| 'whether it can permeate through blood-brain barrier or not' is an inevatible problem inpharmacological study of central nervous diseases. Inflammatory reaction in cerebral ischemia and reperfuion injury is caused by oxygenfree radicals induced by cytotoxicity ingredients,cytokines and preinflammation enzymeprecursors.It can promote convulsion of capillary vessels or arterioles and even theformation of new thrombus and expand injuried brain regions,that is one of the main reasonsof cerbral ischemia and reperfuion injury.Above two points are both basic physiological,pharmacolical and pathological rules of modern life science,also a fundamental problem inthe study of QingKaiLing treating cerebrovascular diseases. This study was supported by a grant(G1999055404) from the National Program for KeyBasic Research Projects(973)-'The effective components and mechanisms of QingKaiLingin depressing cascade reaction of ischemic stroke'.According to characteristic and in vivopharmacolical study of effective components,the research team defined cholic acid,baicalin,geniposide and pinctada martensii digest four effective components from originalQingKaiLing,and composed of a new formula XiaoFang.Purpose To study the expression of related adhesion molecules,proteases and regulating factorsin rat microvasular endothelial cells(MVEC) in vitro ischemia and reperfusion injury,expectto explore the cellular and molecular biological mechanism of QingKaiLing effectivecomponets and formula protecing brain MVEC from injury of inflammatory cascade incerebral ischemia diseases.Method 1.Primary culture of rat cerebral MVEC:To extract rat cerebral MVEC by separatingmicrovessel sections and collagenase enzymatic digesting,taking purifation and serialsubcultivation.Observing cell morphous by inverted microscope and applying Ⅷ factorimmunofluorescence and scanning electron microscope to identify cells. 2.Permeation rate of QingKaiLing effective components through an in vitro blood-brainbarrier:Applying an analog in vitro BBB by using mouse model of microvascular endothelialcells monostratal culture.The permeation rate of QingKaiLing effective components in 24hours is detected by means of LC/MS analysis. 3.Establishment of rat brain MVEC in vitro ischemia reperfusion model: Usingoxygen-glucose deprivation(95%N2,glucose-free Krebs slution) and recovery (air,highglucose DMEM)method,the model is evaluated by morphological changes and MTT. 4.The study of QingKaiLing effective components on inflammation reaction cascade inischemia reperfusion injury: ①Applying MTT colorimetric method to observe the changesof cell survival activity. ②Using nitrate reductase method to study the effect of medicine onNO content of cultured cell supernate fluid. ③Influence on cell adhesion molecules ICAM-1and VCAM-1:to detect the adhesion molecule protein changes by immunocytochemical stainand gene expression changes by RT-PCR analysis.④Effect on cell nuclear factor–kappaB(NF-ΚB): using immunocytochemical stain to observe the nuclear metastasis of positiveexpression.⑤Applying RT-PCR method to semi-quantitatively study the influence ofmedicine on MMP-9mRNA.Result 1.Cultured rat brain MVECs show fusiform or polygon shapes,and confluentmonolayer paving road stone sign;Ⅷ factor is positive,copious microvilli and intercellulartight junctions can be observed by scanning electron microscope.All above prove that thecells are brain MVECs. 2.Geniposide,cholic acid,hyodeoxycholic acid and dexocholic acid can permeatethrough an in vitro BBB, permeation rate is(26.22±0.41)%,(20.28±1.6)%,(13.34±0.95)%,(10.88±0.59)% respectively;Baicalin is not detected owing to its unsteady in this system. 3.Applying oxygen-glucose deprivation(OGD) 4 hours and recovery 12 hours toestablish an in vitro ischemia and reperfusion injury model,there is no difference from modelgroup and normal group when OGD 4 hours(P>0.05)while significant when 12 hours ofreperfusion (P<0.01). 4. In cell survival activity study (MTT),the OD value of geniposide,baicalin,cholic acid,hyocholic acid 1...
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