糖尿病大鼠脊髓组织紧密连接Occludin蛋白表达变化

目的观察糖尿病对大鼠脊髓组织紧密连接Occludin蛋白表达的影响与不同病程阶段其变化趋势,从分子水平探讨糖尿病对血-脊髓屏障(BSCB)损害的机制。方法以链脲佐菌素介导的糖尿病大鼠模型的脊髓组织为研究对象,采用免疫组织化学方法、荧光定量PCR反应和Western Blot方法测定紧密连接Occludin蛋白的表达。结果糖尿病模型1个月组,Occludin蛋白的mRNA水平较正常组显著降低(P<0.05),糖尿病模型3个月组,其值较正常组进一步降低(P<0.01)。糖尿病模型1个月组,Occludin的蛋白质水平较正常组显著降低(P<0.05),糖尿病模型3个月组,其值较正常组降低(P<0.01)。结论糖尿病可以使BSCB紧密连接Occludin蛋白含量降低,随着病程延长其含量有逐渐降低的趋势,导致了糖尿病BSCB的损害。     

糖尿病大鼠血-脊髓屏障紧密连接蛋白claudin-1、claudin-5、ZO-1表达变化

目的动态观察糖尿病大鼠血-脊髓屏障紧密连接蛋白claudin-1、claudin-5、ZO1表达。方法雄性SD大鼠60只,随机分为糖尿病模型组和正常对照组2组,每组30只,以链脲佐菌素(STZ)腹腔注射建立糖尿病大鼠模型,分别于造模后第15天、30天、90天(各时间点均设对照)取脊髓组织(每次各取6只鼠)以Westernblot和实时荧光定量PCR法检测claudin-1、claudin-5、ZO-1蛋白和mRNA表达。结果 (1)claudin 1、claudin-5蛋白相对表达在糖尿病模型组第30天[分别为(0.324±0.01 5)、(0.289±0.032)]、第90天[分别为(0.220±0.029)、(0.242±0.034)]均较对照组[第30天分别为(0.464±0.009)、(0.475±0.035),第90天分别为(0.468±0.031)、(0.489±0.018)]降低(P0.01),且糖尿病模型组第15天、30天、90天三时间点claudin-1、claudin-5蛋白表达逐渐降低,差异有统计学意义(P0.05)。各组均未检出ZO-1表达。(2)claudin-1、claudin-5 mRNA相对表达在糖尿病模型组第30天[分别为(2.67±0.71)×10~(-4) 、(2.87±0.74)×10~(-4)]、第90天[分别为(0.081 4±0.034)×10~(-1)、(0.122±0.027)×10~(-1))]均较对照组[第30天分别为(13.13±3.09)×10~(-1)、(14.81±2.24)×10~(-4),第90天分别为(13.21±1.98)×10 、(1 4.50±2.72)×10~(-4)]降低(P0.01),且糖尿病模型组第15天、第30天、第90天三时间点mRNA相对表达水平逐渐降低,三时间点差异有统计学意义(P0.05)。ZO-1 mRNA表达在糖尿病模型组第15天、第30天及第90天[分别为(4.40±1.94)×10~(-1)、(2.33±0.64)×10~(-1)、(2.32±0.45)×10~(-1)]亦均较对照组[分别为(29.89±6.27)×10~(-1)、(30.68±1 7.64)×10~(-1)、(29.27 ±7.55)×10~(-1)]降低(P0.01)。结论糖尿病可能导致血-脊髓屏障紧密连接蛋白claudin-1、claudin-5、ZO-1表达降低,而且随病程延长claudin-l、claudin-5表达呈逐渐降低趋势。     

