鼠随机分为3组,每组30只,即G-CSF预处理组,于颈总动脉球囊前7天开始皮下注射G-CSF,连续7d后行颈总动脉球囊导管损伤;G-CSF后处理组,于颈总动脉球囊后即刻开始皮下注射G-CSF,连续7d;对照组只行颈总动脉球囊损伤。3组各于术后即刻、3d、7d、14d、21d、28d取损伤血管段,病理组织学检查观察细胞增殖情况,并通过RT-PCR方法分析内皮型一氧化氮合酶(eNOS mRNA)的表达情况,、评价G-CSF预处理及后处理两种不同处理方式对损伤血管的影响;第三阶段:取30只4～6周龄雄性SD大鼠提取骨髓Thy-1.1+干细胞;另取90只雌性SD大鼠随机分为3组,每组30只,即干细胞移植组:于颈总动脉球囊损伤后即刻将约5x 106 Thy-1.1+干细胞注入至损伤血管局部;联合移植组:于颈总动脉球囊前7d开始皮下注射G-CSF,连续7d后行颈总动脉球囊导管损伤,于颈总动脉球囊损伤后即刻将约5x 106 Thy-1.1+干细胞注入至损伤血管局部;对照组:颈总动脉球囊损伤后予等剂量生理盐水局部注入。各组分别于术后即刻、3d、7d、14d、28d取损伤血管段,行病理组织学方法观察细胞增殖情况,原位杂交方法观察移植细胞的定植、分化情况,并通过RT-PCR方法分析内皮型一氧化氮合酶(eNOS mRNA)的表达情况。结果(1)采用Medtronic球囊导管损伤使大鼠颈总动脉内膜剥脱和新生内膜增生,管腔狭窄。术后7d内膜开始增生,14～28d增生最明显,低分子肝素组建模成功率高于普通肝素组,低分子肝素组血栓形成几率低于普通肝素组。低分子肝素组手术成功率93.3%,血栓形成率3.4 %,死亡率3.3%;(2) G-CSF预处理组内膜面积低于G-CSF后处理组及对照组,I/M比值在3组中最低;G-CSF预处理组eNOS mRNA表达明显高于对照组,有统计学差异(P<0.05),G-CSF后处理组与对照组比较eNOS mRNA表达无明显差异;(3)原位杂交发现,干细胞移植组及联合移植组损伤血管壁中均可发现异体细胞;(4)干细胞移植组及联合移植组新生内膜面积、I/M比值比对照组明显减少,eNOSmRNA表达明显高于对照组,联合移植组较干细胞移植组新生内膜面积亦明显减少,二者比较有统计学差异;(5)联合移植组I/M比值低于G-CSF预处理组,eNOS mRNA表达高于G-CSF预处理组。结论(1)在建立大鼠颈动脉球囊损伤模型过程中,采用低分子肝素可提高建模效率,提高建模成功率;(2)G-CSF预处理可促进大鼠颈总动脉球囊损伤后再内皮化的进程,抑制内膜增生过程,对球囊损伤具有修复作用,可预防血管成形术术后再狭窄。G-CSF预处理较后处理有效;(3)Thy-1.1+干细胞局部移植可促进损伤血管的修复;(4)G-CSF与大鼠Thy-1.1+干细胞联合移植可促进大鼠颈总动脉球囊损伤后再内皮化的进程,抑制内膜增生过程,二者具有协同作用;(5)G-CSF及Thy-1.1+干细胞移植促进损伤血管的修复与损伤血管内皮功能重建,eNOSmRNA的表达上调有关。更多还原

【Abstract】 Objective (1) To look for a convenient, quick method of making rat carotid arterial injury model with PTCA balloon, and to investigate the character of intimal thickening by pathology; (2) To compare the effect of low-molecular-weight heparin(LMWH) and unfractionated heparin(UFH) in the process of making rat carotid arterial injury model with Medtronic balloon; (3) To explore the influence of G-CSF on the injured carotid artery ; (4) To evaluate the effect of different treatment of G-CSF on endothelial hyperplasia after saccule injury of rat common carotid artery and the effect on injured blood vessel; (5) To study the effect of Thy-1.1+ stem cells transplant on neointimal, and evaluate the influence of stem cells transplant on restenosis; (6) To study whether recombination G-CSF can cooperate with Thy-1.1+ stem cells to repair the injured artery.Methods The study included three stages. First: 60 female SD rats were equally divided into 2 groups: LMWH group and UFH group, to compare the effect of LMWH and UFH in the process of making carotid arterial injury model. Second: Another 90 female SD rats were randomly divided into 3 groups (30 each group), namely G-CSF pretreated group, G-CSF posttreated group and control group. The G-CSF pretreated group rats were injected daily with 30μg G-CSF /kg for 7 days before carotid artery injury, the G-CSF posttreated group rats were injected daily with 30μg G-CSF /kg for 7 days shortly after carotid artery injury, and the control group was merely underwent carotid artery injury. The animals were killed shortly after injury and 3, 7, 14, 21, 28 days after balloon denudation, and the samples of carotid artery were harvested for pathology and RT-PCR for eNOS