梓葛冻干粉针对大鼠急性脑缺血损伤半暗带神经血管单元的整体保护作用

发布时间：2018-06-28 11:06

本文选题：梓葛冻干粉针 + 缺血性中风　；参考：《西南大学》2016年博士论文

【摘要】：1.研究背景抑制或减轻中风急性期半暗带细胞凋亡或死亡,避免梗死灶扩大,是公认的较为理想的缺血性中风脑保护策略。课题组自主研制的抗中风药物——梓葛冻干粉针(由地黄药效成分梓醇和葛根药效成分葛根素组成),在永久缺血性脑中风模型大鼠和小鼠的非急性期,以及缺血再灌注损伤的脑中风模型大鼠中,均表现出较好的脑保护作用。然而,梓葛冻干粉针对永久缺血性脑中风模型大鼠急性期的脑保护作用尚未观察,其对脑缺血半暗带中神经血管单元细胞的整体保护作用及其可能的通路机制有待研究。本研究获得了国家自然科学基金面上项目(81473549)、教育部博士学科点专项基金项目(博导类:20110182110012)、教育部中央高校基本科研业务费项目(XDJK2014D023)以及重庆市卫生局中医药科研重大项目(渝中医2010[60]2010-1-4)的支持。2.研究目的研究梓葛冻干粉针对永久缺血性脑中风模型大鼠急性期的脑保护作用,以及对脑缺血半暗带中神经血管单元的整体保护作用和机制,为将梓葛冻干粉针开发成治疗缺血性脑中风的候选药物提供基础研究数据。3.方法与结果3.1梓葛冻干粉针对大鼠急性脑缺血损伤整体保护作用研究方法用线栓法梗塞大脑中动脉,复制大鼠中风模型。在造模大鼠苏醒2 h后,采用改良神经功能缺损评分法(m NSS)进行评分并分组。以m NSS评分得4分及以上且各项均有缺损为标准,判定造模成功。将造模成功的64只大鼠随机分为4个组,每组16只:模型组,梓葛冻干粉针65.4 mg·kg-1、32.7 mg·kg-1和16.4 mg·kg-1组。另设假手术组16只。尾静脉注射梓葛冻干粉针或生理盐水,一天2次,给药1天。造模24 h后:(1)用m NSS评分法再次评价大鼠的神经功能,并分析组间差异显著性。(2)每组取8只大鼠麻醉处死,分离新鲜大脑用脑模切片,作TTC染色,拍照后,用软件测算脑梗死面积百分比,并分析比较组间差异显著性。(3)每组另取8只大鼠麻醉,灌流固定后分离大脑,冰冻切片后作HE染色,用显微镜观察并拍照,比较组间脑缺血半暗带组织的损伤情况。结果与假手术组相比,模型组大鼠m NSS得分和脑梗死面积百分比均显著增加(p0.01),缺血区脑组织大面积坏死,部分区皮质域呈高度疏松筛网状结构,细胞结构不清,红色坏死神经元显著增加。与模型组相比,梓葛冻干粉针65.4mg·kg-1、32.7 mg·kg-1和16.4 mg·kg-1组大鼠m NSS得分和脑梗死面积百分比均显著下降(p0.01、p0.05),缺血区脑组织坏死程度显著减轻,红色坏死神经元显著减少。3.2梓葛冻干粉针抗大鼠急性脑缺血损伤半暗带保护机制研究方法造模及分组给药方法同第一章,每组12只大鼠。造模24 h后:(1)每组取6只大鼠,按第一章方法制作大脑冰冻切片,用TUNEL+特异性蛋白双重免疫荧光染色标记脑片,荧光显微镜观察缺血半暗带大脑皮质区神经元、星形胶质细胞和微血管内皮细胞生长形态并拍照,用软件分析3种细胞的凋亡率,并比较组间差异显著性。(2)另取脑片,用免疫组化技术分别标记Bax、Bcl-2、Caspase-3、Cleaved caspase-3和Cyt-C蛋白,用显微镜观察缺血半暗带大脑皮质区并拍照。(3)每组另取6只大鼠麻醉取脑,提取缺血半暗带大脑皮质组织匀浆蛋白,用WB检测匀浆蛋白中Bax、Bcl-2、Caspase-3、Cleaved caspase-3和Cyt-C蛋白表达,并分析比较组间差异显著性。结果与假手术组相比,模型组大鼠缺血半暗带大脑皮质层区域神经元、星形胶质细胞和微血管内皮细胞的凋亡率显著增加(p0.01),细胞生长形态严重受损,促细胞凋亡的Bax、Caspase-3、Cleaved caspase-3和Cyt-C蛋白表达均显著增加(p0.01),抑制细胞凋亡的Bcl-2蛋白表达显著减少(p0.01)。与模型组相比,梓葛冻干粉针65.4 mg·kg-1、32.7 mg·kg-1和16.4 mg·kg-1组大鼠缺血半暗带大脑皮质区域该3种细胞的凋亡率均显著降低(p0.01),其生长形态明显改善,上述促调蛋白表达均显著减少(p0.01,p0.05);其中,梓葛冻干粉针65.4 mg·kg-1组大鼠Bcl-2蛋白表达显著增加(p0.01)。3.3体外脑神经血管单元半暗带损伤模型的建立及梓葛冻干粉针的整体保护作用研究方法(1)大鼠大脑皮层神经元、星形胶质细胞和微血管内皮细胞的原代培养,以及由该3种细胞共培养组成的脑神经血管单元模型的构建,均参照课题组前期方法。(2)用文献报道的半暗带条件培养液,对所建立的脑神经血管单元模型进行损伤,24 h后,以细胞生长状态、数量、轴突长度和细胞跨内皮电阻值显著(p0.05)低于正常培养孔为标准,判定体外脑神经血管单元半暗带损伤模型构建成功。(3)将构建的脑神经血管单元模型随机分为5个组,每组6个孔:正常培养组、损伤模型组(半暗带条件培养液损伤24 h)、梓葛冻干粉针3个剂量组(于更换半暗带条件培养液时加入49.0μg·m L-1、24.5μg·m L-1以及12.25μg·m L-1的梓葛冻干粉针)。用药24 h后,用显微镜观察3种细胞的生长形态并拍照,用台盼蓝计数法检测星形胶质细胞和微血管内皮细胞的活细胞数量,用图像分析软件测量神经元轴突长度,用细胞电阻仪检测模型中跨内皮电阻值,并分析比较组间差异显著性。结果(1)成功分离培养出高纯度的大鼠大脑皮层3种原代细胞,构建了由该3种细胞共培养的脑神经血管单元整体模型。与单细胞培养相比,共培养模型中神经元胞体圆润且立体感强,轴突更粗大且长并形成致密网络;脑微血管内皮细胞和星形胶质细胞胞间接触更紧密,形成致密的单层;脑微血管内皮细胞和星形胶质细胞活细胞数量、神经元轴突长度以及模型跨内皮电阻值均显著增加(p0.05,p0.01)。(2)与正常培养组相比,经半暗带条件培养液损伤的脑神经血管单元整体模型组中,神经元立体感较差,胞体和轴突变小,轴突网络密度降低;脑微血管内皮细胞和星形胶质细胞胞体收缩,部分细胞出现脱落;脑微血管内皮细胞和星形胶质细胞活细胞数量、神经元轴突长度以及跨内皮电阻值均显著降低(p0.01)。与半暗带条件培养液损伤的脑神经血管单元整体模型组相比,梓葛冻干粉针49.0μg·m L-1、24.5μg·m L-1以及12.25μg·m L-1组脑神经血管单元整体模型中,神经元胞体无明显收缩,突触网络仍较紧密;微血管内皮细胞和星形胶质细胞胞体轻微收缩,胞间接触仍较紧密;脑微血管内皮细胞和星形胶质细胞活细胞数量、脑神经血管单元整体跨内皮电阻值均显著增加(p0.01);其中,梓葛冻干粉针49.0μg·m L-1和24.5μg·m L-1组神经元轴突长度显著增加(p0.01)。