【作者】 舒礼良；

【导师】 徐敬；

【作者基本信息】 郑州大学 ， 外科学， 2011， 博士

【摘要】 第一部分人参皂苷Rb1动员自体静脉移植兔骨髓内皮祖细胞增殖的实验研究背景与目的内皮祖细胞(Endothelial progenitor cells, EPCs)是内支细胞的前体细胞,是一群具有游走功能并能增殖分化的幼稚细胞,不仅可以迁移至外周血中分化为成熟内皮细胞,参与胚胎时期的血管生成、出生后的新生血管的生长,还可以存一定条件下转化为平滑肌细胞,并且能够归巢在内皮细胞受损部位,分化为内皮细胞,参与受损内皮细胞的修复,为静脉血管桥的再狭窄的预防提供了新的思路。内皮祖细胞来源广泛,且具有易于分离、增殖能力强等多方面的特点,已成为血管组织工程中一种重要的种子细胞。正常情况下成熟血管内皮细胞与内皮的损伤以及内皮祖细胞对血管内皮的修复处于一种动态的平衡之中。病理状态下,机体储备的内皮祖细胞增殖并释放入外周血,并向内皮损伤部位定向迁移、粘附,协助修复损伤的血管内皮细胞,有效的减轻静脉桥血管的再狭窄。静脉移植术后的再狭窄,末期主要是粥样硬化,而血管内皮损伤是其始动因素,内皮细胞损伤后,巨噬细胞粘附、浸入及平滑肌细胞过度增殖导致受损血管管腔狭窄或者闭塞。促进损伤血管的内皮修复可有效抑制平滑肌细胞增殖及新生内膜的形成,为治疗静脉移植血管的再狭窄提供了一个新的方向。以往的研究发现人参皂苷Rhl能够促进内皮细胞培养基中的内皮相祖细胞的增生,但未见有关于其对骨髓中内皮祖细胞影响的报道,实验通过在建立动物血.管移植模型的基础上,观察动物股骨骨髓中内皮祖细胞表面标志CD34＋、CD133＋和VEGFR2＋的阳性表达情况,探讨人参皂苷Rb1对自体静脉移植兔骨髓内皮祖细胞增殖的影响。材料与方法45只清洁级新西兰兔随机分为实验组、模型组、对照组3组,每组15只。应用"no-touch"技术获取颈外静脉后,剪断颈总动脉,采用连续缝合方法端端吻合颈外静脉一颈总动脉,建立颈外静脉和颈总动脉移植的动物模型。4周后,取移植静脉血管,利用HE染色观察静脉移植血管内膜厚度以及内膜／中膜厚度比,免疫组化观察骨髓内CD34+、CD133和VEGFR2+阳性细胞情况,RT-PCR检测骨髓内CD3、CD133＋和VEGFR2阳性mRNA表达情况。结果1、移植静脉桥血管狭窄程度观察石蜡切片HE染色光镜下观察分析结果显示：移植4周后的实验组、模型组、对照组血管的内膜厚度分别为(46.53±2.50)μm、(50.80±2.68)μm、(43.73±3.24)μm,各组间比较P＜0.05,实验组、模型组、对照组组间比较有统计学意义。3组静脉内膜/中膜厚度比分别为(1.96±0.06)、(2.11±0.07)、(1.81±0.09),各组间比较P＜O.05,实验组、模型组、对照组组间比较有统计学意义。2、骨髓免疫组化CD34+、CD133和VEGFR2＋阳性细胞观察各组移植静脉血管中可见CD34+、CD133和VEGFR2＋阳性细胞表达.4周后,实验组阳性细胞表达高于模型组,模型组阳性细胞表达高于对照组,实验组、模型组、对照组组间比较有统计学意义。3、骨髓RT-PCRCD34+、CD133和VEGFR2基因表达情况观察各组移植静脉血管中可见CD34＋、CD133+和VEGFR2+mRNA表达,CD34+在实验组、模型组、对照组表达的相对系数分别是(2.32±0.08)、(2.08±0.12)、(1.88±0.11),各组间比较P＜0.05,实验组、模型组、对照组组间比较有统计学意义；CD133＋在实验组、模型组、对照组表达的相对系数分别是(2.46±0.12)、(2.24±0.09)、(1.97±0.15),各组间比较0.05,实验组、模型组、对照组组间比较有统计学意义；VEGFR2＋在实验组、模型组、对照组表达的相对系数分别是(1.92±0.14)、(1.79±0.15)、(1.55±0.09),各组间比较0.05,实验组、模型组、对照组组间比较有统计学意义。结论1.自体静脉移植兔骨髓CD34＋、CD133+和VEGFR2+表达增强；2.人参皂苷Rbl能够促进骨髓内皮祖细胞的增生；第二部分人参皂苷Rb1抑制兔自体静脉移植物再狭窄的实验研究背景与目的冠状动脉旁路移植术(coronary artery bypass graft, CABG)是治疗冠状动脉粥样硬化性心脏病的主要方法之一,大隐静脉作为冠脉旁路移植术的主要材料,由于位置表浅,取材方便,并且有足够的长度连接主动脉与远端的冠状动脉,使其在临床上得以广泛应用。但是手术操作造成的血管内膜损伤、血流动力学改变以及炎症因子释放等一系列变化引起静脉桥血管术后再狭窄,使得静脉桥血管在临床上的应用中发现远期效果欠佳。近年来,一系列预防静脉移植血管再狭窄的方法,如药物治疗、基因治疗、血管外支架、扩张液、内皮祖细胞等应用于临床,但是均未取得明确效果。有报道称：人参皂苷Rbl对抑制静脉桥血管再狭窄有一定的积极作用。实验即是在建立自体静脉移植血管动物模型的基础上,通过腹腔注射一定剂量的人参皂苷Rbl成品,观察静脉移植血管内膜厚度的变化以及血管内膜中增殖细胞核抗原(proliferating cell nuclear antigen, PCNA)、转化生长因子(Transforming growth factor, TGF-β)和内皮型一氧化氮合酶(Endometrial nitric oxide synthase, eNOS)内变化,探讨人参皂苷Rbl对自体静脉移植血管再狭窄的抑制作用,为以后的临床应用提供理论依据。材料与方法45只清洁级新西兰兔随机分为实验组、模型组、对照组3组,每组15只。应用"no-touch"技术获取颈外静脉后,剪断颈总动脉,采用连续缝合方法端端吻合颈外静脉和颈总动脉,建立颈外静脉一颈总动脉搭桥的动物模型。4周后,取静脉移植血管,利用HE染色观察静脉移植血管内膜厚度以及内膜／中膜厚度比,免疫组化观察静脉移植血管增殖细胞核抗原(PCNA)阳性细胞指数、TGF-β、eNOS变化情况,RT-PeR检测静脉移植血管中TGF-β、eNOS mRNA基因的表达。结果1、移植静脉桥血管狭窄程度观察石蜡切片HE染色光镜下观察分析结果显示：移植4周后的实验组、模型组、对照组血管的内膜厚度分别为(446.53±2.50)μm、(50.80±2.68)μm、(43.73±3.24)μm,各组间比较P<0.05,实验组、模型组、对照组组间比较有统计学意义。三组静脉内膜/中膜厚度比分别为(1.96±0.06)、(2.11±0.07)、(1.81±0.09),各组间比较P<0.05,实验组、模型组、对照组组间比较有统计学意义。2、移植静脉桥血管免疫组化染色结果观察各组移植静脉血管中可见PCNA、eNOS、TGF-β(?)日性细胞表达。移植4周后的实验组、模型组、对照组PCNA阳性表达指数分别是(40.63±2.30)、(60.45±3.38)、(19.89±2.56),各组间比较P<0.05,实验组、模型组、对照组组间比较有统计学意义。实验组、模型组、对照组TGF-β阳性表达指数分别是(18.15±4.31)、(29.28±6.37)、(12.63±3.72),各组间比较P<0.05,实验组、模型组、对照组组间比较有统计学意义。实验组、模型组、对照组eNOS阳性表达指数分别是(25.64±5.03)、(17.91±3.75)、(10.25±2.31),各组间比较P<0.05,实验组、模型组、对照组组间比较有统计学意义。3、移植静脉桥血管RT-PCR结果观察各组移植静脉血管可见eNOS、TGF-β基因表达。eNOS在实验组、模型组、对照组表达的相对系数分别为(2.12±0.05)、(1.98±0.09)、(1.72±0.09),eNOS在实验组中表达的相对系数高于模型组和对照组,各组间比较P<0.05,实验组、模型组、对照组组间比较有统计学意义。TGF-β在实验组、模型组、对照组表达的相对系数分别为(1.95±0.08)、(2.10±0.01)、(1.82±0.01),TGF-p在模型组中表达的相对系数高于实验组和对照组,各组间比较P<0.05,实验组、模型组、对照组组间比较有统计学意义。结论1内膜损伤是移植静脉血管再狭窄的始动因素,血管壁重塑是静脉血管产生再狭窄的根本原因。2人参皂苷Rb1能够有效的防治移植静脉血管再狭窄。3人参皂苷Rb1能够促进移植血管NOS的基因表达、抑制TGF-β基因表达。更多还原

