人参皂苷Rb_1抑制自体静脉移植物再狭窄的实验研究
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摘要
背景与目的
冠脉搭桥术(coronary artery bypass grafting, CABG)是冠状动脉粥样硬化性心脏病主要的治疗方法之一,尤其适用于多支病变和合并心内畸形的患者。乳内动脉、桡动脉和大隐静脉是临床上常用的桥血管,而大隐静脉作为临床上冠脉搭桥常用的主要材料之一,由于位置表浅,取材方便,并且有足够的长度连接主动脉与冠状动脉的远端,使得其能够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、术后血流动力学改变、炎症因子释放等一系列变化引起静脉桥血管术后再狭窄,使得大隐静脉在临床上的应用受到了一定的限制。近年来,一系列预防静脉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,但是总体效果欠佳。有报道称：人参皂苷Rbl对内皮细胞培养基中血管内皮细胞表达血管血小板衍生生长因子(platelet-derived growth factor, PDGF)和内皮型一氧化氮合酶(endothelial nitric oxide synthase, eNOS)产生一定的影响。本实验即是在建立静脉桥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,观察静脉桥血管内膜厚度的变化以及静脉桥血管PDGF和eNOS阳性表达的变化,从而探讨人参皂营Rb1对移植静脉桥血管再狭窄的抑制作用及其机理,为以后的临床应用提供试验基础和理论依据。
材料与方法
45只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”外科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,建立颈外静脉和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形态及厚度变化,免疫组化观察静脉移植血管血小板衍生生长因子、内皮型一氧化氮合酶阳性表达情况,RT-PCR检测静脉移植血管中PDGF. eNOSmRNA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、对照组静脉桥血管的内膜厚度分别为(44.45±3.10)μm.(51.32±2.61)μm、(41.52±2.62)μm,各组间比较P<0.05。
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PDGF、cNOS阳性细胞表达。移植4周后的实验组、模型组、对照组PDGF阳性表达指数分别是(35.38±2.72)、(40.12±3.77)、(27.17±2.80),各组间比较P<0.05；移植4周后的实验组、模型组、对照组eNOS阳性表达指数分别是(56.92±2.56)、(50.80±3.67)、(43.95±2.60),各组间比较P<0.05,实验组、模型组、对照组组间比较有明显差异。
3、移植静脉桥血管RT-PCR结果观察
各组移植静脉血管可见PDGF.eNOS基因表达。PDGF在实验组、模型组、对照组表达的相对系数分别为(1.97±0.09).(2.19±0.06)、(1.62±0.09),PDGF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、对照组组间比较有明显差异。eNOS在实验组、模型组、对照组表达的相对系数分别为(2.18±0.07)、(1.92±0.06)、(1.63±0.08),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、对照组组间比较有明显差异。
4、相关性分析结果
SPSS简单线性模型结果回示：PDGF与血管狭窄呈相关正相关,相关系数为0.329:eNOS与血管狭窄呈负相关,相关系数为-0.469。
结论
1、静脉桥血管狭窄程度与eNOS基因的表达呈负相关,与PDGF基因的表达呈正相关。
2、人参皂苷Rb1能够促进移植血管eNOS基因的表达、抑制PDGF基因的表达,这与其能够抑制血管再狭窄的作用有关。
Background and Objective
CABG (coronary artery bypass grafting, CABG) is one of the important treatments for coronary heart disease. Especially for patients with multi-vessel or intracardiac malformation. 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 restricted in 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, platelet-derived growth factor (PDGF) and 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 Methods
45 New Zealand rabbits were randomly divided into three groups experimental group, model group, 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. After 4 weeks, grafting vein was obtained as sample, the vein intimal thickness, the expression of PDGF and eNOS gene was examined by RT-PCR.
Results
1. The degree of grafted vein stenosis
The picture under-microscope showed that:After 4 weeks, the vascular intimal thickness of experimental group, model group and control group was(44.45±3.10)μm, (51.32±2.61)u.m,(41.52±2.62)μm, the comparison among groups P＜0.05, experimental group, model group and control group were significantly different among groups.
2. The results observed by immunohistochemical staining
eNOS, PDGF positive cells can be seen in each group grafted vein. After 4 weeks, the experimental group, model group, the control group expression of positive PDGF index were (35.38±2.72),(40.12±3.77),(27.17±2.88), compared among the groups P<0.05. the expression of experimental group, model group and control group were significantly different among groups, the experimental group, model group, the control group expression of positive eNOS index were (56.92±2.56),(50.80±3.67),(43.95±2.60), 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 vein
The expression of eNOS, PDGF gene can be seen in each group grafted vein, the expression of PDGF relative coefficients in experimental group, model group, control group were (1.97±0.09),(2.19±0.06),(1.62±0.09), the expression of PDGF relative coefficient in the model group is higher than 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 among groups. The expression of eNOS relative coefficients in experimental group, model group, control group were (2.18±0.07),(1.92±0.06),(1.63±0.08), the expression of eNOS relative coefficient in the experimental group is higher than in 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 between groups.
4. The results of the correlation
The results calculated by SPSS are that:eNOS is negatively correlated with intimal thickness and that is correlation coefficient is-0.469; PDGF is positive correlated with intimal thickness and that is correlation coefficient is 0.329.
Conclusion
1. Grafted vein stenosis is negative correlationg with eNOS gene expression and positive correlation with PDGF gene expression.
2. Ginsenoside Rbl can promote NOS gene expression and inhibit PDGF gene expression in grafted vein, which related to its effects.
