血流动力学变化对血管重构调控机制初步实验研究
发布时间：2019-06-04 07:06
【摘要】：研究背景许多临床现象显示,机械应激是影响机体修复的重要因素,减轻机械应激可以减轻修复的程度。在临床工作中发现,瘢痕的形成和机械应激密切相关,无机械应激的手术切口瘢痕增生不明显,骨折端的轻微机械应激导致骨痂大量增生,即过度的修复,无机械应激可以获得一期愈合,没有骨痂形成等等,上述事实都说明机械应激是影响组织修复的重要因素。并且很多相关的基础研究也证实此理论,此理论能否在血管上得到证实,血流动力学的改变是否能够导致血管壁结构的重构?本课题组的前期工作中发现：1、喷涂a-氰基丙烯酸酯粘合剂均匀包被血管能够达到固定血管吻合口的目的。2、喷涂粘合剂血管吻合口的狭窄率较低。众多文献报道静脉血管移植到动脉后,静脉血管管壁出现了动脉化的改变。除营养改变外,血流动力学改变应该是导致管壁发生变化的主要因素。另外当前相关研究大多集中于临床较为常见的静脉移到动脉后血流动力学变化引起血管狭窄机制的研究,但对于其它血流动力学变化调控血管重构机制研究较少,本研究以动静脉移植和动静脉瘘两种模型为对象,对血流动力学变化调控血管重构机制进行初步实验研究。目的为了进一步完善血流动力学变化调控血管重构机制理论,并对骨骼肌肉系统出现的“机械应激促进增生,减轻机械应激抑制增生”现象,是否也适用于血流动力学变化调控血管重构进行验证。我们选择了当前研究较少的两种模型进行相关理论初步实验研究,以动静脉移植和动静脉瘘两种模型为对象,对血流动力学参数进行测量,对血管重构中大体、组织、细胞及分子生物水平进行观察研究,并对两要素之间关系进行分析。方法(1)实验动物及血管部位的选择,比格犬和SD大鼠各5只,分别测量两侧颈总动脉和颈静脉以及股动静脉的外径和便于操作有效长度,分析比较适合模型建立的实验动物和血管。(2)动静脉移植模型建立,血流动力学变化调控血管重构机制研究。实验动物为比格犬12只,12-15kg。1将犬左侧颈总动脉1cm切断移植到右侧颈外静脉,结扎左侧颈总动脉,右侧颈总动脉为对照。分别于术后1,2,4周对两组血管进行大体观察;HE染色,病理组织学观测内膜增生厚度；EVG染色,组织病理学观测弹性纤维和胶原纤维含量变化；免疫组化染色观测增值细胞阳性表达;TUNEL技术观测调亡细胞阳性表达;RT-PCR技术观测MMP2,9和eNOS基因表达。(3)动静脉瘘模型建立,血流动力学变化调控血管重构机制研究。实验动物为比格犬12只,12-15kg。1将犬左侧颈总动脉和颈外静脉传统针线吻合,右侧颈总动脉和颈外静脉为对照。分别于术后1,2,4周对两组血管进行大体观察；HE染色,病理组织学观测内膜增生厚度；EVG染色,组织病理学观测弹性纤维和胶原纤维含量变化;免疫组化染色观测增值细胞阳性表达;TUNEL技术观测调亡细胞表达;RT-PCR技术观测MMP2,9和eNOS基因表达。结果(1)实验动物比格犬较SD大鼠颈部血管更有利于手术操作,比格犬更易测量血流动力学变化情况,比格犬血压和人更加接近。(2)动脉移植到静脉后,血流速度和血压明显减小;HE染色观测到内膜厚度减小;EVG染色观测到胶原纤维含量明显减少,弹性纤维含量变化无统计学意义；免疫组化染色,观测到PCNA指数无明显变化;凋亡染色,观测到凋亡细胞明显增加,凋亡指数增大;电镜观察,中膜平滑肌细胞减少;RT-PCR观测到MMP2,9和eNOS均出现基因表达增加。(3)动静脉瘘术后,静脉和动脉段血流速度增加,静脉血压上升,动脉血压小幅度降低;HE染色,测量动脉段内膜厚度增加,静脉段内膜厚度增加明显;EVG染色观测到动脉段胶原纤维含量增加,静脉段胶原纤维含量明显增加,弹性纤维含量变化无统计学意义；免疫组化染色行观测计算,动脉段PCNA指数增大,静脉段PCNA指数明显增大;凋亡染色后观测到动脉段和静脉段凋亡细胞无明显增加,凋亡指数无明显增大；电镜观察,动脉段内膜和中膜平滑肌细胞增多,静脉段内膜和中膜平滑肌细胞明显增加;RT-PCR观测到动脉段MMP2,9和eON9表达减少,静脉段MMP2,9和eONS表达明显减少。结论通过建立动静脉移植模型和动静脉痿模型,从血流动力学变化调控血管重构机制角度进行实验研究,进一步完善相关理论。血流动力学改变后,机械应力信号传导到血管壁细胞,酶(MMP2,9和eONS等)及细胞因子的表达出现变化,血管壁细胞增殖凋亡及迁移机制改变,细胞外基质(胶原纤维和弹性纤维等)代谢机制改变,表现出血管壁结构的变化。进一步证明血流动力学变化对血管重构的调控作用,并且血流动力学改变程度不同,血管重构出现明显不同变化。同时“机械应激促进增生,减轻机械应激抑制增生”理论假设在血管重构过程中进一步得到验证。
[Abstract]:The research background has shown that the mechanical stress is an important factor that affects the repair of the body, and the degree of repair can be reduced by reducing the mechanical stress. During the clinical work, the formation of the mechanical stress and the mechanical stress are closely related, the non-mechanical stress-free operation incision is not obvious, the slight mechanical stress at the end of the fracture leads to a large amount of hyperplasia of the bone, that is, the excessive repair, the non-mechanical stress can obtain the first-stage healing, No bone formation and so on, the fact that mechanical stress is an important factor that affects tissue repair. And many related basic studies have also confirmed this theory, can this theory be confirmed on the blood vessel, whether the change of hemodynamics can lead to the reconstruction of the vessel wall structure? It was found in the earlier work of the research group that:1. The uniform coating of a-cyanoacrylate adhesive on the blood vessel can achieve the purpose of fixing the blood vessel anastomosis. A large number of literature reported that the vascular wall of the vein had an arterial change after the vein graft was transplanted to the artery. In addition to that change in nutrition, the change in hemodynamics should be a major factor in the change in the tube wall. in addition, most of that current study focus on the study of the mechanism of vascular stenosis due to the change in the hemodynamic change of the clinically more common vein to the post-arterial blood flow, but less research on the regulation of the vascular remodeling mechanism for other hemodynamic changes, In this study, two models of the arteriovenous graft and the arteriovenous graft were used as the target, and the mechanism of the blood flow dynamic change and the regulation of the vascular remodeling was studied. Objective In order to further improve the theory of blood flow dynamics and control the mechanism of vascular remodeling, and to study the "Mechanical stress promotes proliferation and relieves mechanical stress and inhibits proliferation" of the skeletal muscle system, it is also applicable to the changes of blood flow dynamics to control the vascular remodeling. In this paper, we selected two models of the current study to study the correlation theory. The two models of the arteriovenous graft and the arteriovenous graft were used as the target, the parameters of the blood flow were measured, and