組織蛋白酶L及高溫對體外血腦屏障緊密連接蛋白claudin-1的影響
發(fā)布時(shí)間：2018-09-04 13:20
【摘要】：[目的]隨著我國人口老齡化和群眾生活方式的改變,近年來缺血性腦血管病(ischemic cerebral vascular disease, ICVD)以其高發(fā)病率、高致殘率和高死亡率嚴(yán)重影響著人們的健康。為此,對其發(fā)病機(jī)制的研究便顯得極為重要。由毛細(xì)血管內(nèi)皮細(xì)胞及其間的緊密連接、完整的基膜、周細(xì)胞以及星形膠質(zhì)細(xì)胞突起構(gòu)成的血腦屏障(blood brain barrier, BBB),在維持中樞神經(jīng)系統(tǒng)的穩(wěn)態(tài)及功能方面極為重要。其中內(nèi)皮細(xì)胞是血腦屏障的主要結(jié)構(gòu)。腦缺血后BBB破壞導(dǎo)致腦水腫、腦出血轉(zhuǎn)化及缺血性腦損傷加重。組織蛋白酶L (cathepsin L, CTSL)以酶原的形式廣泛存在于動(dòng)物細(xì)胞的溶酶體中,腦缺血時(shí)可導(dǎo)致溶酶體膜穩(wěn)定性降低、通透性增高甚至破裂,大量CTSL釋放到胞質(zhì)或組織間隙并被激活,可能對BBB產(chǎn)生破壞。高溫是缺血性腦血管病的常見并發(fā)癥,有研究顯示高溫可加重缺血性腦損傷,但機(jī)制欠清。目前,CTSL是否對體外培養(yǎng)的BBB內(nèi)皮細(xì)胞緊密連接(tight junction, TJ)產(chǎn)生破壞,以及高溫是否通過影響CTSL加劇上述損傷,還無相關(guān)研究。為此,通過體外培養(yǎng)SD大鼠BBB模型,經(jīng)高溫、CTSL以及CTSL+高溫分別處理不同時(shí)間,觀察BBB內(nèi)皮細(xì)胞緊密連接(tight junction,TJ)蛋白claudin-1的變化,從而探索CTSL以及高溫對BBB的影響。 [方法]原代分離、純化,傳代培養(yǎng)大鼠腦微血管內(nèi)皮細(xì)胞和星形膠質(zhì)細(xì)胞。倒置顯微鏡下觀察細(xì)胞形態(tài)。HE染色、腦微血管內(nèi)皮細(xì)胞免疫熒光抗體、星形膠質(zhì)細(xì)胞膠質(zhì)纖維酸性蛋白(glial fibrillary acidic protein, GFAP)抗體鑒定細(xì)胞類型。將兩種細(xì)胞共培養(yǎng)建立體外BBB模型。4h滲漏試驗(yàn)、Lucifer yellow(LY)滲透性測定BBB模型的通透性。經(jīng)鑒定BBB模型成功后,隨機(jī)分為37℃常溫30min、60min及90min組(各5個(gè))、39℃高溫30min、60min及90min組(各5個(gè))、37℃常溫+CTSL30min、60min及90min組(各5個(gè))和39℃高溫+CTSL30min、60mmin及90min組(各5個(gè))。用免疫組化、western blot法檢測BBB模型TJ蛋白claudin-1的表達(dá)。考慮到體外BBB本身可能表達(dá)CTSL,同時(shí)用免疫組化及western blot法檢測了37-C常溫30min、60min及90min組(各5個(gè))及39℃高溫30min、60min及90min組(各5個(gè))BBB模型CTSL的表達(dá)。 [結(jié)果]1、經(jīng)HE染色、免疫熒光法鑒定所培養(yǎng)的細(xì)胞為大鼠腦微血管內(nèi)皮細(xì)胞和星形膠質(zhì)細(xì)胞。2、建立與12孔板相匹配的非接觸BBB模型。鏡下細(xì)胞融合完整的模型行4h滲漏試驗(yàn),結(jié)果成陽性。模型具有低通透系數(shù)(permeability coefficient, Pe),其值為1.08±0.16×10-3cm/min.3、在相同時(shí)間點(diǎn),39℃高溫組星形膠質(zhì)細(xì)胞CTSL陽性表達(dá)細(xì)胞數(shù)較37℃常溫組增高,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。在高溫組中,隨著時(shí)間的延長,星形膠質(zhì)細(xì)胞CTSL陽性表達(dá)細(xì)胞數(shù)亦增加,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。4、Western blot法顯示,在相同時(shí)間點(diǎn),39℃高溫組星形膠質(zhì)細(xì)胞CTSL表達(dá)明顯高于37℃常溫組。在高溫組中,隨著高溫時(shí)間的延長,星形膠質(zhì)細(xì)胞CTSL表達(dá)亦有增加,差異均具有統(tǒng)計(jì)學(xué)意義(P0.05)。5、在不同時(shí)間點(diǎn),37℃常溫+CTSL組內(nèi)皮細(xì)胞claudin-1陽性表達(dá)細(xì)胞數(shù)量較37℃常溫組下降,并隨時(shí)間的延長而加劇,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。39℃高溫情況類似。6、在不同時(shí)間點(diǎn),western blot法顯示37℃常溫+CTSL組內(nèi)皮細(xì)胞claudin-1表達(dá)較37℃常溫組顯著降低,并隨時(shí)間的延長而加劇,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。7、免疫組化法顯示,在相同時(shí)間點(diǎn),39℃高溫+CTSL組內(nèi)皮細(xì)胞claudin-1陽性表達(dá)細(xì)胞數(shù)量均較37℃常溫+CTSL組明顯下降,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。8、western blot法顯示,在不同時(shí)間點(diǎn),39℃高溫+CTSL組內(nèi)皮細(xì)胞claudin-1的表達(dá)均較37℃常溫+CTSL組明顯下降,并隨時(shí)間的延長而有進(jìn)一步下降的趨勢,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 [結(jié)論]1、構(gòu)建的BBB體外模型在形態(tài)學(xué)和通透性方面具備了BBB的基本特性。2、體外BBB模型膠質(zhì)細(xì)胞可表達(dá)CTSL,高溫可上調(diào)體外BBB模型膠質(zhì)細(xì)胞CTSL表達(dá)。3、常溫情況下,CTSL可破壞體外BBB模型內(nèi)皮細(xì)胞TJ蛋白,并隨時(shí)間的延長有加劇。4、高溫可加劇CTSL對體外BBB模型內(nèi)皮細(xì)胞TJ的破壞,并隨時(shí)間的延長而加劇。5、高溫通過上調(diào)CTSL表達(dá)從而加劇體外BBB模型的破壞。
[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.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：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.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R743.3
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 陳yN招,徐如祥,黃柒金,徐中俊,姜曉丹,蔡穎謙;高溫對血腦屏障內(nèi)皮細(xì)胞緊密連接的影響[J];第一軍醫(yī)大學(xué)學(xué)報(bào);2003年01期
2 趙康峰;王，
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