大鼠重型颅脑损伤急性期水通道蛋白4的表达

目的探讨水通道蛋白（AQP4）在大鼠重型脑外伤急性期的表达变化及其与脑水肿间的关系。方法49只成年雄性SD大鼠,随机分为对照组及实验组（伤后4h、8h、12h、24h、5d共5组）。制作重度冲击加速性损伤模型,分别于伤后4h、8h、12h、24h、72h、5d采用干湿比重法测脑组织含水量,原子吸收分光光度法测定Na^＋、K^＋含量,Evans Blue（EB）测定法观察大鼠血-脑屏障（BBB）通透性变化,半定量逆转录聚合酶链反应（RT-PCR）检测脑组织AQP4 mRNA表达及其变化。结果脑组织AQP4 mRNA在伤后4h开始表达上调,8h、12h依次增高,24h达到峰值（P〈0.05）,3d时仍维持较高水平,伤后5d有所降低。脑含水量、Na^＋含量的变化与AQP4 mRNA表达变化一致。经相关性分析,AQP4 mRNA的表达与脑含水量及脑EB含量均呈正相关（P〈0.05）。结论重型脑损伤急性期,AQP4 mRNA表达的变化与颅脑损伤后BBB的破坏及脑水肿的形成和发展密切相关。AQP4可能参与重型脑损伤后脑水肿的形成并起重要作用。     

糖尿病对脑缺血大鼠细胞间黏附分子-1表达及脑组织和血-脑屏障损害的影响

目的:探讨糖尿病对脑缺血大鼠细胞间黏附分子-1(ICAM-1)表达及脑组织和血-脑屏障(BBB)损害的影响.方法:120只雄性SD大鼠,随机分为糖尿病组、正常对照组、糖尿病伴脑缺血组和脑缺血组,每组30只大鼠.后两组又分为脑缺血1h、3h、6h、12h、24h亚组,每亚组6只大鼠.用链脲佐菌素腹腔注射制作糖尿病大鼠模型,用线栓法制作局灶性脑缺血模型.ICAM-1mRNA的表达水平用逆转录PCR检测,分别用TTC染色法和伊文思蓝注射法检测大鼠脑梗死体积及BBB破坏的程度.结果:脑缺血组各时间点亚组缺血侧脑组织ICAM-1mRNA表达均显著高于正常对照组(均P<0.05);糖尿病伴脑缺血组各时间点亚组ICAM-1mRNA表达均明显高于脑缺血组(均P<0.05).糖尿病伴脑缺血组大鼠脑梗死体积及BBB开放程度均明显大于脑缺血组(均P<0.05).结论:糖尿病使脑缺血大鼠ICAM-1表达上调,并可加重脑组织及BBB损害,提示ICAM-1表达上调可能是糖尿病加重脑缺血损伤的机制之一.     

水孔蛋白-4及其表达变化在脑水肿中的作用

本文介绍了水孔蛋白4在结构、分布以及定位方面的最新研究成果,分析文献,指出水孔蛋白4既参与水肿的形成,又参与水肿的消退;它的表达变化是组织缺氧、内环境紊乱及血脑屏障破坏的结果,对机体既有保护作用,也产生损害;同时提出一种新的治疗脑水肿的设想。     

实验性自身免疫性脑脊髓炎小鼠血脑屏障通透性与紧密连接蛋白-5的变化

目的观察实验性自身免疫性脑脊髓炎(EAE)小鼠血脑屏障(BBB)通透性与紧密连接蛋白-5(claudin-5)表达的变化。方法 40只C57BL/6小鼠随机分为EAE组和正常对照组,应用髓鞘少突胶质细胞糖蛋白35-55诱导制作小鼠EAE模型,每日进行神经功能缺损评分。采用苏木精-伊红、髓鞘染色和免疫组化法观察炎症细胞浸润、髓鞘脱失及轴索损坏程度。伊文思蓝染色和Western blot法观察BBB通透性和claudin-5表达的变化。结果与正常对照组比较,EAE组小鼠神经功能缺损评分、炎症细胞浸润、髓鞘脱失、轴索损坏和BBB通透性均明显增加;而claudin-5表达显著降低(均P0.01)。BBB通透性的变化与平均神经功能缺损评分、炎症细胞浸润和轴索损坏均呈正相关(P0.01);而与claudin-5表达水平呈负相关(P0.05)。结论 EAE发病过程中claudin-5表达减少,可能导致BBB通透性增加,使外周大量的炎症反应细胞向中枢迁移,继而进一步加重中枢的炎症反应。     