the general, the tissue, the cell and the molecular biological level in the blood vessel reconstruction were observed and studied. And the relationship between the two elements is analyzed. Methods (1) The external diameter of the common carotid artery and the jugular vein and the femoral vein and the effective length of the femoral vein were measured respectively in the experimental animals and the blood vessel, and the experimental animals and the blood vessels established by the model were compared. (2) The establishment of the arteriovenous graft model, the regulation of the blood flow dynamics and the mechanism of the reconstruction of the blood vessel. The experimental animals were 12-15kg beagle dogs.1 cm of the left common carotid artery of the dog was cut and transplanted to the right external jugular vein, and the left common carotid artery and the right common carotid artery were ligated. The two groups of blood vessels were generally observed at 1,2 and 4 weeks after operation, and the thickness of the intimal hyperplasia was observed by HE staining and pathology. The changes of the content of the elastic fibers and the collagen fibers were observed by the histopathological observation, and the positive expression of the value-added cells was observed by immunohistochemical staining. The expression of MMP2,9 and eNOS gene was observed by RT-PCR. (3) The establishment of the arteriovenous blood vessel model, the regulation of the blood flow dynamics and the mechanism of the reconstruction of the blood vessel. The experimental animals were 12-15kg beagle dogs. The left common carotid artery and the external jugular vein of the dog were matched with the traditional needle-and-needle, and the right-hand common carotid artery and the external jugular vein were used as the control. The two groups of blood vessels were generally observed at 1,2 and 4 weeks after operation, and the thickness of the intimal hyperplasia was observed by HE staining and pathology. The changes of the content of the elastic fibers and the collagen fibers were observed by the histopathological observation, and the positive expression of the value-added cells was observed by immunohistochemical staining. The expression of MMP2,9 and eNOS gene was observed by RT-PCR. Results (1) The experimental animal beagle dog was more beneficial to the operation of the neck vessel in the SD rat, and the beagle dogs were more likely to measure the hemodynamic changes, and the beagle dogs were closer to the blood pressure and human blood pressure. (2) The blood flow velocity and blood pressure were significantly reduced after the artery was transplanted to the vein; the thickness of the intima was observed with HE staining; the content of the collagen fibers was significantly reduced by the EVG staining, and the content of the elastic fibers was not statistically significant; and the expression of PCNA was observed without significant change in the immunohistochemical staining. The apoptotic cells increased significantly and the apoptotic index increased. The expression of MMP2,9 and eNOS in MMP2,9 and eNOS was increased by RT-PCR. (3) The blood flow velocity of the vein and the arterial segment increased, the blood pressure of the vein and the small amplitude of the arterial blood pressure were decreased after the operation of the arteriovenous system, the thickness of the intima of the arterial segment was increased, the thickness of the inner membrane of the vein was increased, and the content of the collagen fiber in the arterial segment was observed by the EVG staining. The content of collagen fibers in the vein segment was significantly increased, and the content of the elastic fibers was not statistically significant; the expression of PCNA in the arterial segment increased with the observation of the immunohistochemical staining, and the PCNA index of the venous segment increased significantly; the apoptotic cells observed in the arterial segment and the vein segment did not increase significantly after the apoptosis was stained. The expression of MMP2,9 and eON9 in the arterial segment was decreased, and the expression of MMP2,9 and eON9 in the venous segment was significantly reduced. Conclusion By establishing the model of arteriovenous graft and the model of arteriovenous malformation, the angle of the vascular remodeling is controlled by the change of blood flow dynamics, and the relevant theory is further improved. after the