基于PLGF探讨通心络对脑缺血后微血管内皮介导神经元保护的分子机制

发布时间：2018-05-08 21:12

本文选题：通心络 + 脑微血管内皮细胞　；参考：《北京中医药大学》2014年博士论文

【摘要】：脑血管病是导致人类死亡的三大疾病之一,具有发病率高、致残率高、死亡率高和复发率高的特点。其中缺血性脑血管病(Ischemic Cerebral Vascular Disease,ICVD)在世界范围内约占脑血管病的55%～80%,其发病率约是出血性脑血管疾病的3倍。因此,有效地防治缺血性脑血管病一直是医学领域的重要研究课题之一。现代医学对于本病的治疗主要为溶栓及神经元保护两个方面。但迄今仍无公认的具有显著临床疗效的药物,这就迫使我们在缺血性脑血管病的治疗中寻找新出路。随着人们应用中医药对缺血性脑病治疗的不断探索,近年来的研究重点立足于神经血管单元的概念,通过保护血管内皮细胞来减轻脑组织损伤。基于中医络病理论研发的通心络在基础与临床研究中都具有保护缺血区微血管完整性等作用,为脑缺血治疗开辟了一个新途径。而现代研究已发现脑微血管内皮细胞(Brain microvascular endothelial cells,BMEC)的旁分泌功能对神经元的存活有重要调节作用,不同脑微血管内皮细胞条件培养液中的差异蛋白可能是实现其调节作用的物质基础,如胎盘生长因子(Placenta growthfactor,PLGF)对缺氧缺糖神经元有明显的保护作用。本研究以PLGF为切入点,分别从整体和离体研究探讨脑缺血缺氧后在通心络作用下PLGF在脑微血管内皮细胞和脑组织分泌表达的规律及其保护神经元的分子生物学机制,为通心络治疗缺血性脑病提供科学依据。研究目标本研究在"营卫交会、由络以通"理论指导下,以PLGF为切入点,观察通心络对实验性大鼠局灶性脑缺血损伤的保护作用及PLGF表达的影响;探讨脑缺血状态下脑微血管内皮细胞条件液对神经元活性及PLGF含量的影响,进而揭示PLGF保护神经元的分子生物学机制及内皮细胞介导的脑损伤保护环节,围绕神经血管单元探讨通心络干预脑梗死的作用机制,揭示既往提出的"缺血区微血管保护—脑梗死治疗新靶点"的科学内涵,对于指导治疗脑血管疾病的中医药现代化研究具有重要启示。研究方法和内容1.线栓法建立大鼠大脑中动脉阻塞的脑缺血模型。实验动物随机分为假手术组、模型组、通心络组、丁苯酞组(阳性对照药),存活期为1d、3d。各组动物在手术清醒后、术后1d、3d及取材前分别进行神经功能评分,分数越高损伤越重,前后两者比值表示神经功能恢复情况;采用HE染色观察脑组织的病理变化;免疫荧光技术检测MAP-2的表达以观察神经元的变化,确证通心络的疗效和对神经元的保护作用。2.动物分组及存活期同上。免疫组化法检测脑组织中PLGF表达,免疫荧光双标方法(PLGF+CD31、PLGF+MAP-2)分析PLGF在脑血管内皮细胞和神经元的表达规律;酶联免疫吸附法(Elisa法)检测大鼠脑组织匀浆及血清中PLGF的含量,探索脑缺血损伤大鼠脑组织中的PLGF表达部位及细胞类型。3.原代培养脑微血管内皮细胞,采用CCK-8法观察不同浓度通心络含药血清在作用不同时间后对脑微血管内皮细胞活性的影响以确定其给药的最佳浓度和时间。建立体外脑微血管内皮细胞氧糖剥夺模型,采用CCK-8法观察通心络含药血清对体外缺血损伤内皮细胞活性的影响;利用硝酸银还原法检测其上清液中一氧化氮(nitric oxide,NO)、Elisa法检测血管性血友病因子(von Willebrand factor,vWF)、PLGF的含量,分析通心络对脑微血管内皮细胞功能及分泌PLGF的影响。4.原代培养大鼠皮层神经元,采用氧糖剥夺法建立体外神经元缺血模型。收集通心络含药血清干预下脑微血管内皮细胞不同条件液作用于培养的神经元,CCK-8法检测神经元存活率、WST法检测超氧化物歧化酶(Superoxide Dismutase,SOD)活性、脂质氧化检测法检测丙二醛(malondialdehyde,MDA)含量变化,以探索通心络可能通过调节内皮细胞的旁分泌功能保护神经元的机制。5.原代培养大鼠皮层神经元并建立体外神经元缺血模型。在MAPK信号通路阻断剂和PI-3K信号通路阻断剂作用下,CCK-8法检测PLGF干预前后神经元活性及存活率,Tunel法检测神经元凋亡,免疫蛋白印记法检测不同信号通路上相关蛋白Bax、Bcl2和p-ERK1/2的表达变化,进一步从细胞信号转导通路的角度探索PLGF在缺血状态下抑制神经元凋亡的分子生物学机制。结果1.整体实验部分(1)动物造模醒后各组神经功能评分无明显差异;给药后,各时间点通心络组与模型组相比评分降低;将取材前与造模醒后评分的比值进行分析,相对于1d模型组,通心络组的两次评分比值显著下降。相对于3d模型组,通心络组和阳性对照药组的两次评分比值显著下降。(2)HE染色结果显示假手术组脑组织结构完整,细胞排列整齐。模型组缺血侧脑组织皮层组织结构紊乱,神经元细胞核固缩、浓染,部分神经元消失。通心络组各时间点的脑组织病理变化均较模型组有不同程度的减轻,存活神经元数目有所增加,丁苯酞组皮层缺血区的损伤比通心络组严重。(3)假手术组脑组织MAP-2荧光强度高。模型组患侧皮层MAP-2的表达明显减少,尤以梗死区为甚,可见较大范围的弱荧光区;与模型组比较,通心络组大鼠患侧皮层的MAP-2荧光表达强度增加,丁苯酞荧光强度稍有增加。(4)Elisa结果显示PLGF在大鼠缺血脑损伤1d时血清中含量开始显著升高,3d含量更高,大鼠脑组织匀浆中PLGF的表达呈现相同的表达规律;通心络组PLGF在血清及匀浆中的表达均明显高于假手术组和模型组,并随时间延长而增高。(5)免疫组化结果显示PLGF主要表达在缺血半暗带和梗死中心的神经元、血管内皮细胞,脑缺血后血管内皮细胞表达显著增多。双重免疫荧光标记法显示正常状态下PLGF主要表达于神经元,而缺血状态下血管内皮细胞也大量表达PLGF。2.离体实验部分(1)通心络含药血清干预后,10%含药血清作用24h后BMEC活性明显提高即为通心络含药血清最佳浓度及作用时间。体外拟缺血模型组内皮细胞上清液中NO、vWF含量显著升高,而通心络含药血清可明显降低NO、vWF的水平。(2)正常内皮细胞条件培养液对正常神经元和拟缺血损伤神经元的活性无明显影响,但通心络含药血清干预的正常内皮细胞条件培养液对损伤的神经元有明显的保护作用;拟缺血损伤的内皮细胞条件液可使正常和损伤神经元的活性和SOD活力降低、MDA的产生增加,但通心络含药血清干预的拟缺血内皮细胞条件培养液可明显抑制这种降低,并使MDA的产生减少。拟缺血模型组的条件液中PLGF表达显著升高,经过通心络含药血清干预后PLGF较正常组有明显升高。(3)相对于正常对照组,拟缺血损伤神经元(OGD组神经元)的存活率显著下降,凋亡率显著升高,相关凋亡蛋白Bcl-2的表达明显减低而Bax显著升高;与模型组相比,PLGF干预后神经元存活率明显提高,凋亡率显著降低,Bcl-2明显升高而Bax显著降低;与OGD+PLGF组比,MAPK信号通路阻断剂PD98059组神经元存活率明显降低,凋亡率显著升高,Bcl-2明显减低而Bax显著升高,而PI-3K信号通路阻断剂LY294002组细胞存活率等均无明显差异。另一方面,OGD组可明显降低p-ERK1/2表达,PLGF干预后可提高拟缺血神经元p-ERK1/2的表达。结论1.通心络可以促进大鼠脑缺血后神经功能恢复、保护神经元及增加PLGF的表达和分泌量。正常状态下脑组织微血管内皮细胞仅释放少量PLGF,相反,受到缺血刺激而被激活的血管内皮细胞可释放大量的PLGF。故本研究证实了通心络对实验性大鼠局灶性脑缺血模型具有确切可靠的保护作用,并提出这种保护作用可能与PLGF相关。2.本研究首次观察到通心络干预下的脑微血管内皮细胞的旁分泌功能对拟缺血神经元的存活及氧化应激反应有重要的改善作用,并发现通心络干预的脑微血管内皮细胞分泌的PLGF在受到缺血缺氧刺激后会反应性升高。3.PLGF可以提高拟缺血神经元的存活率,本研究首次发现它可降低拟缺血神经元凋亡率及调节凋亡相关蛋白的表达,并发现这种作用与MAPK信号通路相关。总之,通心络治疗脑缺血的新靶点可能是以神经血管单元为核心改善微血管功能以介导脑神经保护的作用,而该作用可能与PLGF相关,PLGF可能通过MAPK信号通路发挥保护神经元的作用。