3.4梓葛冻干粉针抗体外脑神经血管单元模型半暗带损伤的机制研究方法(1)大鼠大脑皮层3种原代细胞的培养、脑神经血管单元模型的构建及体外脑神经血管单元模型半暗带损伤方法同第3章。(2)将构建的脑神经血管单元模型随机分为7个组,每组6孔:正常培养组:在正常培养条件下培养;损伤模型组:半暗带条件培养液损伤24 h;梓葛冻干粉针3个剂量组:在更换半暗带条件培养液时添加梓葛冻干粉针,使其终浓度分别为49.0μg·m L-1、24.5μg·m L-1、12.25μg·mm L-1;抑制剂MG132组:在更换半暗带条件培养液时添加MG132,使其终浓度为10μM;49.0μg·mm L-1梓葛冻干粉针+MG132组:在更换半暗带条件培养液时,添加梓葛冻干粉针(49.0μg·m L-1)和MG132(10μM)。(3)用药后,用流式细胞仪检测不同组别脑神经血管单元细胞共培养模型中3种细胞凋亡率,并分析比较组间差异显著性。(4)用试剂盒提取不同组别脑神经血管单元模型细胞总蛋白,蛋白定量后,按WB操作步骤,检测分析Bax、Bcl-2、Caspase3、Cleaved caspase-3、Caspase-9、Cleaved caspase-9、Cyt-C、XIAP的蛋白表达,并分析比较组间差异显著性。结果与正常培养组相比,经半暗带条件培养液损伤的脑神经血管单元模型组中,神经元、星形胶质细胞和微血管内皮细胞凋亡率均显著增加(p0.01),细胞Bax、Cyt-C、Cleaved caspase-9、Caspase-3和Cleaved caspase-3蛋白表达均显著增加(p0.01),Bcl-2和XIAP蛋白表达显著降低(p0.01)。与半暗带条件培养液损伤的脑神经血管单元整体模型组相比,梓葛冻干粉针49.0μg.m L-1及24.50μg.m L-1组该3种细胞的凋亡率均显著降低(p0.01),Cyt-C、Cleaved caspase-9和Cleaved caspase-3的蛋白表达均显著降低(p0.01),Bcl-2和XIAP的蛋白表达均显著升高(p0.01),其中梓葛冻干粉针49.0μg·m L-1组Bax蛋白表达显著降低(p0.01)。与梓葛冻干粉针49.0μg·m L-1组相比,MG132+梓葛冻干粉针49.0μg·m L-1组XIAP蛋白与Cleaved caspase-3蛋白表达均显著增加(p0.01,p0.05)。4.研究结论4.1梓葛冻干粉针能减轻大鼠脑缺血区域受损细胞形态变化,减少细胞死亡,减少缺血区脑组织梗死灶面积,改善大鼠神经功能,对大鼠急性脑缺血损伤发挥整体保护作用。4.2梓葛冻干粉针对大鼠急性脑缺血损伤的整体保护作用,主要是通过抑制缺血半暗带区神经元、星形胶质细胞和微血管内皮细胞凋亡实现的,其机制与调节线粒体凋亡通路有关。4.3梓葛冻干粉针对急性脑缺血半暗带神经血管单元细胞线粒体凋亡通路的调节作用,与促进XIAP蛋白表达有关。4.4本研究中,体外脑神经血管单元半暗带模型损伤变化及梓葛冻干粉针的逆转作用,与在体实验结果具有较高一致性。提示该模型可用于缺血半暗带脑神经血管单元细胞损伤机制研究及其保护药物效果的体外评价。
[Abstract]:1. the research background suppresses or reduces the apoptosis or death of the cells in the acute phase of the apoplexy, and avoids the enlargement of the infarct. It is an ideal cerebral protection strategy for ischemic stroke. The cerebral apoplexy model rats and mice, as well as the cerebral apoplexy model rats with ischemia-reperfusion injury, showed better cerebral protective effect. However, the protective effect of Catalpol dry powder on the acute ischemic cerebral apoplexy rat model was not yet observed, and its neurovascular cell cells in the cerebral ischemic penumbra The overall protection and its possible pathway mechanisms need to be studied. This study obtained the National Natural Science Foundation Project (81473549), the special fund project of the doctoral program of the Ministry of Education (20110182110012), the basic scientific research service fee project (XDJK2014D023) of the central colleges and universities of the Ministry of Education and the importance of Chinese medicine in the Chongqing Municipal Health Bureau. The project (Chongqing Chinese medicine 2010[60]2010-1-4) supports.2. research to study the protective effect of Catalpol dry powder on the acute stage of acute ischemic cerebral apoplexy in rats, as well as the overall protective effect and mechanism of the neurovascular units in the cerebral ischemic penumbra, in order to develop a candidate drug for the treatment of ischemic stroke. The basic research data.3. method and results 3.1 The Study on the overall protective effect of Catalpol freeze-dried powder on acute cerebral ischemia injury in rats, the middle cerebral artery was infarcted by the thread emboli method, and the rat model of stroke was replicated. After the rats were awakened to 2 h, the improved neural function defect scoring (m NSS) was used to score and group. The score of M NSS score was 4 points. 