hemodynamic changes, the mechanical stress signal is conducted to the blood vessel wall cells, the expression of the enzymes (MMP2,9 and eONS, etc.) and the cytokines changes, the proliferation and apoptosis of the vascular wall cells and the change of the migration mechanism, the metabolic mechanisms of the extracellular matrix (collagen fibers and elastic fibers, etc.) are changed, Showing the change in the structure of the bleeding tube wall. The effect of the changes of hemodynamics on the vascular remodeling was further demonstrated, and the degree of the change of the hemodynamics was different, and the changes of the blood vessel remodeling were significantly different. At the same time, the theoretical hypothesis of the "Mechanical stress promotes proliferation and relieves mechanical stress and inhibits proliferation" is further verified in the course of blood vessel reconstruction.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R654.3
[Abstract]:The research background has shown that the mechanical stress is an important factor that affects the repair of the body, and the degree of repair can be reduced by reducing the mechanical stress. During the clinical work, the formation of the mechanical stress and the mechanical stress are closely related, the non-mechanical stress-free operation incision is not obvious, the slight mechanical stress at the end of the fracture leads to a large amount of hyperplasia of the bone, that is, the excessive repair, the non-mechanical stress can obtain the first-stage healing, No bone formation and so on, the fact that mechanical stress is an important factor that affects tissue repair. And many related basic studies have also confirmed this theory, can this theory be confirmed on the blood vessel, whether the change of hemodynamics can lead to the reconstruction of the vessel wall structure? It was found in the earlier work of the research group that:1. The uniform coating of a-cyanoacrylate adhesive on the blood vessel can achieve the purpose of fixing the blood vessel anastomosis. A large number of literature reported that the vascular wall of the vein had an arterial change after the vein graft was transplanted to the artery. In addition to that change in nutrition, the change in hemodynamics should be a major factor in the change in the tube wall. in addition, most of that current study focus on the study of the mechanism of vascular stenosis due to the change in the hemodynamic change of the clinically more common vein to the post-arterial blood flow, but less research on the regulation of the vascular remodeling mechanism for other hemodynamic changes, In this study, two models of the arteriovenous graft and the arteriovenous graft were used as the target, and the mechanism of the blood flow dynamic change and the regulation of the vascular remodeling was studied. Objective In order to further improve the theory of blood flow dynamics and control the mechanism of vascular remodeling, and to study the "Mechanical stress promotes proliferation and relieves mechanical stress and inhibits proliferation" of the skeletal muscle system, it is also applicable to the changes of blood flow dynamics to control the vascular remodeling. In this paper, we selected two models of the current study to study the correlation theory. The two models of the arteriovenous graft and the arteriovenous graft were used as the target, the parameters of the blood flow were measured, and the general, the tissue, the cell and the molecular biological level in the blood vessel reconstruction were observed and studied. And the relationship between the two elements is analyzed. Methods (1) The external diameter of the common carotid artery and the jugular vein and the femoral vein and the effective length of the femoral vein were measured respectively in the experimental animals and the blood vessel, and the experimental animals and the blood vessels established by the model were compared. (2) The establishment of the arteriovenous graft model, the regulation of the blood flow dynamics and the mechanism of the reconstruction of the blood vessel. The experimental animals were 12-15kg beagle dogs.1 cm of the left common carotid artery of the dog was cut and transplanted to the right external jugular vein, and the left common carotid artery and the right common carotid artery were ligated. The two groups of blood vessels were generally observed at 1,2 and 4 weeks after operation, and the thickness