肝豆灵对Wilson病患者脑血流动力学和血管损伤因子及TX小鼠血管损伤病理研究
发布时间：2018-08-27 14:21
【摘要】：目的探讨WD患者是否存脑血流改变和血管损害;观察肝豆灵对Wilson病患者脑血管损害的影响;观察肝豆灵对TX小鼠脑血管组织的病理形态学变化。方法临床部分:60例WD患者与健康组比较,观察2组超声检测(超声脑血管功能检测仪、经颅多普勒彩超)、磁共振灌注成像、血管损伤因子(血管性血友病因子(v WF)、血栓调节蛋白(TM)、内皮细胞蛋白C受体(EPCR)、同型半胱氨酸(HCY))变化,上述60例WD患者随机分为治疗组和对照组,治疗组采用肝豆灵联合西药二巯基丙磺酸钠(DMPS)治疗,对照组仅使用DMPS治疗,观察2组治疗前后上述指标变化。实验部分:TX小鼠随机分为模型组、肝豆灵组、青霉胺组,另DL小鼠设为对照组,4组经不同处理后,检测上述血管损伤因子变化,观察光镜下脑血管病理变化,观察电镜下细胞超微结构变化,免疫组化观察ICAM-1和VCAM-1在脑血管上的表达。结果临床部分:(1)超声脑血管功能检测结果:WD组双侧颈总动脉最大流速、最小流速平均流速、平均流量低于健康组(P0.05);WD组双侧颈总动脉动态阻力、双侧外周阻力、脉搏波速、特性阻抗数值高于健康组(P0.05)。治疗组治疗后与治疗前比较双侧颈总动脉最大流速、最小流速、平均流速、平均血流量均提升(P0.05);治疗组治疗后与治疗前比较双侧外周阻力、动态阻力、脉搏波速、特性阻抗改变无统计学意义(P0.05)。对照组双侧颈总动脉各项指标改变均无统计学意义(P0.05)。(2)TCD结果:WD组LMCA、LACA血管收缩期峰值流速低于健康组(P0.05);RMCA、RACA血管收缩期峰值流速低于健康组(P0.01);RPCA血管舒张末期血流速度低于健康组(P0.05);LMCA、LACA、LPCA、RMCA、RACA、血管舒张末期血流速度低于健康组(P0.01),LMCA、LACA、RMCA、RACA血管平均流速低于健康组(P0.01);LPCA、RPCA管平均流速低于健康组(P0.05);WD组各血管PI值与健康组比较差异无统计学意义(P0.05)。经治疗后,治疗组LMCA、LACA、RMCA、RACA的收缩期峰值速度较治疗前升高(P0.01),LPCA、RPCA的收缩期峰值速度较治疗前升高(P0.05),LMCA、LACA、RMCA、RACA的舒张末期速度较治疗前升高(P0.01),LPCA、RPCA的舒张末期速度较治疗前升高(P0.05);LMCA、LACA、RMCA、RACA、LPCA、RPCA的平均血流速度较治疗前升高(P0.01);各血管PI值疗前疗后比较无统计学意义(P0.05)。对照组各血管收缩期峰值速度、舒张末期速度、平均血流速度、PI值疗前疗后无统计学意义(P0.05)。治疗组治疗后LACA、RMCA收缩期峰值速度和平均速度与对照组治疗后比较(P0.05);治疗组治疗后RACA收缩期峰值速度和平均速度与对照组治疗后比较(P0.01)。(3)磁共振灌注成像结果:与健康组比较,WD组双侧丘脑r CBF无统计学意义(P0.05),双侧尾状核区域、双侧尾状核头区域r CBF数值降低(P0.01)。治疗后治疗组双侧尾状核头区域、双侧豆状核区域r CBF较治疗前升高(P0.05);对照组各区域数值变化无统计学意义(P0.05)。治疗组治疗后右侧豆状核区域r CBF数值较对照组治疗后明显升高(P0.01);左侧豆状核区域r CBF数值较对照组治疗后升高(P0.05)。(4)血管损伤因子结果:WD组HCY、v WF、TM、EPCR与健康组比较数值均升高(P0.01)。经治疗后,治疗组Hcy下降有显著性差异(P0.05),v WF、TM、EPCR数值均下降有极显著性差异(P0.01)。治疗组治疗后v WF、EPCR数值均低于于对照组治疗后(P0.01);TM数值低于对照组治疗(P0.05)。实验部分:(1)血管损伤因子测定:与对照组比较,模型组Hcy、v WF、TM、EPCR数值明显升高(P0.01)。与模型组比较,肝豆灵组Hcy、v WF、EPCR数值下降(P0.05),TM数值明显下降(P0.01)。与模型组比较,青霉胺组Hcy、v WF、TM、EPCR数值下降,无统计学意义(P0.05)。与青霉胺组比较,肝豆灵组Hcy、v WF、TM、EPCR数值下降(P0.05)。(2)脑血管病理学改变:1H-E染色:可见模型组、肝豆灵组、青霉胺组脑血管内皮细胞有不同程度水肿变性,神经元细胞有不同程度变性及坏死,改变以模型组为著,青霉胺组、肝豆灵组与模型组相比损伤轻。2透射电镜观测:脑组织可见脑血管内皮细胞和神经元不同程度变性及坏死,线粒体空泡化,嵴模糊甚至消失,以模型组损伤最重,治疗后青霉胺组、肝豆灵组改善,且肝豆灵组改善较明显。(3)免疫组化结果:1ICAM-1免疫染色阳性血管的变化:模型组与对照组比较ICAM-1表达增多(P0.01);肝豆灵组与模型组比较表达减少(P0.01);青霉胺组与模型组比较表达减少(P0.05);肝豆灵组与青霉胺组比较表达较少(P0.05)。2VCAM-1免疫染色阳性血管的变化:模型组与对照组比较ICAM-1表达增多(P0.01);肝豆灵组与模型组比较表达较少(P0.01);青霉胺组与模型组比较表达减少(P0.01);肝豆灵组与青霉胺组比较表达减少(P0.05)。结论(1)WD患者存在血管损伤;表现为脑中小动脉血流速度下降;豆状核区域、尾状核头区域局部脑血流量减低。血管损伤因子(Hcy、v WF、TM、EPCR)异常增高。而大血管颈总动脉血流动力学改变不明显。(2)肝豆灵能够提高痰瘀互结型WD患者中小血管血流,增加豆状核区域、尾状核头区域脑血流量;减少血管损害因子(Hcy、v WF、TM、EPCR)水平。(3)WD模型TX小鼠同样血管存在损伤,血管内皮细胞和神经元不同程度变性及坏死,导致血管损伤因子(Hcy、v WF、TM、EPCR、ICAM-1、VCAM-1)水平升高。(4)肝豆灵能够改善TX小鼠血管损伤,减轻内皮细胞及神经元病理损伤,减少血管损伤因子(Hcy、v WF、TM、EPCR、ICAM-1、VCAM-1)表达,肝豆灵对WD模型TX小鼠的血管具有一定的保护作用。
[Abstract]:Objective To investigate whether there are cerebral blood flow changes and vascular damage in patients with WD, to observe the effect of Gandouling on cerebral vascular damage in patients with Wilson disease, and to observe the pathomorphological changes of cerebrovascular tissue in TX mice. Puller color Doppler ultrasound, magnetic resonance perfusion imaging, vascular injury factor (von Willebrand factor (v WF), thrombomodulin (TM), endothelial cell