64 rats were randomly divided into 4 groups, 16 rats in each group, with 16 rats in each group: model group, Catalpol freeze dry powder 65.4 mg. Kg-1,32.7 mg. Kg-1 and 16.4 mg. Kg-1. There were 16 in the sham operation group. The tail vein was injected Catalpol dry powder needle or saline, 2 times a day and 1 days. The model 24 was 24. After H: (1) the m NSS scoring method was used to reevaluate the nerve function of the rats and to analyze the difference between the groups. (2) 8 rats in each group were killed, and the fresh brain was separated by the brain model section to make TTC staining. The percentage of cerebral infarction was measured by software and the difference was statistically significant. (3) another 8 rats in each group were anaesthetized and perfusion. After the fixation, the brain was separated and the frozen section was stained with HE. The damage of the ischemic penumbra in the group was compared with the microscope. Results compared with the sham group, the scores of M NSS and the percentage of cerebral infarction increased significantly (P0.01), the cerebral tissue in the ischemic area was large and the cortical area was highly sparse. In the group of 65.4mg. Kg-1,32.7 mg. Kg-1 and 16.4 mg. Kg-1, the scores of M NSS and the percentage of infarcted area decreased significantly (P0.01, P0.05), and the necrosis degree of cerebral tissue in the ischemic area was significantly reduced and the red necrotic neurons were significant compared with the model group. To reduce the protection mechanism of.3.2 Catalpol freeze-dried powder for the protection of semi dark zone of acute cerebral ischemia injury in rats, the method of modeling and group administration is the same as the first chapter, 12 rats in each group. After 24 h, the model of cerebral frozen section is made in each group, and the brain slices are marked with TUNEL+ specific protein double immunofluorescence staining. Microscopically, the neurons, astrocytes and microvascular endothelial cells in the cerebral cortex of the ischemic penumbra were observed and photographed. The apoptosis rate of the 3 kinds of cells was analyzed with software. (2) the brain slices were taken and the Bax, Bcl-2, Caspase-3, Cleaved caspase-3 and Cyt-C protein were marked with immunohistochemical technique. The cerebral cortex area of ischemic half dark zone was observed and photographed. (3) another 6 rats in each group were taken to take the brain, and the cerebral cortex homogenate protein was extracted from the ischemic