组织蛋白酶L及高温对体外血脑屏障紧密连接蛋白claudin-1的影响
发布时间：2018-09-04 13:20
【摘要】：[目的]随着我国人口老龄化和群众生活方式的改变,近年来缺血性脑血管病(ischemic cerebral vascular disease, ICVD)以其高发病率、高致残率和高死亡率严重影响着人们的健康。为此,对其发病机制的研究便显得极为重要。由毛细血管内皮细胞及其间的紧密连接、完整的基膜、周细胞以及星形胶质细胞突起构成的血脑屏障(blood brain barrier, BBB),在维持中枢神经系统的稳态及功能方面极为重要。其中内皮细胞是血脑屏障的主要结构。脑缺血后BBB破坏导致脑水肿、脑出血转化及缺血性脑损伤加重。组织蛋白酶L (cathepsin L, CTSL)以酶原的形式广泛存在于动物细胞的溶酶体中,脑缺血时可导致溶酶体膜稳定性降低、通透性增高甚至破裂,大量CTSL释放到胞质或组织间隙并被激活,可能对BBB产生破坏。高温是缺血性脑血管病的常见并发症,有研究显示高温可加重缺血性脑损伤,但机制欠清。目前,CTSL是否对体外培养的BBB内皮细胞紧密连接(tight junction, TJ)产生破坏,以及高温是否通过影响CTSL加剧上述损伤,还无相关研究。为此,通过体外培养SD大鼠BBB模型,经高温、CTSL以及CTSL+高温分别处理不同时间,观察BBB内皮细胞紧密连接(tight junction,TJ)蛋白claudin-1的变化,从而探索CTSL以及高温对BBB的影响。 [方法]原代分离、纯化,传代培养大鼠脑微血管内皮细胞和星形胶质细胞。倒置显微镜下观察细胞形态。HE染色、脑微血管内皮细胞免疫荧光抗体、星形胶质细胞胶质纤维酸性蛋白(glial fibrillary acidic protein, GFAP)抗体鉴定细胞类型。将两种细胞共培养建立体外BBB模型。4h渗漏试验、Lucifer yellow(LY)渗透性测定BBB模型的通透性。经鉴定BBB模型成功后,随机分为37℃常温30min、60min及90min组(各5个)、39℃高温30min、60min及90min组(各5个)、37℃常温+CTSL30min、60min及90min组(各5个)和39℃高温+CTSL30min、60mmin及90min组(各5个)。用免疫组化、western blot法检测BBB模型TJ蛋白claudin-1的表达。考虑到体外BBB本身可能表达CTSL,同时用免疫组化及western blot法检测了37-C常温30min、60min及90min组(各5个)及39℃高温30min、60min及90min组(各5个)BBB模型CTSL的表达。 [结果]1、经HE染色、免疫荧光法鉴定所培养的细胞为大鼠脑微血管内皮细胞和星形胶质细胞。2、建立与12孔板相匹配的非接触BBB模型。镜下细胞融合完整的模型行4h渗漏试验,结果成阳性。模型具有低通透系数(permeability coefficient, Pe),其值为1.08±0.16×10-3cm/min.3、在相同时间点,39℃高温组星形胶质细胞CTSL阳性表达细胞数较37℃常温组增高,差异具有统计学意义(P0.05)。在高温组中,随着时间的延长,星形胶质细胞CTSL阳性表达细胞数亦增加,差异具有统计学意义(P0.05)。4、Western blot法显示,在相同时间点,39℃高温组星形胶质细胞CTSL表达明显高于37℃常温组。在高温组中,随着高温时间的延长,星形胶质细胞CTSL表达亦有增加,差异均具有统计学意义(P0.05)。5、在不同时间点,37℃常温+CTSL组内皮细胞claudin-1阳性表达细胞数量较37℃常温组下降,并随时间的延长而加剧,差异有统计学意义(P0.05)。39℃高温情况类似。6、在不同时间点,western blot法显示37℃常温+CTSL组内皮细胞claudin-1表达较37℃常温组显著降低,并随时间的延长而加剧,差异有统计学意义(P0.05)。7、免疫组化法显示,在相同时间点,39℃高温+CTSL组内皮细胞claudin-1阳性表达细胞数量均较37℃常温+CTSL组明显下降,差异有统计学意义(P0.05)。8、western blot法显示,在不同时间点,39℃高温+CTSL组内皮细胞claudin-1的表达均较37℃常温+CTSL组明显下降,并随时间的延长而有进一步下降的趋势,差异有统计学意义(P0.05)。 [结论]1、构建的BBB体外模型在形态学和通透性方面具备了BBB的基本特性。2、体外BBB模型胶质细胞可表达CTSL,高温可上调体外BBB模型胶质细胞CTSL表达。3、常温情况下,CTSL可破坏体外BBB模型内皮细胞TJ蛋白,并随时间的延长有加剧。4、高温可加剧CTSL对体外BBB模型内皮细胞TJ的破坏,并随时间的延长而加剧。5、高温通过上调CTSL表达从而加剧体外BBB模型的破坏。
[Abstract]:[Objective] With the aging of population and the change of people's life style, ischemic cerebrovascular disease (ICVD) has seriously affected people's health in recent years because of its high incidence, high disability rate and high mortality rate. Therefore, it is very important to study the pathogenesis of ICVD. The blood brain barrier (BBB), which is composed of complete basement membrane, pericytes and astrocyte processes, plays an important role in maintaining the stability and function of the central nervous system. Cathepsin L (CTSL) is ubiquitous in lysosomes of animal cells in the form of enzymes. Cerebral ischemia can result in decreased stability, increased permeability and even rupture of lysosome membranes. Large amounts of CTSL are released into cytoplasm or tissue spaces and activated, which may destroy BBB. Hyperthermia is ischemic cerebral blood. Some studies have shown that hyperthermia can aggravate ischemic brain damage, but the mechanism is unclear. At present, whether CTSL can destroy the tight junction (TJ) of BBB endothelial cells cultured in vitro and whether hyperthermia can aggravate the above-mentioned damage by affecting CTSL have not been studied. The changes of Claudin-1 in BBB endothelial cell tight junction (TJ) were observed at different time after high temperature, CTSL and CTSL + treatment.