[Abstract]:Cerebrovascular disease is one of the three major diseases that cause human death. It has the characteristics of high incidence, high disability rate, high mortality and high recurrence rate. Among them, ischemic cerebrovascular disease (Ischemic Cerebral Vascular Disease, ICVD) accounts for about 55% to 80% of cerebrovascular disease worldwide, and its incidence is about 3 times of hemorrhagic cerebrovascular disease. Effective prevention and control of ischemic cerebrovascular disease is one of the important research topics in the medical field. The treatment of modern medicine for this disease is mainly two aspects of thrombolytic and neuron protection. However, there are still no recognized drugs with significant clinical efficacy so far, which compels us to find a new way out in the treatment of ischemic cerebrovascular disease. In recent years, people use traditional Chinese medicine to explore the treatment of ischemic encephalopathy. In recent years, the focus of the research is based on the concept of neurovascular unit and the protection of vascular endothelial cells to reduce the injury of brain tissue. It has opened up a new approach for the treatment of cerebral ischemia. The paracrine function of Brain microvascular endothelial cells (BMEC) has been found to play an important role in regulating the survival of neurons in the modern study. The differential protein in the conditioned medium of different cerebral microvascular endothelial cells may be a substance to realize its regulation. Placenta growthfactor (PLGF), such as placental growth factor (PLGF), has an obvious protective effect on anoxic glucose and glucose deficiency neurons. In this study, the secretory expression of PLGF in the cerebral microvascular endothelial cells and brain tissue and the protection of neurons under the action of Tongxinluo on cerebral ischemia and hypoxia were studied with PLGF as the breakthrough point. The molecular biological mechanism provides a scientific basis for the treatment of ischemic encephalopathy by Tongxinluo. Under the guidance of the theory of "Ying Wei rendezvous, by collaterals" theory, the protective effect of Tongxinluo on focal cerebral ischemia injury in experimental rats and the effect of PLGF on cerebral ischemia were observed under the guidance of the theory of "Wei Wei rendezvous," PLGF as the breakthrough point. The effect of cell conditioned solution on the activity of neurons and the content of PLGF, and then to reveal the molecular biological mechanism of PLGF protective neurons and the protective link of brain damage mediated by endothelial cells, and to explore the mechanism of the intervention of Tongxinluo on cerebral infarction by the neurovascular unit, and reveal the new target of "microvascular protection in ischemic area - a new target for cerebral infarction" The scientific connotation of "point" has important enlightenment for the study of the modernization of Chinese medicine for the treatment