penumbra. The expression of Bax, Bcl-2, Caspase-3, Cleaved caspase-3 and Cyt-C protein in the homogenate protein was detected by WB, and the difference between the groups was statistically significant. The results were compared with the sham group, the model group was larger than the sham group. The apoptosis rate of astrocytes and microvascular endothelial cells increased significantly (P0.01), and the cell growth morphology was severely damaged. The expression of Bax, Caspase-3, Cleaved caspase-3 and Cyt-C protein increased significantly (P0.01), and the expression of Bcl-2 protein inhibited apoptosis significantly decreased (P0 (P0). .01). Compared with the model group, the apoptosis rate of the 3 cells in the ischemic half dark zone of the rats of 65.4 mg. Kg-1,32.7 mg. Kg-1 and 16.4 mg kg-1 group were significantly decreased (P0.01), and the expression of the TGP was significantly reduced (P0.01, P0.05). Among them, the Catalpol dry powder needle was 65.4 mg. The expression of Bcl-2 protein in rats increased significantly (P0.01) the damage model of the subdark zone of the neurovascular unit of the.3.3 body and the study method of the overall protection of the dry powder of Catalpol (1) the primary culture of the cerebral cortex neurons, astrocytes and microvascular endothelial cells in the rats, and the cerebral nerve blood composed of the 3 kinds of cells co culture. The construction of the tube unit model referred to the preliminary method of the project group. (2) using the semi dark zone conditioned medium reported in the literature, the brain nerve cell model was damaged. After 24 h, the cell growth state, the number, the axon length and the cell cross endothelial resistance value were significantly lower than the normal culture pore, and the brain nerve was determined in vitro. The model of vascular unit semi dark zone damage model was successfully constructed. (3) the constructed brain nerve cell model was randomly divided into 5 groups, with 6 holes in each group: normal culture group, injury model group (24 h damage of semi dark zone conditioned medium), and 3 dose groups of Catalpol freeze-dried powder (49 mu m L-1,24.5 G. M L-1 when replacing the semi dark zone conditioned medium. After 24 h, the growth morphology of 3 cells was observed with microscope and the number of living cells of astrocytes and microvascular endothelial cells were detected by trypan blue counting, and the length of the axon was measured by the image analysis software, and the resistance value of the endothelium in the model was detected by the cell resistance instrument, and the resistance value of the endothelium in the model was detected by the cell resistance instrument, and the resistance value of the 12.25 h L-1 was detected, and the resistance value of the model was detected by the cell resistance instrument. Results (1) 3 primary cells of the