上皮膜蛋白1在实验性糖尿病大鼠模型中额叶皮质血—脑屏障上的表达

目的测定不同时间点实验性糖尿病大鼠额叶皮质血—脑屏障(BBB)上皮膜蛋白1(EMP1)的表达。方法选成年健康雄性Sprague-Dawley大鼠随机分为链脲佐菌素(STZ)诱导糖尿病组(STZ组)和对照组(CON组),每组分为2w、4w、8w 3个亚组,采用免疫组织化学法检测EMP1的表达。结果STZ大鼠与CON大鼠比较,4w组中STZ大鼠上EMP1的表达显著增多(P0.01),2w组和8w组中差异无统计学意义;EMP1的表达在STZ大鼠各亚组之间及CON大鼠各亚组之间比较差异无统计学意义(P0.05)。结论 EMP1在实验性糖尿病大鼠额叶皮质BBB上的表达改变,在模型建立成功后4w时表达显著增加。     

脑出血后血脑屏障紧密连接蛋白occludin的表达变化

目的 探讨脑出血后血脑屏障微血管内皮细胞间紧密连接蛋白occludin的表达变化. 方法 将SD雄性大鼠按随机数字表法分为正常对照组和脑出血组,再按时间因素将脑出血组分为出血后6h、24h、48h、72h、7d、14d6个亚组.采用脑内注入自体血法制作脑出血模型.HE染色观察脑出血后血肿周围脑组织的形态学改变;透射电镜观察脑出血后血肿周围紧密连接的超微结构改变;免疫荧光染色检测脑出血后血肿周围紧密连接蛋白occludin的表达分布状况;定量RT-PCR检测脑出血后血肿周围脑组织中occludin mRNA的表达状况. 结果 与正常对照组相比,脑出血组血肿周围脑组织出现水肿,在48 h左右尤为明显,局部可见明显脑细胞坏死及炎细胞浸润.脑出血后血肿周围紧密连接发生明显破坏,内皮细胞间出现裂隙.免疫荧光染色结果显示:正常对照组紧密连接蛋白occludin呈强阳性表达.脑出血后6hoccludin的表达即开始下降,呈阳性表达;脑出血后24～72h occludin的表达维持在较低水平,呈弱阳性表达.定量RT-PCR结果显示:脑出血后血肿周围脑组织中occludin mRNA相对含量明显降低,在6～72 h持续维持在较低水平,与正常对照组比较差异均有统计学意义(P＜0.05). 结论 脑出血发生后,紧密连接蛋白occludin 的表达下降,这可能是脑出血发生后血脑屏障破坏及脑水肿发生发展的重要分子基础之一.     

高血压脑出血患者血肿周围血-脑屏障葡萄糖转运蛋白1表达的研究

目的观察高血压脑出血(ICH)患者血肿周围血-脑屏障葡萄糖转运蛋白1(GLUT1)表达的变化。方法采用免疫组化方法检测28例高血压ICH患者不同病程血肿周围脑组织GLUT1的表达情况。结果与对照组相比,ICH组发病12 h GLUT1表达开始增强,24 h、48 h表达最强(均P<0.01);72 h表达明显下降(P<0.01)。结论ICH早期血肿周围GLUT1表达增多,有助于恢复脑的能量代谢。     

脑出血大鼠血肿周围组织水孔蛋白-4表达分析

目的研究大鼠脑出血(ICH)后皮质、CPu血肿周围组织水孔蛋白-4(AQP4)表达的变化,以探讨其在脑水肿形成过程中的作用。方法采用立体定向技术,经尾动脉采血制作大鼠CPu出血模型。采用免疫组化技术对皮质及血肿周围组织AQP4蛋白进行动态检测。结果ICH6 h后CPu血肿周围组织邻近毛细血管的AQP4蛋白表达开始增高,于出血第1~3天达高峰期,之后逐渐下降,至第7天仍高于正常水平。在出血侧皮质AQP4蛋白表达亦相应增加,但不如血肿周围组织明显。结论ICH时血肿成分可诱导AQP4蛋白表达增加,脑毛细血管周围星形胶质细胞终足表达增强的AQP4蛋白可能促进水通过血-脑脊液屏障向脑实质流动,直接参与ICH后血管源性脑水肿的形成。     