【Abstract】 Because of the improving of living standard and increased elderly population, the morbality of coronary atherosclerotic heart disease increased significantly in recent years, so more and more patients need coronary artery bypass graft. The materials for coronary bypass graft include arteries and venous, but because the limited of arteries and more serious hurt to get arteries graft than venous, the great saphenous vein is widely used in clinical practice as the primary material of CABG, because the great saphenous vein is superficial and easily obtained, and has enough length to connect the aorta and the far coronary artery.The autogenous vein will be restenosis after vein grafts because of endometrial injury by operative, hemodynamic changes and a series of inflammatory factors release.That make the saphenous vein is restrictedin the clinical application.In recent years,a series of methods was used for preventing vein restenosis in clinical,It was reported that Ginsenoside Rbl has some positive effect on preventing vein restenosis.After established the animal models of vein grafting, we used a certain dose of Ginsenoside Rbl by intraperitoneal injection for the animal models. By observed the changes of vein past-grafting and endothelial progenitor cells proliferation of bone marrow, to investigate the influence of ginsenoside Rb 1 on vein graft restenosis in autogenous vein graft rabbits. Part one Expertment investigation on Ginsenoside Rbl mobilizing the endothelial progenitor cell proliferation of bone marrow in autogenous vein graft rabbitsBackground and ObjectiveEndothelial progenitor cells(Endothelial progenitor cells, EPCs) are a group of precursor cells which functions with migration and proliferation and differentiation of naive cells can not only migrate to the peripheral blood to differentiate into mature endothelial cells involved in angiogenesis during embryonic, postnatal angiogenesis growth, under certain conditions can also be transformed into smooth muscle cells, for the vein to prevent restenosis in the bridge provides a new way of thinking. Source of progenitor cells wide, and has easy to separate, proliferation ability, and many other features, has become an important vascular tissue engineering of seed cells. Under normal circumstances mature endothelial cells and endothelial damage and endothelial progenitor cells to repair in a dynamic balance. Pathological state, the body reserves the release of endothelial progenitor cells to endothelial damage to the peripheral parts of the directional migration, adhesion, assist in the repair of vascular endothelial cell injury, effective to reduce restenosis in vein grafts. Restenosis after coronary artery bypass grafting, mainly late atherosclerosis, endothelial injury is the initial factor, endothelial cell injury, macrophage adhesion, immersion and the excessive proliferation of smooth muscle cells led to stenosis or occlusion of target vessel. Promote vascular endothelial damage repair can effectively inhibit smooth muscle cell proliferation and neointimal formation, for the treatment of restenosis vein provides a new target.Previous studies found that ginsenoside Rbl can promote endothelial cell culture medium in the progenitor cell proliferation, this study based on animal vessel model based on the observation of animals, the femur bone marrow endothelial cell surface markers CD34+, CD 133+ and VEGFR2+ positive expression, to investigate the influence of ginsenoside Rbl on endothelial progenitor cells proliferation of bone marrow in autogenous vein graft rabbits.Materials and Methods45 New Zealand rabbits were randomly divided into