of the intimal hyperplasia was observed by HE staining and pathology. The changes of the content of the elastic fibers and the collagen fibers were observed by the histopathological observation, and the positive expression of the value-added cells was observed by immunohistochemical staining. The expression of MMP2,9 and eNOS gene was observed by RT-PCR. (3) The establishment of the arteriovenous blood vessel model, the regulation of the blood flow dynamics and the mechanism of the reconstruction of the blood vessel. The experimental animals were 12-15kg beagle dogs. The left common carotid artery and the external jugular vein of the dog were matched with the traditional needle-and-needle, and the right-hand common carotid artery and the external jugular vein were used as the control. The two groups of blood vessels were generally observed at 1,2 and 4 weeks after operation, and the thickness of the intimal hyperplasia was observed by HE staining and pathology. The changes of the content of the elastic fibers and the collagen fibers were observed by the histopathological observation, and the positive expression of the value-added cells was observed by immunohistochemical staining. The expression of MMP2,9 and eNOS gene was observed by RT-PCR. Results (1) The experimental animal beagle dog was more beneficial to the operation of the neck vessel in the SD rat, and the beagle dogs were more likely to measure the hemodynamic changes, and the beagle dogs were closer to the blood pressure and human blood pressure. (2) The blood flow velocity and blood pressure were significantly reduced after the artery was transplanted to the vein; the thickness of the intima was observed with HE staining; the content of the collagen fibers was significantly reduced by the EVG staining, and the content of the elastic fibers was not statistically significant; and the expression of PCNA was observed without significant change in the immunohistochemical staining. The apoptotic cells increased significantly and the apoptotic index increased. The expression of MMP2,9 and eNOS in MMP2,9 and eNOS was increased by RT-PCR. (3) The blood flow velocity of the vein and the arterial segment increased, the blood pressure of the vein and the small amplitude of the arterial blood pressure were decreased after the operation of the arteriovenous system, the thickness of the intima of the arterial segment was increased, the thickness of the inner membrane of the vein was increased, and the content of the collagen fiber in the arterial segment was observed by the EVG staining. The content of collagen fibers in the vein segment was significantly increased, and the content of the elastic fibers was not statistically significant; the expression of PCNA in the arterial segment increased with the observation of the immunohistochemical staining, and the PCNA index of the venous segment increased significantly; the apoptotic cells observed in the arterial segment and the vein segment did not increase significantly after the apoptosis was stained. The expression of MMP2,9 and eON9 in the arterial segment was decreased, and the expression of MMP2,9 and eON9 in the venous segment was significantly reduced. Conclusion By establishing the model of arteriovenous graft and the model of arteriovenous malformation, the angle of the vascular remodeling is controlled by the change of blood flow dynamics, and the relevant theory is further improved. after the hemodynamic changes, the mechanical stress signal is conducted to the blood vessel wall cells, the expression of the enzymes (MMP2,9 and eONS, etc.) and the cytokines changes, the proliferation and apoptosis of the vascular wall cells and the change of the migration mechanism, the metabolic mechanisms of the extracellular matrix (collagen fibers and elastic fibers, etc.) are changed, Showing the change in the structure of the bleeding tube wall. The effect of the changes of hemodynamics on the vascular remodeling was further demonstrated, and the degree of the change of the hemodynamics was different, and the changes of the blood vessel remodeling were significantly different. At the same time, the theoretical hypothesis of the "Mechanical stress promotes proliferation and relieves mechanical stress and inhibits proliferation" is further verified in the course of blood vessel reconstruction.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R654.3
【参考文献】
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