[Methods] Rat brain microvascular endothelial cells and astrocytes were isolated, purified and subcultured. Cell morphology was observed under inverted microscope. HE staining, immunofluorescent antibody of brain microvascular endothelial cells and glial fibrillary acidic protein (GFAP) antibody were used to identify cell types. BBB model was established by co-culture in vitro. The permeability of BBB model was determined by Lucifer yellow (LY) permeability test. After the successful identification of BBB model, BBB model was randomly divided into 37 C room temperature 30 min, 60 min and 90 min groups (each 5), 39 C high temperature 30 min, 60 min and 90 min groups (each 5), 37 C room temperature + CTSL 30 min, 60 min and 90 min groups (each 5) and 39 C high temperature + CTSL 30 m. The expression of TJ protein Claudin-1 in BBB model was detected by immunohistochemistry and Western blot. The expression of CTSL in BBB model was detected by immunohistochemistry and Western blot at 37-C room temperature for 30 min, 60 min and 90 min (5 in each group) and 39-C high temperature for 30 min, 60 min and 90 min (5 in each group). Da.
[Results] 1. The cultured cells were identified as rat brain microvascular endothelial cells and astrocytes by HE staining and immunofluorescence assay. 2. A non-contact BBB model matched with 12-well plate was established. The model with complete cell fusion under microscope was tested for 4 hours and the results were positive. The model had low permeability coefficient (Pe) and its value. At the same time point, the number of CTSL positive astrocytes in 39 C group was significantly higher than that in 37 C group (P 0.05). At the same time, the expression of CTSL in astrocytes of 39 C hyperthermia group was significantly higher than that of 37 C normothermia group. The expression of CTSL in astrocytes of 39 C hyperthermia group increased with the prolongation of hyperthermia time, and the difference was statistically significant (P 0.05). At different time points, the expression of Claudin-1 in endothelial cells of 37 C normothermia + CTSL group was significantly higher than that of 37 The number of endothelial cells decreased and aggravated with the prolongation of time. The difference was statistically significant (P 0.05). The high temperature of 39 C was similar. 6. At different time points, Western blot showed that the expression of Claudin-1 in endothelial cells of 37 C room temperature + CTSL group was significantly lower than that of 37 C room temperature group, and increased with the prolongation of time, the difference was statistically significant (P 0.05). At the same time point, the number of claudin-1 positive cells in the high temperature + CTSL group was significantly lower than that in the normal temperature + CTSL group (P 0.05). 8. Western blot showed that at different time points, the expression of Claudin-1 in the high temperature + CTSL group was significantly lower than that in the normal temperature + CTSL group (P 0.05). Obviously decreased, and the trend of further decline with the extension of time, the difference was statistically significant (P0.05).