experimental group, model group, control group 3 groups of 15 rats. Application of "no-touch" technology access after the external jugular vein, cut the carotid artery, using a continuous suture anastomosis external jugular vein and carotid artery, the establishment of external jugular vein and carotid artery bypass model. After 4 weeks, immunohistochemistry to detect bone marrow CD34+, CD133+ and VEGFR2+ positive cells in the situation, RT-PCR detection of bone marrow CD34+, CD133+ and VEGFR2+ positive mRNA expression.Results1. The degree of stenosis of grafted veinThe picture under-microscope showed that:After 4 weeks, the vascular intimal thickness of experimental group, model group and control group was(46.53±2.50)μm,(50.80±2.68)μm,(43.73±3.24)μm, the comparison among groups P<0.05, experimental group, model group and control group were significantly different among groups. The intimal/medial thickness ratio of three groups was (1.96±0.06), (2.11±0.07), (1.81±0.09), the comparison between groups P<0.05, the ratio of experimental group, model group and control group was significantly difference between groups.2. Immunohistochemistry of bone marrow CD34+, CD133+ and VEGFR2+ positive cells were observed Vein graft in each group shows CD34+, CD133+ and VEGFR2+.4 weeks after positive cells, positive cells in the experimental group was higher than the model group, model group, positive cells in the control group.3. bone marrow RT-PCRCD34+, CD133+ and VEGFR2+ gene expression observed The group transplanted veins visible CD34+, CD133+ and VEGFR2+ mRNA expression, CD34+ in the experimental group, model group and control group expressed by the relative coefficient is (2.32±0,08), (2.08±0.12), (1.88±0.11), the comparison between groups P<0.05, experimental group, model group, control group between the two groups were significantly different; CD133+ in the experimental group, model group, control the expression of the relative coefficient is (2.46±0.12), (2.24±0.09) and (1.97＝0.15), the comparison between groups P<0.05, experimental group, model group, control group between the two groups were significantly different; VEGFR2+ in the experimental group, model group, control the expression of the relative coefficient is (1.92±0.14), (1.79±0.15) and (1.55±0.09), the comparison between groups P<0.05, experimental group, model group and control group were significantly different between groups.Conclusion1.The expression of CD34+, CD133+ and VEGFR2+ increased significantly in autogenous graft vein rabbits.2. Ginsenoside Rbl can promote the endothelial progenitor cells proliferation of bone marrow.Part two Expertment investigation on Ginsenoside Rbl to prevent the restenosis of autogenous vein graft in rabbitsBackground and ObjectiveCABG (coronary artery bypass grafting, CABG) is one of the important treatments for coronary heart disease.the great saphenous vein is widely used in clinical practice as the primary material of CABG, because the the great saphenous vein is superficial and easily obtained, and has enough length to connect the aorta and the far coronary artery.The autogenous vein will be restenosis after vein grafts because of endometrial injury by operative, hemodynamic changes and a series of inflammatory factors release.That make the saphenous vein is restrictedin the clinical application.In recent years,a series of methods was used for preventing vein restenosis in clinical, such as drug treatment, gene therapy, extravascular stent, the improve expansion fluid and endothelial progenitor cells, but the effect was so poor. It was reported that:Ginsenoside