基于炎症反应分析高脂血症条件下脑缺血大鼠模型脑组织病理学变化

发布时间：2018-06-07 20:26

本文选题：高脂血症 + 脑缺血　；参考：《北京中医药大学》2012年博士论文

【摘要】：1.背景单纯脑缺血在临床上发病率较低,仅见于房颤附壁血栓脱落、感染性脓栓、脂肪性栓子、癌细胞栓子、寄生虫卵栓子等引起的栓塞。大多数脑缺血是在某些基础病变条件下发生的,如高血压、高脂血症、糖尿病等；这些基础性病变本身就是缺血性中风的高危因素,在其影响或作用下,脑缺血性虽然伴随脑水肿、血液高凝状态、自由基损伤、炎症反应、神经细胞坏死及凋亡等共同的病理生理过程,但是也存在一些差异,如缺血程度、炎症反应、神经功能缺损的程度及神经细胞死亡的方式等并不完全一样。这些差异可能在整个病理过程中起重要作用；而采用同一的治疗方案很难达到满意的治疗效果。目前,已经上市的治疗中风的新药很少是针对带有某种基础性病变而研发的。中医对缺血性中风有着丰富的认识,其病因主要涉及“风、火、痰、瘀、虚”；病因不同,中风后表现亦有区别；针对不同证型,采用对症的方药进行治疗,疗效显著；中医对中风的辩证施治,体现了中医在治疗复杂疾病中的优势。本研究拟把中医病机理论与现代医学病理、病理生理理论系统结合起来,针对在某些基础病变(证型)条件下构建脑缺血复合模型,相对接近临床实际,为探讨中药复方的药理机制和对证治疗疗效评价提供规范的模型基础。 2.目的 1)研究高脂血症大鼠模型炎症反应状态及脑组织的影响,比较高脂血症大鼠模型和正常大鼠脑组织的病理性差异,分析高脂血症大鼠模型脑组织的病理学特点。 2)比较复合脑缺血模型和单纯脑缺血模型在不同时间炎症反应变化及对神经-血管单元的影响,研究在高脂血症条件下脑缺血病变的特异性变化,分析高脂血症炎症因素对缺血脑组织的影响。 3.方法 1)造模：采用高脂饲料饲养大鼠,复制经典的高脂血症动物模型,检测大鼠血清中血脂的含量以确定模型成功；复制大脑中动脉缺血模型,观察大鼠脑部缺血损伤情况。 2)分组：将实验大鼠分为正常组、假手术组、单纯脑缺血3d组、单纯脑缺血7d组与高脂血症组、高脂血症假手术组、复合脑缺血3d组、复合脑缺血7d组,采用神经功能评分和TTC染色的方法,检测不同组间动物的行为功能,并观察脑组织梗死体积。 3)断尾取血,酶联免疫法(ELISA)检测血清中ET-1、NO、6-keto-FGF1a、TX-B2、MCP-1、TNF-α、CRP、SOD、MDA和vWF含量。 4)免疫组织化学方法比较各组大鼠脑组织MMP-2、ICAM-1、Bax、Bcl-2和CD34表达。 5)HE染色的方法观察各组大鼠脑组织梗死边缘区组织病理学改变。 6)利用透射电镜技术,观察各组大鼠脑组织梗死边缘区细胞超微结构改变。 4.结果 1)高脂血症组与正常组比较 ①高脂血症组血清中NO、6-keto-FGF1a含量减少,差异显著(0.01p0.05)；TX-B2含量增多,差异显著(p0.05)；MCP-1、TNF-α、CRP、vWF、MDA含量明显增多,差异显著(p0.01)；脑组织中MMP-2、ICAM-1和CD34的表达增高,差异显著性(0.01P0.05)。 ②高脂血症组神经元有少量萎缩,线粒体嵴变短,粗面内质网减少；皮质区毛细血管增多,血管内皮细胞线粒体及线粒体嵴增多；神经胶质细胞增多明显,线粒体嵴丰富；神经突触地突触小泡减少,线粒体嵴变短。 2)复合脑缺血组与单纯脑缺血组相比较： ①两模型组在缺血3d、7d神经功能评分无差异,但造模大鼠苏醒后和缺血3d、7d神经功能评分的差值比较,复合脑缺血组缺血7d差值减小,差异显著(p0.05)。 ②复合脑缺血组缺血7d梗死体积缩小,差异显著(p0.05),缺血3d无差异(P0.05)。 ③NO、6-keto-FGF1a在复合脑缺血组和单纯脑缺血组与相应假手术组组内比较,脑缺血组在缺血3d、7d血清含量均高于相应的假手术组,差异显著(p0.05)；复合脑缺血组与单纯脑缺血组组间比较,复合脑缺血组在缺血3d、7d血清含量均减少,但在缺血3d组间比较无明显差异(P0.05),缺血7d组间比较差异显著(p0.05)。 ④ET-1在脑缺血模型各时间点血清含量呈增多趋势,但复合脑缺血组和单纯脑缺血组与相应假手术组组内及组间比较无显著性差异(P0.05)。TX-B2在脑缺血模型各时间点血清含量呈增多趋势,单纯脑缺血组与相应假手术组组内比较,在缺血3d、7d血清含量增多,差异显著(p0.05),复合脑缺血组与相应假手术组组内比较,在缺血3d、7d血清含量呈增多趋势,但差异无显著性(P0.05)；复合脑缺血组与单纯脑缺血组组间比较,复合脑缺血组血清含量减少,缺血3d、7d差异均显著(p0.05)。 ⑤vWF因子在脑缺血模型各时间点血清含量呈增多趋势,单纯脑缺血组与相应假手术组组内比较,单纯脑缺血组再缺血7d血清含量增多,差异显著(p0.05)；复合脑缺血组与相应假手术组组内比较,复合脑缺血组在缺血3 d血清含量增多,差异显著(p0.05)；复合脑缺血组与单纯脑缺血组组间比较,复合脑缺血组在缺血3d、7d血清含量均增多,差异显著(p0.05)。 ⑥MCP-1、TNF-α和CRP在复合脑缺血组和单纯脑缺血组与相应假手术组组内比较,脑缺血组在缺血3d、7d血清含量均多于相应的假手术组,差异显著(p0.05)；复合脑缺血组与单纯脑缺血组组间比较,复合脑缺血组在缺血3d、7d血清含量均增多,差异显著(p0.05,0.01p0.05)。 ⑦MMP-2、ICAM-1、CD34在复合脑缺血组和单纯脑缺血组与相应假手术组组内比较,脑缺血组在缺血3d、7d脑组织的表达均高于相应的假手术组,差异显著(p0.05)；复合脑缺血组与单纯脑缺血组组间比较,复合脑缺血组缺血3d、7d脑组织中表达均增高,差异显著(p0.05,0.01P0.05)。 ⑧MDA在单纯脑缺血组与假手术组组内比较,缺血3d血清中含量增多,差异显著(p0.05)；复合脑缺血组与高脂血症假手术组组内比较,缺血3d、7d血清中含量均减少,差异显著(p0.05)；复合脑缺血组与单纯脑缺血组组间比较,复合脑缺血组在缺血3d、7d血清中含量均减少,差异显著(p0.05)。SOD在复合脑缺血组和单纯脑缺血组与相应假手术组组内比较,脑缺血组在缺血3d、7d血清含量均减少,差异显著(p0.05)；复合脑缺血组与单纯脑缺血组组间比较,复合脑缺血组在缺血3d血清含量减少,差异显著(p0.05)。 ⑨Bcl-2、Bax在复合脑缺血组和单纯脑缺血组与相应假手术组组内比较,脑缺血组在缺血3d、7d在脑组织中的表达均增高,差异显著(p0.05)；复合脑缺血组与单纯脑缺血组组间比较,Bax在复合脑缺血组脑组织中表达3d、7d均下降,差异显著(p0.05)；Bcl-2在缺血3d表达增高,差异显著(p0.05)；Bcl-2/Bax比值在复合脑缺血组3d、7d均升高,差异显著(p0.05)。 ⑩缺血边缘组织学观察,复合脑缺血组与单纯脑缺血组比较,缺血3d均有大量神经元变性、坏死,核染色质稀疏,线粒体嵴变短或消失、嵴间腔扩大,粗面内质网明显减少；血管内皮细胞肿胀,细胞连接破坏,线粒体及线粒体嵴减少；神经胶质细胞反应增生明显,线粒体嵴变短或消失；有小胶质细胞增生；神经突触地突触小泡减少,线粒体嵴变短或消失。缺血7d上述损伤减轻,复合脑缺血组与单纯脑缺血组比较神经元变性、坏死减少,水肿减轻,线粒体损伤恢复；神经胶质细胞增多明显,线粒体嵴丰富；内皮细胞水肿减轻。 5.结论 1)高脂血症病变过程中,病变损伤血管内皮细胞,诱发脑组织的炎症反应,并对血管-神经元产生一定的影响； 2)炎症及炎症相关因子可能是痰湿、瘀血中医病机的部分生物学基础； 3)高脂血症条件下,炎症因子在缺血不同时期既有累积现象,又有特异性的表达,这可能在脑缺血恢复期有助于损伤的修复； 4)在炎症反应提前预刺激下,内源性的脑保护机制可能被启动。
[Abstract]:1. background
The incidence of simple cerebral ischemia is low in clinical, only in the embolus caused by atrial fibrillation, septic suppository, fatty embolus, cancer cell embolus, and parasitic oocyte embolus. Most cerebral ischemia occurs under certain basic pathological conditions, such as hypertension, hyperlipidemia, diabetes and so on; these basic lesions themselves are The high risk factors of ischemic apoplexy, in which the ischemic stroke is associated with brain edema, hypercoagulability, free radical damage, inflammation, necrosis of the nerve cells and apoptosis, but there are also some differences, such as the degree of ischemia, the inflammation, the degree of nerve function defect and the death of the nerve cells. The ways of dying are not exactly the same. These differences may play an important role in the whole pathological process; and the same treatment is difficult to achieve satisfactory results.
