| [Objective] Stroke is a leading cause of death and disability in the world, its pathogenesis is very complex. Ischemic stroke is the most common type accouting for 87%. The blood-brain barrier (BBB) plays a key role in ischemic stroke.BBB is a selective permeability barrier system between the blood and brain tissue, strictly control the material translocation on both sides of the vessel, so as to maintain the central nervous system (CNS) in a relatively stable environment. BBB disruption directly affect the central nervous system function after stroke, resulting in vasogenic brain edema, hemorrhagic transformation and neuronal apoptosis, aggravation of the neurological function injury. The activation of matrix metalloproteinases (MMPs) is considered to be an important molecular mechanism for damage to structure and function of BBB. After cerebral ischemia, matrix metalloproteinase -2 (MMP-2) may be through the degradation of endothelial cells tight junction protein and basement membrane components for damage to the integrity of vessel wall. Fever is a common complication in the early stage of stroke patients. Some studies have found that fever can worsen the prognosis of patients with stroke, but the specific mechanism has not been elucidated.Therefore, it has a certain practical significance to explore whether fever increase the brain damage after stroke, and upregulate MMP-2 activity exacerbating the damage to tight junctions protein of BBB. In this article, we use the primary culture of brain micro vascular endothelial cells and astrocytes of SD neonatal rat to establish a simple, easy vitro cell model of BBB. Given the exogenous addition of MMP-2 and fever treatment, We analyze the changes of tight junctions protein occludin and explore the possible mechanism of MMP-2 and fever about BBB damage after cerebral ischemia, providing a theoretical basis for the prevention and treatment of complications in patients with ischemic stroke.[Methods] This article use SD neonatal rats as experimental material and enzyme digestion of primary isolation, purification and culture brain microvascular endothelial cells and astrocytes.The morphology of these two kinds of cells were observed using inverted microscope. After HE staining, immunofluorescence identify cell type and cell purity, endothelial cells and astrocytes passaged 3 subcultures were co-cultured by Transwell chamber, to build in vitro BBB contact model. And this article through leakage tests and Lucifer yellow (LY) permeability determination to identificate model barrier function.The built successful models were randomly divided into four groups:37℃,39℃ fever,37℃+MMP-2 and 39℃ fever +MMP-2. Then this article use immunocytochemical method and western blotting to test the changes of tight junctions protein occludin in each group. At the same time, the expression of astrocytes MMP-2 was detected in 37℃ and 39℃ fever groups by immunocytochemical method.[Results] 1. The cultured brain microvascular endothelial cells and astrocytes were observed, showing their typical growth characteristics. By HE staining and immunofluorescence identified, two kinds of cells purity were more than 95%, meetting the experiment requirements for co-culture.2. Two kinds of cells were co-cultured with transwell chambers, to establish BBB contact model, matching with the 12 holes culture plate. Selecting the model of complete confluent cells under the microscope,4h leakage test was positive. LY permeability checked the models with low permeability coefficient (Pe), and its value is 0.95±0.06×10-3cm/min.3. Immunocytochemical method showed, the number of positive cells of astrocytes MMP-2,39℃ fever groups were higher than 37℃ normal groups at the same time, the differences were statistically significant (P<0.05).39℃ fever group along with the prolonging of treatment time, the number of MMP-2 positive cells were also increased, the difference was statistically significant (P<0.05).4. Immunocytochemistry and western blotting showed, compared with 37℃ normal groups at the same time point, the expression of endothelial cells occludin were decreased for 60 minutes and 90 minutes at 37℃+MMP-2 groups, the difference was statistically significant (P<0.05). And with the extension of time, the expression of endothelial cells occludin had a clear downward trend at 37℃+MMP-2 groups, the difference was statistically significant (P<0.05).5. Immunocytochemistry and western blotting showed, compared with 39℃ fever groups at the same time point, the expression of endothelial cells occludin were decreased for 60 minutes and 90 minutes at 39℃+MMP-2 groups, the difference was statistically significant (P<0.05). And with the extension of time, the expression of endothelial cells occludin had a clear downward trend at 39℃+MMP-2 groups, the difference was statistically significant (P<0.05).6. Immunocytochemistry and western blotting showed, compared with 37℃ normal groups at the same time point, the expression of endothelial cells occludin were decreased at 39℃ fever groups, the difference was statistically significant (P<0.05). And with the extension of time, the expression of endothelial cells occludin had a clear downward trend at 39℃ fever groups, the difference was statistically significant (P<0.05).7. Immunocytochemistry and western blotting also showed, compared with 37℃+MMP-2 groups at the same time point, the expression of endothelial cells occludin were decreased significantly at 39℃+MMP-2 fever groups, the difference was statistically significant (P<0.05).[Conclusions] 1. Brain microvascular endothelial cells and astrocytes of SD neonatal rat by primary isolation and culturing, and successfully established in vitro BBB contact models to meet the experiment requirements.2. In vitro cultured astrocytes express MMP-2, fever can up-regulate the expression of astrocytes MMP-2.3. At 37℃ normal conditions, addition of MMP-2 can reduce the expression of endothelial cells tight junction protein occludin of BBB contact models in vitro, and with the extension of time was aggravated.4. At 39℃ fever conditions, it’s down regulation which the expression of endothelial cells tight junction protein occludin of BBB models, and with the extension of time was aggravated.5. Fever further aggravated the down regulation of MMP-2 to endothelial cells tight junction protein occludin of BBB models in vitro, and with the extension of time was aggravated.6. Fever may aggravate the damage of BBB models in vitro through up-regulate the expression of MMP-2. |
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