protein C receptor (EPCR), homocysteine (HCY) changes, the above 60 cases of WD patients were randomly divided into treatment group and control group, the treatment group was treated with Gandouling combined with Western medicine sodium dimercaptopropionate (DMPS), on. The experimental part: TX mice were randomly divided into model group, Gandouling group, Penicillin group, and DL mice as control group. After different treatments, the changes of above-mentioned vascular injury factors were detected, the pathological changes of cerebrovascular under light microscope were observed, and the ultrastructural changes of cells under electron microscope were observed. Results: (1) Ultrasound cerebrovascular function test showed that the maximum and minimum common carotid artery flow velocity in WD group were lower than those in healthy group (P 0.05), and the dynamic resistance, peripheral resistance, pulse wave velocity and characteristic impedance of common carotid artery in WD group were lower than those in healthy group (P 0.05). After treatment, the maximum, minimum, average and average blood flow of the bilateral common carotid artery were increased (P 0.05), while the peripheral resistance, dynamic resistance, pulse wave velocity and characteristic impedance of the treatment group were not significantly different from those before treatment (P 0.05). TCD results: The peak systolic velocity of LMCA and LACA in WD group was lower than that of healthy group (P 0.05); the peak systolic velocity of RMCA and RACA was lower than that of healthy group (P 0.01); the late diastolic velocity of RPCA was lower than that of healthy group (P 0.05); LMCA, LACA, LPCA, RMCA, RACA, and the end diastolic velocity of blood vessels were lower than that of healthy group (P 0.05). The mean velocity of LMCA, LACA, RMCA and RACA was lower than that of healthy group (P 0.01), LPCA and RPCA were lower than that of healthy group (P 0.05), and the PI value of each vessel in WD group was not significantly different from that of healthy group (P 0.05). The peak systolic velocity of LMCA, LACA, RMCA, RACA was higher than that before treatment (P 0.05), and the end diastolic velocity of LPCA, RPCA was higher than that before treatment (P 0.01); the mean blood flow velocity of LMCA, LACA, RMCA, RACA, LPCA, RPCA was higher than that before treatment (P 0.01); there was no significant difference between pre-and post-treatment (P 0.01). The peak systolic velocity, end-diastolic velocity, mean blood flow velocity and PI value of the control group had no statistical significance before and after treatment (P 0.05). Comparison after treatment (P 0.01). (3) Magnetic resonance perfusion imaging results: Compared with the healthy group, there was no significant difference in bilateral thalamus R CBF (P 0.05), bilateral caudate nucleus region, bilateral caudate nucleus head region R CBF decreased (P 0.01). There was no significant difference in the domain values (P 0.05). After treatment, the R CBF values in the right lenticular nucleus region in the treatment group were significantly higher than