Chinese medicine has a rich understanding of ischemic stroke, its cause is mainly involved in "wind, fire, phlegm, stasis, deficiency"; the cause is different and the performance is different after the stroke; the curative effect is remarkable for the different syndromes, using the prescriptions of the symptomatic, the dialectical treatment of stroke, embodies the advantages of traditional Chinese medicine in the treatment of complex diseases.
This study is to combine the theory of pathogenesis of traditional Chinese medicine with the modern medical pathology and pathophysiology system, and to construct a complex model of cerebral ischemia under certain basic pathological conditions (syndrome type), which is relatively close to the clinical practice, and provides a standard model basis for the study of the pharmacological mechanism of Chinese medicine compound and the evaluation of the therapeutic effect of the syndrome treatment.
2. purposes
1) to study the effect of inflammatory reaction and brain tissue in hyperlipidemia rat model, compare the pathological difference between hyperlipidemia rat model and normal rat brain tissue, and analyze the pathological characteristics of brain tissue of hyperlipidemia rat model.
2) to compare the changes of the inflammatory response and the effects on the neurovascular units of the complex cerebral ischemia model and the simple cerebral ischemia model at different time and to study the specific changes of the cerebral ischemia in the hyperlipidemia condition, and to analyze the effect of the inflammatory factors of hyperlipidemia to the ischemic brain tissue.
3. method
1) model: feeding rats with high fat feed, replicating the classic hyperlipidemia animal model, detecting the serum lipid content in rats to determine the model success, replicating the cerebral ischemia model of the middle cerebral artery, and observing the cerebral ischemia injury in the rat.
2) group: the experimental rats were divided into normal group, sham operation group, simple cerebral ischemia 3D group, simple cerebral ischemia 7d group and hyperlipidemia group, hyperlipidemia sham group, compound cerebral ischemia 3D group, complex cerebral ischemia 7d group, neural function score and TTC staining method, the behavioral function of different groups were detected and the infarct volume of brain tissue was observed.