of cerebrovascular diseases. The model of cerebral ischemia in rats with middle cerebral artery occlusion was established by the method and content of 1. lines. The experimental animals were randomly divided into sham operation group, model group, Tongxinluo group and butylphthalide group (positive control medicine), the survival period was 1D, 3d. groups After the operation was sober, the nerve function was scored after the operation of 1D, 3D and material after the operation. The higher the score, the heavier the injury. The ratio of the back and back showed the recovery of nerve function. The pathological changes of the brain tissue were observed by HE staining. The expression of MAP-2 was detected by immunofluorescence technique to observe the changes of neurons, and the effect of Tongxinluo and the God was confirmed to confirm the effect of the Tongxinluo and the God. The.2. group and the survival time were equal. The expression of PLGF in the brain tissue was detected by immunohistochemistry, and the expression of PLGF in the cerebral vascular endothelial cells and neurons was analyzed by PLGF+CD31 (PLGF+MAP-2). The content of PLGF in the homogenate and serum of the rat brain was detected by enzyme linked immunosorbent assay (Elisa). The expression of PLGF in brain tissue of rats with cerebral ischemia injury and cell type.3. primary culture of cerebral microvascular endothelial cells. The effect of different concentration of Tongxinluo serum on the activity of cerebral microvascular endothelial cells after different time was observed by CCK-8 method to determine the best concentration and time of the drug administration by CCK-8 method. The cell oxygen glucose deprivation model was used to observe the effect of Tongxinluo serum on the activity of endothelial cells of ischemic injury in vitro, the nitric oxide (nitric oxide, NO) in the supernatant was detected by the silver nitrate reduction method, and the content of von Willebrand factor, vWF, PLGF was detected by Elisa method, and the effect of Tongxinluo on cerebral Microblood was analyzed. The function of endothelial cells and the effect of PLGF secretion in the primary cultured cortical neurons of.4. rats, the neuron ischemia model in vitro was established by oxygen glucose deprivation. The neuron survival rate was detected by CCK-8 method and the neuron survival rate was detected by CCK-8 method. The WST method was used to detect the superoxide dismutase. The activity of Superoxide Dismutase (SOD) and the changes in the content of malondialdehyde (MDA) were detected by the method of lipid oxidation. In order to explore the mechanism of Tongxinluo to protect the neurons by regulating the paracrine function of the endothelial cells, the primary cultured rat cortical neurons were cultured and the neuron ischemic model in vitro was established. The blocking of the MAPK signal pathway was blocked. Under the action of agent and PI-3K signaling pathway blocker, CCK-8 assay was used to detect the activity and survival rate of neurons before and after PLGF intervention, Tunel method was used to detect neuronal apoptosis. The expression of Bax, Bcl2 and p-ERK1/2 in different signal pathways was detected by immunoglobulin imprint, and PLGF was further