those in the control group (P 0.01); the R CBF values in the left lenticular nucleus region were significantly higher than those in the control group (P 0.05). (4) Vascular injury factor results: HCY, V WF, TM, EPCR values in WD group were higher than those in the healthy group (P 0.01). After treatment, the values of VWF, TM and EPCR in the treatment group were lower than those in the control group (P 0.01), and the values of TM were lower than those in the control group (P 0.05). Compared with the model group, the values of Hcy, V WF, EPCR decreased (P 0.05), TM decreased significantly (P 0.01). Compared with the model group, the values of Hcy, V WF, TM and EPCR in the penicillamine group decreased, with no statistical significance (P 0.05). Compared with the penicillamine group, the values of Hcy, V WF, TM and EPCR in the Gandouling group decreased (P 0.05). Changes: 1H-E staining: It was observed that the cerebral vascular endothelial cells of model group, Gandouling group and Penicillin group had edema and degeneration in different degrees, and neurons had degeneration and necrosis in different degrees. Mitochondrial vacuolation, cristae blurred and even disappeared in different degrees of degeneration and necrosis. Injury in model group was the most serious. After treatment, penicillamine group and Gandouling group were improved, and Gandouling group was improved significantly. (3) Immunohistochemical results: 1 ICAM-1 immunostaining positive vascular changes: The expression of ICAM-1 increased in model group compared with control group (P 0.01); Gandouling group and Gandouling group were improved significantly. (3) Immunohistochemical results: 1 ICAM-1 The expression of ICAM-1 was decreased in model group (P 0.01), decreased in penicillamine group and model group (P 0.05), less in Gandouling group and penicillamine group (P 0.05). The expression of ICAM-1 was increased in model group and control group (P 0.01), less in Gandouling group and model group (P 0.01), less in Penicillamine group and penicillamine group (P 0.05). CONCLUSION (1) Vascular injury exists in WD patients, which is manifested by decreased blood flow velocity of small and medium cerebral arteries, decreased regional cerebral blood flow in the lentiform nucleus region and caudate nucleus region, abnormal increase of vascular injury factors (Hcy, V WF, TM, EPCR), and abnormal increase of common carotid artery in large vessels. (2) Gandouling could increase the blood flow of small and medium vessels, increase the cerebral blood flow in the area of lenticular nucleus and caudate nucleus, and decrease the levels of vascular damage factors (Hcy, V WF, TM, EPCR). (3) TX mice of WD model also had vascular damage, vascular endothelial cells and neurons degenerated and deteriorated in different degrees. (4) Gandouling can improve the vascular injury of TX mice, alleviate the pathological injury of endothelial cells and neurons, reduce the expression of vascular injury factors (Hcy, V WF, TM, EPCR, ICAM-1, VCAM-1). Gandouling has a protective effect on the blood vessels of WD model TX mice.