mRNA, to evaluate the effect of different treatment of G-CSF on endothelial hyperplasia. Third: 30 male 4-6w old SD rats were killed for Thy-1.1+ stem cells. Another 90 female SD rats were randomized divided into stem cells transplant group, combination transplant group and control group with 30 rats in every group. The stem cells transplant group rats were injected about 5x106 Thy-1.1+ stem cells into the injured artery after carotid artery injury; the combination transplant group rats were injected daily with 30μg G-CSF /kg for 7 days before carotid artery injury and were injected about 5x106 Thy-1.1+ stem cells into the injured artery shortly after the artery injury, and the control group was underwent carotid artery injury and injected the same amount of saline. The animals were killed shortly after injury and 3, 7, 14, 21, 28 days after balloon denudation, and the samples of carotid artery were harvested for pathology, RT-PCR and in situ hybridzation(ISH) to detect the transplanted cells in the injured artery.Results (1) Irregular intimal thickening and stenosis of arterial cavity were observed since 1 week after balloon denudation. At 2 to 4 week, the intimal thickening reached peak. More rats in LMWH group accomplished carotid arterial injury model than UFH group, and the thrombosis is more frequently in UFH group. (2) It was found that the intimal thickness was thinner in G-CSF pretreated group, and eNOS mRNA expression was higher in G-CSF pretreated Group compared with that in control Group(P <0.05), I/M ratio was the smallest in G-CSF pretreated Group. (3) ISH can trace the transplanted cells in the rats of the stem cells transplant group and combination stem cells transplant group. (4) The intimal thickness was thinner in stem cells transplant group and combination stem cells transplant group, and eNOS mRNA expression was much higher compared with those in control group. I/M ratio was lower in combination stem cells transplant group compared with that in stem cells transplant group. (5) The intimal thickness was lower in combination stem cells transplant group compared with that in G-CSF pretreated Group, and eNOS mRNA expression was higher in combination stem cells transplant group.Conclusions (1) LMWH is more effective in the process of making carotid arterial injury model than UFH. (2) G-CSF pretreatment accelerated reendothelialization and decreased neointimal formation following vascular injury, which suggest that exogenous G-CSF may be a feasible treatment to prevent restenosis after PCI. The pretreatment G-CSF is more effective than posttreatment G-CSF.(3)Thy-1.1+ stem cells transplant can promote the repairing of the injured artery. (4) G-CSF combined with Thy-1.1+ stem cells transplant can accelerate reendothelialization and decrease neointimal formation following vascular injury, G-CSF can cooperate with Thy-1.1+ stem cells to repair the injured artery. (5) The repairing of G-CSF and Thy-1.1+ is associated with upregulation of eNOSmRNA expression.更多还原