3) blood samples were taken from the tail, and the levels of ET-1, NO, 6-keto-FGF1a, TX-B2, MCP-1, TNF-, CRP, SOD, MDA and vWF in serum were detected by enzyme linked immunosorbent assay (ELISA).
4) immunohistochemical method was used to compare the expression of MMP-2, ICAM-1, Bax, Bcl-2 and CD34 in brain tissue of each group.
5) HE staining method was used to observe the histopathological changes in the marginal area of cerebral infarction in each group.
6) transmission electron microscopy was used to observe the ultrastructural changes in the marginal area of infarct of rats in each group.
4. results
1) comparison of hyperlipidemia group and normal group
(1) the levels of NO and 6-keto-FGF1a in the serum of hyperlipidemia group were decreased (0.01p0.05), and the content of TX-B2 increased significantly (P0.05); MCP-1, TNF- a, CRP, vWF and MDA increased significantly (P0.01).
2. The neurons in the hyperlipidemia group had a small amount of atrophy, the mitochondrial crista shortened, the rough endoplasmic reticulum decreased, the capillary vessels in the cortex increased, the mitochondria and mitochondrial crista of the vascular endothelial cells increased, the glial cells increased obviously, the mitochondrial crista was rich, the synapse vesicles decreased and the mitochondrial crista became shorter.
2) the compound cerebral ischemia group was compared with the simple cerebral ischemia group.
(1) there was no difference between the two model group in the ischemic 3D and the 7d nerve function score, but the difference between the model rats after awakening and the ischemic 3D, and the difference of the 7d nerve function score, the difference of the 7d difference between the compound cerebral ischemia group and the ischemic group was significantly decreased (P0.05).
(2) the infarct volume of 7D in the combined cerebral ischemia group was significantly reduced (P0.05), and there was no difference in ischemic 3D (P0.05).
(3) NO, 6-keto-FGF1a was compared in the complex cerebral ischemia group and the simple cerebral ischemia group and the corresponding sham operation group. The serum content of 3D in the ischemic group was higher than that in the corresponding sham operation group, and the difference was significant (P0.05). The ischemic group and the simple cerebral ischemia group were compared with the ischemic group in the ischemic group, and the serum content of 7D decreased in the ischemic 3D and 7d in the ischemic group, but in the ischemic group, the serum content of 7D was decreased, but in the ischemic group, the serum content of 7D was reduced. There was no significant difference in ischemic 3D between groups (P0.05), but there was a significant difference between ischemic 7d groups (P0.05).
(4) the serum content of ET-1 was increased in each time point of cerebral ischemia model, but there was no significant difference between the combined cerebral ischemia group and the group of simple cerebral ischemia and the corresponding sham operation group (P0.05). The serum content of.TX-B2 in the cerebral ischemia model increased, and the ischemia group and the corresponding sham operation group were compared with the corresponding sham operation group, and in the ischemia group, the.TX-B2 was in ischemia. The serum content of 3D and 7d increased significantly (P0.05). Compared with the corresponding sham operation group, the serum content of 7D in the ischemic group and the corresponding sham operation group was increased, but there was no significant difference (P0.05) in the ischemic 3D, but in the compound cerebral ischemia group and the simple cerebral ischemia group, the serum content of the complex cerebral ischemia group decreased, and the difference of 3D and 7d in ischemia was significant (P0.05).
(5) the serum content of vWF factor increased in the cerebral ischemia model at all time points. Compared with the corresponding sham operation group, the serum content of 7D increased significantly in the simple cerebral ischemia group, and the difference was significant (P0.05). In the compound cerebral ischemia group and the corresponding sham operation group, the serum content of 3 D in the complex cerebral ischemia group increased, and the difference between the cerebral ischemia group and the corresponding sham operation group increased. Significant (P0.05); compared with the simple cerebral ischemia group, the serum levels of 3D and 7d in the combined cerebral ischemia group increased significantly (P0.05).