【学位授予单位】：安徽中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R742.4
本文编号：2207547
[Abstract]:Objective To investigate whether there are cerebral blood flow changes and vascular damage in patients with WD, to observe the effect of Gandouling on cerebral vascular damage in patients with Wilson disease, and to observe the pathomorphological changes of cerebrovascular tissue in TX mice. Puller color Doppler ultrasound, magnetic resonance perfusion imaging, vascular injury factor (von Willebrand factor (v WF), thrombomodulin (TM), endothelial cell protein C receptor (EPCR), homocysteine (HCY) changes, the above 60 cases of WD patients were randomly divided into treatment group and control group, the treatment group was treated with Gandouling combined with Western medicine sodium dimercaptopropionate (DMPS), on. The experimental part: TX mice were randomly divided into model group, Gandouling group, Penicillin group, and DL mice as control group. After different treatments, the changes of above-mentioned vascular injury factors were detected, the pathological changes of cerebrovascular under light microscope were observed, and the ultrastructural changes of cells under electron microscope were observed. Results: (1) Ultrasound cerebrovascular function test showed that the maximum and minimum common carotid artery flow velocity in WD group were lower than those in healthy group (P 0.05), and the dynamic resistance, peripheral resistance, pulse wave velocity and characteristic impedance of common carotid artery in WD group were lower than those in healthy group (P 0.05). After treatment, the maximum, minimum, average and average blood flow of the bilateral common carotid artery were increased (P 0.05), while the peripheral resistance, dynamic resistance, pulse wave velocity and characteristic impedance of the treatment group were not significantly different from those before treatment (P 0.05). TCD results: The peak systolic velocity of LMCA and LACA in WD group was lower than that of healthy group (P 0.05); the peak systolic velocity of RMCA and RACA was lower than that of healthy group (P 0.01); the late diastolic velocity of RPCA was lower than that of healthy group (P 0.05); LMCA, LACA, LPCA, RMCA, RACA, and the end diastolic velocity of blood vessels were lower than that of healthy group (P 0.05). The mean velocity of LMCA, LACA, RMCA and RACA was lower than that of healthy group (P 0.01), LPCA and RPCA were lower than that of healthy group (P 0.05), and the PI value of each vessel in WD group was not significantly different from that of healthy group (P 0.05). The peak systolic velocity of LMCA, LACA, RMCA, RACA was higher than that before treatment (P 0.05), and the end diastolic velocity of LPCA, RPCA was higher than that before treatment (P 0.01); the mean blood flow velocity of LMCA, LACA, RMCA, RACA, LPCA, RPCA was higher than that before treatment (P 0.01); there was no significant difference between pre-and post-treatment (P 0.01). The peak systolic velocity, end-diastolic velocity, mean blood flow velocity and PI value of the control group had no statistical significance before and after treatment (P 0.05). Comparison after treatment (P 0.01). (3) Magnetic resonance perfusion imaging results: Compared with the healthy group, there was no significant difference in bilateral thalamus R CBF (P 0.05), bilateral caudate nucleus region, bilateral caudate nucleus head region R CBF decreased (P 0.01). There was no significant difference in the domain values (P 0.05). After treatment, the R CBF values in the right lenticular nucleus region in the treatment group were significantly higher than those in the control group (P 0.01); the R CBF values in the left lenticular nucleus region were significantly higher than those in the control group (P 0.05). (4) Vascular injury factor results: HCY, V WF, TM, EPCR values in WD group were higher than those in the healthy group (P 0.01). After treatment, the values of VWF, TM and EPCR in the treatment group were lower than those in the control group (P 0.01), and the values of TM were lower than those in the control group (P 0.05). Compared with the model group, the values of Hcy, V WF, EPCR decreased (P 0.05), TM decreased significantly (P 0.01). Compared with the model group, the values of Hcy, V WF, TM and EPCR in the penicillamine group decreased, with no statistical significance (P 0.05). Compared with the penicillamine group, the values of Hcy, V WF, TM and EPCR in the Gandouling group decreased (P 0.05). Changes: 1H-E staining: It was observed that the cerebral vascular endothelial cells of model group, Gandouling group and Penicillin group had edema and degeneration in different degrees, and neurons had degeneration and necrosis in different degrees. Mitochondrial vacuolation, cristae blurred and even disappeared in different degrees of degeneration and necrosis. Injury in model group was the most serious. After treatment, penicillamine group and Gandouling group were improved, and Gandouling group was improved significantly. (3) Immunohistochemical results: 1 ICAM-1 immunostaining positive vascular changes: The expression of ICAM-1 increased in model group compared with control group (P 0.01); Gandouling group and Gandouling group were improved significantly. (3) Immunohistochemical results: 1 ICAM-1 The expression of ICAM-1 was decreased in model group (P 0.01), decreased in penicillamine group and model group (P 0.05), less in Gandouling group and penicillamine group (P 0.05). The expression of ICAM-1 was increased in model group and control group (P 0.01), less in Gandouling group and model group (P 0.01), less in Penicillamine group and penicillamine group (P 0.05). CONCLUSION (1) Vascular injury exists in WD patients, which is manifested by decreased blood flow velocity of small and medium cerebral arteries, decreased regional cerebral blood flow in the lentiform nucleus region and caudate nucleus region, abnormal increase of vascular injury factors (Hcy, V WF, TM, EPCR), and abnormal increase of common carotid artery in large vessels. (2) Gandouling could increase the blood flow of small and medium vessels, increase the cerebral blood flow in the area of lenticular nucleus and caudate nucleus, and decrease the levels of vascular damage factors (Hcy, V WF, TM, EPCR). (3) TX mice of WD model also had vascular damage, vascular endothelial cells and neurons degenerated and deteriorated in different degrees. (4) Gandouling can improve the vascular injury of TX mice, alleviate the pathological injury of endothelial cells and neurons, reduce the expression of vascular injury factors (Hcy, V WF, TM, EPCR, ICAM-1, VCAM-1). Gandouling has a protective effect on the blood vessels of WD model TX mice.
【学位授予单位】：安徽中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R742.4
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