explored in the ischemic state from the angle of cell signal transduction pathway. The molecular biological mechanism of inhibiting neuron apoptosis. Results 1. the whole experiment part (1) there was no significant difference in the score of the nerve function of each group after the animal model awakening. After the administration, the score of the Tongxinluo group was lower than the model group, and the ratio of the score before and after the model was analyzed. Compared with the 1D model group, the two times of the Tongxinluo group were evaluated. Compared with the 3D model group, the score ratio of the two times of the Tongxinluo group and the positive control group decreased significantly. (2) the results of HE staining showed that the brain tissue structure of the sham operation group was complete and the cells arranged neatly. The cortical tissue structure of the ischemic brain tissue in the model group was disorganized, the nuclei of the nerve cell nuclei were condensed, concentrated and some neurons disappeared. The pathological changes of brain tissue at each time point in the collaterals group were all less than the model group, the number of surviving neurons was increased, the damage of cortical ischemic area in the butylphthalide group was more serious than that of the Tongxinluo group. (3) the MAP-2 fluorescence intensity of the brain tissue in the sham operation group was high. The expression of MAP-2 in the lateral cortex of the model group was significantly reduced, especially in the infarct area. Compared with the model group, the intensity of MAP-2 fluorescence expression in the lateral cortex of Tongxinluo group increased and the fluorescence intensity of butylphthalide increased slightly. (4) Elisa results showed that the serum content of PLGF in the ischemic brain injury of rats increased significantly and the content of 3D was higher, and the expression of PLGF in the homogenate of the rat brain presented the same table. The expression of PLGF in the serum and homogenate of Tongxinluo group was significantly higher than that in sham operation group and model group, and increased with time. (5) the results of immunohistochemistry showed that PLGF mainly expressed in the neurons of the ischemic penumbra and infarct center, vascular endothelial cells, the expression of vascular endothelial cells after cerebral ischemia increased significantly. Double immunofluorescence The labeling method showed that under normal state, PLGF was mainly expressed in the neurons, and the vascular endothelial cells in the ischemic state also expressed a large number of PLGF.2. in vitro experiment part (1) the prognosis of the blood serum of Tongxinluo. The BMEC activity was significantly improved after the action of 24h in the drug serum, which was the best concentration and time of action of Tongxinluo serum. The content of NO and vWF in the supernatant of endothelial cells increased significantly, while Tongxinluo serum could significantly reduce the level of NO and vWF. (2) the normal endothelial cell conditioned medium had no obvious effect on the activity of normal neurons and the ischemic injured neurons, but the normal endothelial cell conditioned medium of Tongxinluo serum containing drug