冠脉搭桥术(coronary artery bypass grafting, CABG)是冠状动脉粥样硬化性心脏病主要的治疗方法之一,尤其适用于多支病变和合并心内畸形的患者。乳内动脉、桡动脉和大隐静脉是临床上常用的桥血管,而大隐静脉作为临床上冠脉搭桥常用的主要材料之一,由于位置表浅,取材方便,并且有足够的长度连接主动脉与冠状动脉的远端,使得其能够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、术后血流动力学改变、炎症因子释放等一系列变化引起静脉桥血管术后再狭窄,使得大隐静脉在临床上的应用受到了一定的限制。近年来,一系列预防静脉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,但是总体效果欠佳。有报道称：人参皂苷Rbl对内皮细胞培养基中血管内皮细胞表达血管血小板衍生生长因子(platelet-derived growth factor, PDGF)和内皮型一氧化氮合酶(endothelial nitric oxide synthase, eNOS)产生一定的影响。本实验即是在建立静脉桥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,观察静脉桥血管内膜厚度的变化以及静脉桥血管PDGF和eNOS阳性表达的变化,从而探讨人参皂营Rb1对移植静脉桥血管再狭窄的抑制作用及其机理,为以后的临床应用提供试验基础和理论依据。
材料与方法
45只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”外科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,建立颈外静脉和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形态及厚度变化,免疫组化观察静脉移植血管血小板衍生生长因子、内皮型一氧化氮合酶阳性表达情况,RT-PCR检测静脉移植血管中PDGF. eNOSmRNA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、对照组静脉桥血管的内膜厚度分别为(44.45±3.10)μm.(51.32±2.61)μm、(41.52±2.62)μm,各组间比较P<0.05。
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PDGF、cNOS阳性细胞表达。移植4周后的实验组、模型组、对照组PDGF阳性表达指数分别是(35.38±2.72)、(40.12±3.77)、(27.17±2.80),各组间比较P<0.05；移植4周后的实验组、模型组、对照组eNOS阳性表达指数分别是(56.92±2.56)、(50.80±3.67)、(43.95±2.60),各组间比较P<0.05,实验组、模型组、对照组组间比较有明显差异。
3、移植静脉桥血管RT-PCR结果观察
各组移植静脉血管可见PDGF.eNOS基因表达。PDGF在实验组、模型组、对照组表达的相对系数分别为(1.97±0.09).(2.19±0.06)、(1.62±0.09),PDGF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、对照组组间比较有明显差异。eNOS在实验组、模型组、对照组表达的相对系数分别为(2.18±0.07)、(1.92±0.06)、(1.63±0.08),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、对照组组间比较有明显差异。
4、相关性分析结果
SPSS简单线性模型结果回示：PDGF与血管狭窄呈相关正相关,相关系数为0.329:eNOS与血管狭窄呈负相关,相关系数为-0.469。
结论
1、静脉桥血管狭窄程度与eNOS基因的表达呈负相关,与PDGF基因的表达呈正相关。
2、人参皂苷Rb1能够促进移植血管eNOS基因的表达、抑制PDGF基因的表达,这与其能够抑制血管再狭窄的作用有关。
Background and Objective
CABG (coronary artery bypass grafting, CABG) is one of the important treatments for coronary heart disease. Especially for patients with multi-vessel or intracardiac malformation. 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 restricted in 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, platelet-derived growth factor (PDGF) and 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 Methods
45 New Zealand rabbits were randomly divided into three groups experimental group, model group, 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. After 4 weeks, grafting vein was obtained as sample, the vein intimal thickness, the expression of PDGF and eNOS gene was examined by RT-PCR.
Results
1. The degree of grafted vein stenosis
The picture under-microscope showed that:After 4 weeks, the vascular intimal thickness of experimental group, model group and control group was(44.45±3.10)μm, (51.32±2.61)u.m,(41.52±2.62)μm, the comparison among groups P＜0.05, experimental group, model group and control group were significantly different among groups.
2. The results observed by immunohistochemical staining
eNOS, PDGF positive cells can be seen in each group grafted vein. After 4 weeks, the experimental group, model group, the control group expression of positive PDGF index were (35.38±2.72),(40.12±3.77),(27.17±2.88), compared among the groups P<0.05. the expression of experimental group, model group and control group were significantly different among groups, the experimental group, model group, the control group expression of positive eNOS index were (56.92±2.56),(50.80±3.67),(43.95±2.60), 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 vein
The expression of eNOS, PDGF gene can be seen in each group grafted vein, the expression of PDGF relative coefficients in experimental group, model group, control group were (1.97±0.09),(2.19±0.06),(1.62±0.09), the expression of PDGF relative coefficient in the model group is higher than 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 among groups. The expression of eNOS relative coefficients in experimental group, model group, control group were (2.18±0.07),(1.92±0.06),(1.63±0.08), the expression of eNOS relative coefficient in the experimental group is higher than in 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 between groups.
4. The results of the correlation
The results calculated by SPSS are that:eNOS is negatively correlated with intimal thickness and that is correlation coefficient is-0.469; PDGF is positive correlated with intimal thickness and that is correlation coefficient is 0.329.
Conclusion
1. Grafted vein stenosis is negative correlationg with eNOS gene expression and positive correlation with PDGF gene expression.
2. Ginsenoside Rbl can promote NOS gene expression and inhibit PDGF gene expression in grafted vein, which related to its effects.
引文
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