cerebral cortex of high purity rat were successfully isolated and cultured. The whole model of cerebral nerve cell unit was constructed by the 3 cells. Compared with the single cell culture, the cell body was round and strong in the co culture model, and the axon was thicker and more dense and dense. The intercellular contact between the cerebral microvascular endothelial cells and astrocytes was closer, forming a compact monolayer; the number of living cells in the brain microvascular endothelial cells and astrocytes, the length of the axon and the resistance value of the model were significantly increased (P0.05, P0.01). (2) compared with the normal culture group, the semi dark zone conditioned medium injury was damaged. In the whole model group of cerebral neurovascular unit, the stereoscopic sense of neuron was poor, the cell body and axon became smaller, the density of the axon network decreased, the cerebral microvascular endothelial cells and astrocytes were contracted, the part of the cell appeared to fall off, the number of the brain microvascular endothelial cells and astrocytes, the length of the neuron axon and the cross endothelial electricity. Compared with the whole model group of brain neurovascular unit damaged by semi dark zone conditioned medium, 49 mu g m L-1,24.5 G. M L-1 and 12.25 mu g. M L-1 group were compared with the whole model group. The neuronal cell body was not obviously contracted and the synapse network was still tight; microvascular endothelial cells and stars were still relatively close. The cell bodies of glial cells were slightly contracted and the intercellular contact was still close, the number of living cells in the cerebral microvascular endothelial cells and astrocytes, the overall cross endothelial resistance value of the brain neurovascular unit increased significantly (P0.01), and the length of the axon of the neurons of 49 mu g / m L-1 and the 24.5 mu g. L-1 group increased significantly (P0.01).3.4 Catalpol The mechanism of the semi dark zone damage of the freeze-dried powder needle antibody external brain neurovascular unit model (1) the cultivation of 3 primary cells in the cerebral cortex of the rats, the construction of the brain nerve cell model and the method of the damage of the semi dark band in the brain nerve cell model in the same third chapters. (2) the neural vascular unit model was randomly divided into 7 groups. Each group of 6 holes: normal culture group: cultured under normal culture conditions; damage model group: semi dark zone conditioned medium injury 24 h; zigge freeze-dried powder 3 doses group: adding Catalpol freeze-dried powder when replacing semi dark zone conditioned medium, the final concentration was 49 mu. M L-1,24.5, m L-1,12.25 mu g. Mm L-1; inhibitor MG132 group: in addition, inhibitor MG132 group: in more Adding MG132 to the medium dark zone conditioned