serum was used for the injured neurons. The endothelial cell conditioned medium for ischemic injury can reduce the activity of normal and damaged neurons and the activity of SOD, and increase the production of MDA. However, this reduction can be inhibited and the production of MDA can be reduced by the intervention of Tongxinluo serum containing serum, and the production of MDA in the quasi ischemic model group is PLG. The expression of F was significantly higher than that in the normal group. (3) compared with the normal control group, the survival rate of the ischemic injured neurons (group OGD neurons) decreased significantly, the apoptosis rate increased significantly, the expression of apoptosis protein Bcl-2 decreased and Bax significantly increased. Compared with the model group, the prognosis of PLGF was compared with the model group. The survival rate of neurons was significantly increased, the rate of apoptosis decreased significantly, the Bcl-2 increased significantly, and the Bax decreased significantly. Compared with the OGD+PLGF group, the survival rate of the neurons in the PD98059 group of the MAPK signaling pathway blocker PD98059 was significantly reduced, the apoptosis rate was significantly increased, the Bcl-2 decreased significantly, and the Bax was significantly increased, but the survival rate of the LY294002 group of PI-3K signaling blockers was not clear. On the other hand, the expression of p-ERK1/2 was obviously reduced in group OGD, and the expression of p-ERK1/2 in ischemic neurons could be improved by PLGF. Conclusion 1. Tongxinluo can promote the recovery of nerve function after cerebral ischemia in rats, protect the neurons and increase the expression and secretion of PLGF. The microvascular endothelial cells in the brain tissue can release only a small amount of PLGF in normal state. On the contrary, vascular endothelial cells stimulated by ischemia can release a large number of PLGF.. This study confirms that Tongxinluo has a definite and reliable protective effect on experimental rat focal cerebral ischemia model, and it is suggested that this protective effect may be the first time to observe the cerebral microvascular endothelium under the intervention of Tongxinluo in the PLGF related.2. study. The paracrine function of the cells has an important improvement in the survival and oxidative stress response of ischemic neurons, and it is found that the reactivity of PLGF secreted by the microvascular endothelial cells in the cerebral microvascular endothelial cells intervened by Tongxinluo can increase the survival rate of.3.PLGF after ischemia and hypoxia. This study can be found for the first time that it can reduce the pseudo ischemic neuron. The apoptosis rate of ischemic neurons and the expression of apoptosis related proteins are associated with the MAPK signaling pathway. In conclusion, the new target of Tongxinluo in the treatment of cerebral ischemia may be the role of neurovascular unit as the core to improve the function of microvascular to mediate the protection of brain nerve, which may be related to PLGF, and PLGF may pass through MAPK Signaling pathways play a role in protecting neurons.

【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R743.3

【相似文献】