| [Objective] With the aging of our population and the change of life style, in recent years, ischemic cerebral vascular disease (ischemic cerebral vascular disease, ICVD) is seriously affecting people’s health by its high incidence, high morbidity and high mortality. Therefore, it becomes very important to clarify its mechanism. Blood brain barrier formed by capillary endothelial and its tight junction, complete basement membrane, pericytes and astrocyte foot processes (blood brain barrier, BBB), is very important in the maintenance of homeostasis and function of the central nervous system. The endothelium is the main structure of the blood brain barrier. BBB damage cause cerebral edema, cerebral hemorrhage transformation and cerebral ischemic injury aggravated after cerebral ischemia. Cathepsin L (cathepsin L, CTSL) with the form of zymogen exists widely in animal cell lysosomes, and cerebral ischemia can reduce the stability of lysosomal membrane permeability and even rupture. A large amount of CTSL released into the cytoplasm or interstitial and activated, may destruct BBB. High temperature is a common complication of ICVD. Researches have shown that high temperature may aggravate the cerebral ischemic injury, but the mechanism is not clear completely. At present, whether CTSL is closely connected to the cultured BBB endothelial cells TJ damage and whether high temperature takes a part in the effect of CTSL to damage cultured BBB endothelial cell protein TJ, and increases the BBB damage, there is no related research. Therefore,we want to culture of SD rat BBB model in vitro. After high temperature, CTSL and CTSL+high temperature treatment in different time, we can observe the changes of BBB endothelial cell tight junction (tight junction, TJ) protein claudin-l.we can explore the effects of CTSL and high temperature to BBB more.[Methods] We get brain microvascular endothelial cells and astrocytes from SD rats by Primary isolation, purification and culturing. Cell morphology is observed under inverted microscope. HE staining of brain microvascular endothelial cells, vascular fluorescent antibody, astrocytic glial fibrillary acidic protein (glial fibrillary acidic protein, GFAP) cell antibody identification type. The two kinds of cells were cultured in vitro BBB model.4h leakage test, Lucifer yellow permeability determination of permeability of BBB model. The identification of BBB model is successfully, The models were randomly divided into normal groups (n=5),30min groups (n=5), high temperature heat of60min groups (n=5),90min groups (n=5), the heating temperature of+CTSL30min groups (n=5),60min (n=5) and90min groups (n=5) and high temperature (n=5),60min (+CTSL30min n=5) and90min groups (n=5). Using immunohistochemistry to investigate the expression of western, blot method was used to detect the TJ protein claudin-1BBB model. The model itself and high temperature may result in CTSL, the model can be divided into normal temperature group (n=5) and30min group (n=5), high temperature heat of60min group (n=5),90min group (n=5), high temperature with immunohistochemistry to investigate the expression of Western, Blot method for detection of CTSL.[Results]1, The cells we cultured are brain microvascular endothelial cells and astrocytes by HE staining, immunofluorescence identification.2, The non-contact BBB model matched12holes plate is the. Complete fusion model under microscope, cells.4H leakage test results were positive. The model has lower the permeability coefficient (permeability coefficient, Pe). The value is1.08±0.1610-3cm/min.3, At the same time point, The number of CTSL positive cells in astrocytes with39℃high temperatureing groups were higher than37℃normal groups. The differences were statistically significant (P<0.05). In high temperatureing groups, with the extension of time, the number of CTSL positive cells in astrocytes also increased. The differences were statistically significant (P<0.05).4, Western blot showed that at the same time point,the CTSL expression of astrocytes in high temperatureing groups were significantly higher than that in normal temperature groups, with the high temperatureing time increased, the differences were statistically significant (P<0.05).5, At different time points, the number of endothelial cells claudin-1positive cells at37℃temperature groups+CTSL were higher decreased than37℃temperature groups. With the time prolonged the number of endothelial cells claudin-1positive cells were decreased more, the differences were statistically significant (P<0.05). It is Similar to39℃high temperatureing groups.6, At the different time points, the Western blot showed that the claudin-1’expression of endothelial cells at37℃+CTSL groups were lower than37℃groups. They were significantly. With the time prolonged the claudin-1’expression of endothelial cells were decreased more, the differences were statistically significant (P<0.05).7, Immunohistochemistry showed that at the same time point, the number of endothelial cells claudin-1positive cells at39℃+CTSL high temperatureing groups were lower than37℃+CTSL groups.The differences were statistically significant (P<0.05).8, Western blot showed that at different time points, the claudin-1expression of endothelial cells at39℃+CTSL high temperatureing groups were lower than37℃+CTSL groups. They were further decreased with the extension of time. The differences were statistically significant (P<0.05).[Conclusions]1,The BBB models in vitro we made obrain the basic characteristics of BBB on the morphology and permeability.2, The glial cells of BBB models in vitro can express CTSL. High temperature may up the expression of CTSL at glial cells in vitro BBB model.3, CTSL can damage the endothelial cell protein TJ of BBB models in vitro at37℃. The damage were aggravated with the time prolonging.4,4, High temperatureing may exacerbate the CTSL on the BBB models in vitro endothelial cells TJ’damage. They were increased with the time prolonged.5,High temperature can promote BBB’damage through uping regulation of CTSL. |
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