medium, the final concentration was 10 mu M; 49 G. Mm L-1 catalpa freeze-dried powder needle +MG132 group: adding Catalpol freeze-dried powder needle (49 mu g. M L-1) and MG132 (10 mu M) when replacing the semi dark zone conditioned medium. (3) using flow cytometry to detect the co culture model of cerebral nerve cell cell cell in different groups 3 (4) the total protein of the brain nerve cell model of different groups was extracted with the kit. After the WB operation, the protein expression of Bax, Bcl-2, Caspase3, Cleaved Caspase-3, Caspase-9, Cleaved caspase-9, Cyt-C, XIAP were detected and analyzed, and the differences between the groups were analyzed and compared. The apoptosis rate of neurons, astrocytes and microvascular endothelial cells increased significantly (P0.01), and the expression of Bax, Cyt-C, Cleaved caspase-9, Caspase-3 and Cleaved caspase-3 proteins increased significantly (P0.01), Bcl-2 and X, compared with the normal culture group. The expression of IAP protein was significantly decreased (P0.01). Compared with the whole model group, the apoptosis rates of the 3 kinds of cells were significantly decreased (P0.01), and the protein expressions of Cyt-C, Cleaved caspase-9 and Cleaved caspase-3 were significantly decreased. The expression of protein in XIAP and XIAP increased significantly (P0.01), and the expression of Bax protein in the 49 u g / m L-1 group was significantly decreased (P0.01). Compared with the 49 mu g / m L-1 group of Catalpol freeze-dried powder, the expression of 49 mu g. GL dry powder can reduce the morphological changes of damaged cells in the cerebral ischemia area of rats, reduce cell death, reduce the area of cerebral infarction in the ischemic area, improve the nerve function of rats, and play an integral protective effect on the acute cerebral ischemia injury in rats. The main protective effect of.4.2 Catalpol dry powder on acute cerebral ischemia injury in rats is mainly through the protection. Inhibiting the apoptosis of neurons, astrocytes and microvascular endothelial cells in the ischemic penumbra region, its mechanism is related to regulation of mitochondrial apoptosis pathway related to the regulation of.4.3 Catalpol dry powder on the mitochondrial apoptosis pathway in the acute cerebral ischemic penumbra cell cell cells, which is related to the.4.4 study of promoting the expression of XIAP protein. The changes in the damage of the semi dark zone model of the neurovascular unit of the outer brain and the reversal effect of Catalpol freeze-dried powder needle are in good agreement with the experimental results, suggesting that the model can be used to study the mechanism of cell damage in the ischemic penumbra and the evaluation of the drug effect in vitro.
【学位授予单位】：西南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R285.5

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