Rbl has some positive effect on preventing vein restenosis. After established the animal models of vein grafting, we used a certain dose of Ginsenoside Rbl by intraperitoneal injection for the animal models.By observed the changes of vein past-grafting, proliferating cell nuclear antigen (PCNA), transforming growth factor (Transforming growth factor, TGF-P) and nitric oxide synthase(Endothelial nitric oxide synthase, eNOS)in endometrial cells,we found that the ginsenoside Rbl has some positive effect on the vein graft restenosis inhibition. This was a basis theoretical for future clinical application.Materials and Methods45 New Zealand rabbits were randomly divided into three groups:the experimental group, the model group and the control group,15 rabbits in each group, "no-touch" technology was used for getting the external jugular vein. Establish the animal model with using a continuous way anastomosis external jugular vein to ipsilateral carotid artery by end-to-end fashion.4 weeks later, grafting vein was obtained as sample, the vein intimal thickness,and the intima/media thickness ratio, and the expression of TGF-βand eNOS gene was examined by RT-PCR.Results1. The degree of stenosis of grafted veinThe picture under-microscope showed that:After 4 weeks, the vascular intimal thickness of experimental group, model group and control group was(46.53±2.50) um,(50.80±2.68)μm,(43.73±3.24)μm, the comparison among groups P<0.05, experimental group, model group and control group were significantly different among groups. The intimal/medial thickness ratio of three groups was (1.96±0.06), (2.11±0.07), (1.81±0.09), the comparison between groups P<0.05, the ratio of experimental group, model group and control group was significantly difference between groups.2.The results observed by immunohistochemical stainingPCNA, eNOS, TGF-P positive cells can be seen in each group grafted vein. After 4 weeks.the experimental group, model group, the control group expression of positive PCNA index were (40.63±2.30),(60.45±3.38),(19.89±2.56), The expression of TGF-p were (18.15±4.31), (29.28±6.37), (12.63±3.72) respectively, The expression of eNOS were (25.64±5.03),(17.91±3.75), (10.25±2.31)respectively, compared among the groups P<0.05. the expression of experimental group, model group and control group were significantly different among groups.3 The results observed by RT-PCR of grafted veinThe expression of eNOS, TGF-βgene can be seen in each group grafted vein. the expression of eNOS relative coefficients in experimental group, model group, control group were (2.12±0.05),(1.98±0.09),(1.72±0.09), the expression of eNOS relative coefficient in the experimental group is higher than the model group and control group, compared among groups P<0.05, the expression of experimental group, model group and control group were significantly different among groups. The expression of TGF-βrelative coefficients in experimental group, model group, control group were (1.95±0.08),(2.10±0.01),(1.82±0.01), the expression of TGF-βrelative coefficient in the model group is higher than in the experimental group and control group, compared among groups P<0.05, the expression of experimental group, model group and control group were significantly different between groups.Conclusion1. Grafted vein intimal injury is the initial factor of restenosis.The remodeling of vascular is the most important reason of restenosis.2. Ginsenoside Rbl can prevent restenosis of vein graft effectively.3. Ginsenoside Rbl can promote NOS gene expression in grafted vein, inhibited TGF-βgene expression in grafted vein.更多还原