[Conclusion] 1. The BBB model in vitro has the basic characteristics of BBB in morphology and permeability. 2. The BBB model glial cells can express CTSL in vitro. High temperature can up-regulate the expression of CTSL in BBB model glial cells in vitro. 3. Under normal temperature, CTSL can destroy the TJ protein of BBB model endothelial cells in vitro and increase with time. 4. High temperature can up-regulate the expression of CTSL in BBB model glial cells. CTSL aggravated the destruction of TJ in BBB model endothelial cells in vitro, and increased with time. 5. Hyperthermia aggravated the destruction of BBB model in vitro by up-regulating the expression of CTSL.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.3
[Abstract]:[Objective] With the aging of population and the change of people's life style, ischemic cerebrovascular disease (ICVD) has seriously affected people's health in recent years because of its high incidence, high disability rate and high mortality rate. Therefore, it is very important to study the pathogenesis of ICVD. The blood brain barrier (BBB), which is composed of complete basement membrane, pericytes and astrocyte processes, plays an important role in maintaining the stability and function of the central nervous system. Cathepsin L (CTSL) is ubiquitous in lysosomes of animal cells in the form of enzymes. Cerebral ischemia can result in decreased stability, increased permeability and even rupture of lysosome membranes. Large amounts of CTSL are released into cytoplasm or tissue spaces and activated, which may destroy BBB. Hyperthermia is ischemic cerebral blood. Some studies have shown that hyperthermia can aggravate ischemic brain damage, but the mechanism is unclear. At present, whether CTSL can destroy the tight junction (TJ) of BBB endothelial cells cultured in vitro and whether hyperthermia can aggravate the above-mentioned damage by affecting CTSL have not been studied. The changes of Claudin-1 in BBB endothelial cell tight junction (TJ) were observed at different time after high temperature, CTSL and CTSL + treatment.
[Methods] Rat brain microvascular endothelial cells and astrocytes were isolated, purified and subcultured. Cell morphology was observed under inverted microscope. HE staining, immunofluorescent antibody of brain microvascular endothelial cells and glial fibrillary acidic protein (GFAP) antibody were used to identify cell types. BBB model was established by co-culture in vitro. The permeability of BBB model was determined by Lucifer yellow (LY) permeability test. After the successful identification of BBB model, BBB model was randomly divided into 37 C room temperature 30 min, 60 min and 90 min groups (each 5), 39 C high temperature 30 min, 60 min and 90 min groups (each 5), 37 C room temperature + CTSL 30 min, 60 min and 90 min groups (each 5) and 39 C high temperature + CTSL 30 m. The expression of TJ protein Claudin-1 in BBB model was detected by immunohistochemistry and Western blot. The expression of CTSL in BBB model was detected by immunohistochemistry and Western blot at 37-C room temperature for 30 min, 60 min and 90 min (5 in each group) and 39-C high temperature for 30 min, 60 min and 90 min (5 in each group). Da.
[Results] 1. The cultured cells were identified as rat brain microvascular endothelial cells and astrocytes by HE staining and immunofluorescence assay. 2. A non-contact BBB model matched with 12-well plate was established. The model with complete cell fusion under microscope was tested for 4 hours and the results were positive. The model had low permeability coefficient (Pe) and its value. At the same time point, the number of CTSL positive astrocytes in 39 C group was significantly higher than that in 37 C group (P 0.05). At the same time, the expression of CTSL in astrocytes of 39 C hyperthermia group was significantly higher than that of 37 C normothermia group. The expression of CTSL in astrocytes of 39 C hyperthermia group increased with the prolongation of hyperthermia time, and the difference was statistically significant (P 0.05). At different time points, the expression of Claudin-1 in endothelial cells of 37 C normothermia + CTSL group was significantly higher than that of 37 The number of endothelial cells decreased and aggravated with the prolongation of time. The difference was statistically significant (P 0.05). The high temperature of 39 C was similar. 6. At different time points, Western blot showed that the expression of Claudin-1 in endothelial cells of 37 C room temperature + CTSL group was significantly lower than that of 37 C room temperature group, and increased with the prolongation of time, the difference was statistically significant (P 0.05). At the same time point, the number of claudin-1 positive cells in the high temperature + CTSL group was significantly lower than that in the normal temperature + CTSL group (P 0.05). 8. Western blot showed that at different time points, the expression of Claudin-1 in the high temperature + CTSL group was significantly lower than that in the normal temperature + CTSL group (P 0.05). Obviously decreased, and the trend of further decline with the extension of time, the difference was statistically significant (P0.05).
[Conclusion] 1. The BBB model in vitro has the basic characteristics of BBB in morphology and permeability. 2. The BBB model glial cells can express CTSL in vitro. High temperature can up-regulate the expression of CTSL in BBB model glial cells in vitro. 3. Under normal temperature, CTSL can destroy the TJ protein of BBB model endothelial cells in vitro and increase with time. 4. High temperature can up-regulate the expression of CTSL in BBB model glial cells. CTSL aggravated the destruction of TJ in BBB model endothelial cells in vitro, and increased with time. 5. Hyperthermia aggravated the destruction of BBB model in vitro by up-regulating the expression of CTSL.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.3
【参考文献】
相关期刊论文 前10条
1 陈yN招,徐如祥,黄柒金,徐中俊,姜晓丹,蔡颖谦;高温对血脑屏障内皮细胞紧密连接的影响[J];第一军医大学学报;2003年01期
2 赵康峰;王，
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