(6) MCP-1, TNF- alpha and CRP were compared in the complex cerebral ischemia group and the simple cerebral ischemia group and the corresponding sham operation group. The serum content of the ischemic group was more than that of the corresponding sham operation group, and the serum content of 7D was significantly higher than that of the corresponding sham operation group (P0.05). The complex cerebral ischemia group was compared with the simple cerebral ischemia group, and the serum content of the ischemic group was increased in the ischemic group of 3D, and the serum level of 7D was more than that of the group of cerebral ischemia. The serum content of 7D in the complex cerebral ischemia group increased and the serum level of 7D was increased. Different significance (p0.05,0.01p0.05).
MMP-2, ICAM-1, CD34 were compared in the group of ischemic cerebral ischemia and simple cerebral ischemia group and the corresponding sham operation group. The expression of 3D in ischemic group was higher than that in the corresponding sham operation group, and the difference was significant (P0.05). The expression of 3D in the compound cerebral ischemia group was more than that in the ischemic group, and the expression of 3D in the complex cerebral ischemia group was increased and the expression of 7D brain tissue increased. The difference was significant (p0.05,0.01P0.05).
Compared with the group of pure cerebral ischemia and sham operation group, the content of ischemic 3D in the serum was increased, the difference was significant (P0.05), and the content of ischemic 3D and 7d in the compound cerebral ischemia group and the hyperlipidemic sham group were decreased, the difference was significant (P0.05), and the complex cerebral ischemia group was less than the group of simple cerebral ischemia group, and the complex cerebral ischemia group was deficient in the group of cerebral ischemia. The blood 3D, 7d serum content decreased significantly (P0.05).SOD in the complex cerebral ischemia group and the Dan Chunnao ischemia group and the corresponding sham group, the cerebral ischemia group was in the ischemic 3D, 7d serum content decreased, the difference was significant (P0.05), the compound cerebral ischemia group was compared with the simple cerebral ischemia group, the complex cerebral ischemia group decreased the serum level of ischemic 3D in the ischemic group. The difference was significant (P0.05).
Bcl-2, Bax in cerebral ischemia group and simple cerebral ischemia group and the corresponding sham operation group, cerebral ischemia group in ischemic 3D, 7d expression in the brain tissue increased, the difference was significant (P0.05), Bax in the cerebral ischemia group compared with the simple cerebral ischemia group, Bax in the brain tissue of the complex brain tissue expression 3D, 7d decreased significantly (P0.05). The expression of Bcl-2 in ischemic 3D increased significantly (P0.05), and Bcl-2/Bax ratio increased significantly in 3D group and 7d group (P0.05).
Compared with the simple cerebral ischemia group, the ischemic 3D had a large number of neurons denatured, necrotic, the nucleus chromatin was sparse, the mitochondrial crista shortened or disappeared, the intercristal cavity enlarged and the rough endoplasmic reticulum decreased obviously; the vascular endothelial cells were swollen, the cell connection was destroyed, the mitochondria and mitochondrial crista were reduced; nerve gum was reduced. The mitochondrial crista became short or disappearing in the mitochondrial crista; there were microgliosis, the synaptic vesicles decreased, the mitochondrial crista shortened or disappeared. The ischemic 7d was reduced, and the complex cerebral ischemia group compared with the simple cerebral ischemia group, the neuron degeneration, the decrease of necrosis, the decrease of edema, the recovery of mitochondrial damage and the glia. The number of cells increased obviously, the mitochondria cristae were abundant, and the edema of endothelial cells was alleviated.
5. conclusion
1) during hyperlipidemia, lesions can damage vascular endothelial cells, induce inflammatory reaction in the brain tissue, and have a certain effect on blood vessels and neurons.
2) inflammation and inflammation related factors may be part of the biological basis of TCM pathogenesis of phlegm dampness and blood stasis.
3) under the condition of hyperlipidemia, the inflammatory factors have both accumulation and specific expression in different periods of ischemia, which may contribute to the repair of injury in the recovery period of cerebral ischemia.
4) the endogenous brain protection mechanism may be activated under the pre stimulation of inflammation.
【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】：2012
【分类号】：R589.2;R-332

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