黄体酮对脑缺血再灌注大鼠紧密连接蛋白ZO-1和occludin表达的影响

目的探讨黄体酮对大鼠局灶性脑缺血再灌注后血脑屏障紧密连接蛋白ZO-1、occludin表达及血脑屏障通透性的影响。 方法将42只健康雄性SD大鼠按随机数字表法分为假手术组（6只）和缺血再灌注组,后者再按再灌注时间分为缺血2h再灌注3h、6h、12h、24 h、48 h及72h组（各6只）。缺血再灌注组用线栓法制备成大鼠大脑中动脉缺血再灌注模型。采用荧光分光光度法测定缺血侧脑组织中伊文氏蓝(EB)含量来评价血脑屏障的通透性,Western blotting法检测脑组织ZO-1和occludin的表达。取EB漏出最多组的时间点,增设黄体酮干预组和溶剂对照组（各6只）,与相同时间点的缺血再灌注组比较,观察黄体酮对ZO-1、occludin表达及血脑屏障通透性的影响。 结果 缺血2h再灌注3h时脑组织EB含量开始增加,再灌注24 h时达高峰;ZO-1、occludin的表达在缺血2h再灌注3h时开始下降,再灌注24 h时达最低。黄体酮干预组EB含量明显低于缺血2h再灌注24 h组,差异有统计学意义(P＜0.05)。黄体酮干预组ZO-1和occludin的表达水平均明显高于缺血2h再灌注24 h组,差异有统计学意义(P＜0.05)。 结论 黄体酮町抑制缺血再灌注大鼠紧密连接蛋白ZO-1和occludin表达的降低,从而起到保护血脑屏障的作用。     

大鼠局灶性脑缺血再灌注血脑屏障超微结构及紧密连接蛋白Occludin变化的研究

目的研究大鼠局灶性脑缺血再灌注模型血脑屏障(BBB)超微结构和Occludin的变化,探讨BBB的结构改变及Occludin的表达异常在再灌注损伤中的作用。方法雄性Wistar大鼠,随机分成假手术组、缺血2h再灌注3h、12h、24h、72h组,应用透射电镜、RT-PCR、免疫组化和Western Blot等方法观察再灌注后不同时相缺血区皮质BBB的超微结构,Occludin mRNA和蛋白水平的变化。结果局灶性脑缺血再灌注后,缺血区皮质BBB的超微结构受损,Occludin mRNA和蛋白表达水平下调。上述变化开始于再灌注后3h,再灌注24h达到高峰,72h开始减弱。结论脑缺血再灌注过程中,BBB的超微结构损伤及Occludin的表达下降加重了缺血再灌注损伤。     

HIV-1 Tat protein alters tight junction protein expression and distribution in cultured brain endothelial cells

Disruption of the blood-brain barrier (BBB) is widely believed to be the main route of human immunodeficiency virus (HIV) entry into the central nervous system (CNS). Although mechanisms of this process are not fully understood, alterations of tight junction protein expression can contribute, at least in part, to this phenomenon. Tight junctions are critical structural and functional elements of cerebral microvascular endothelial cells and the BBB. The aim of the present study was to examine the effects of HIV-1 Tat protein on expression of tight junction proteins. Primary cultures of brain microvascular endothelial cells (BMEC) were employed in these experiments. A 24-hr exposure of BMEC to Tat(1-72) resulted in a decrease of claudin-1, claudin-5, and zonula occludens (ZO)-2 expression, whereas total levels of occludin and ZO-1 remained unchanged. In addition, a short (3-hr) exposure of BMEC to Tat(1-72) induced cellular redistribution of claudin-5 immunoreactivity. Tat(1-72)-induced alterations of claudin-5 expression also were confirmed in vivo where Tat(1-72) was injected into the right hippocampus of mice. These findings indicate that HIV-1 Tat protein can markedly affect expression and distribution of specific tight junction proteins in brain endothelium. Alterations of only distinct tight junction proteins suggest a finely tuned effect of Tat(1-72) on the BBB. Because tight junction proteins are critical for the barrier function of the BBB, such alterations can lead to disturbances of the BBB integrity and contribute to HIV trafficking into the brain.     

Hypoxia-induced changes in tight junction permeability of brain capillary endothelial cells are associated with IL-1beta and nitric oxide

We examined whether hypoxia alone could produce changes in the permeability of brain capillary endothelial cells (EC) and whether a stimulation of hypoxic status alters the gene expression of occludin and glucose transporter 1 (GLUT1). Exposure of EC to hypoxia resulted in increased permeability, with the greatest decrease in transendothelial electrical resistance (TER) at 40 h. Moreover, hypoxia alone induced the expression of both mRNA in EC. Furthermore, we found that interleukin-1 (IL-1)beta, glutamate, hydrogen peroxide (H2O2), and sodium nitroprusside (SNP) induced the expression of mRNA for occludin and GULT1 under normoxic condition. The decrease in TER due to hypoxia was inhibited on addition of an anti-IL1 antibody and nitric oxide synthase (NOS) inhibitor in EC. These results indicate that the expression of occludin and GLUT1 mRNA is sensitive to exposure to hypoxia and that the changes of permeability in EC are associated with IL-1beta and NO.     

Occludin expression in microvessels of neoplastic and non-neoplastic human brain

The tight junction protein occludin 'glues' normal, adjacent brain microvessel endothelial cells together. Malignant brain tumours cause cerebral oedema because they have leaky endothelial tight junctions, which allow plasma fluid to enter the brain from the microvessel lumen. In order to identify molecular abnormalities in tumour endothelial tight junctions, we investigated occludin expression in microvessels from adult human non-neoplastic brain tissue using immunohistochemistry and immunoblotting. The proportions of microvessels immunolabelling for occludin were >2/3 in 5/5 non-neoplastic brain tissue samples, >1/3 in 5/5 low grade (Daumas-Duport I or II) astrocytomas and <1/3 in 5/5 high grade (III or IV) astrocytomas and 6/6 metastatic adenocarcinomas. Six non-neoplastic brain tissue immunoblots gave a 55-kDa occludin band, three low-grade astrocytomas gave 55-kDa and 60-kDa bands, 13 high-grade astrocytomas gave 60-kDa or no band and four adenocarcinomas did not give an occludin band. Expression of 55-kDa occludin inversely correlated with the presence of contrast enhancement on computed tomograms (P < 0.001). Electron microscopy showed open endothelial tight junctions in 0/2 non-neoplastic human brain specimens and 2/2 high-grade astrocytomas. We suggest that loss of 55-kDa occludin expression in human brain tumours may contribute to endothelial tight junction opening. Characterizing the molecular pathology of brain endothelial tight junctions may facilitate the design of novel drugs against cerebral oedema.     

缺血后处理对大鼠脑缺血再灌注后紧密连接的保护作用及ZO-1蛋白表达的影响

目的观察缺血后处理对脑缺血再灌注后紧密连接的保护作用及相关蛋白ZO-1表达的影响。方法 45只Wistar雄性大鼠随机分为假手术(Sham)组、缺血再灌注(I/R)组、缺血后处理(IP)组。采用线栓法建立大鼠大脑中动脉缺血模型,脑缺血2 h后,I/R组予再灌注,IP组给予缺血后处理之后予再灌注。于脑缺血再灌注后24 h行TTC染色观察脑梗死体积,应用电镜观察紧密连接蛋白结构改变,Western blot观察ZO-1蛋白表达的变化。结果 IP组脑梗死体积明显小于I/R组,与Sham组相比,I/R组血脑屏障紧密连接开放,ZO-1蛋白表达明显减少;与I/R组比较,IP组紧密连接开放程度减轻及ZO-1表达增加。结论缺血后处理减小脑梗死体积;缺血后处理能够减轻紧密连接破坏,其保护机制可能与ZO-1蛋白表达增加有关。     

缬沙坦对糖尿病大鼠脑组织氧化应激的影响

目的探讨缬沙坦对糖尿病大鼠脑组织氧化应激的影响。方法Wistar大鼠24只,随机分为正常对照组(NC组)、糖尿病组(DM1组)、糖尿病缬沙坦治疗组(DM2组),每组8只。用链脲佐菌素诱发糖尿病大鼠模型制模成功后,DM2组予以缬沙坦40mg/(kg.d)灌胃,共12周。12周后测定各组质量、血糖,Morris水迷宫试验观察大鼠认知功能,比色法测定脑组织中羟自由基(OH-)、丙二醛(MDA)含量及超氧化物歧化酶(SOD)活性,荧光实时定量反转录-聚合酶链反应检测尼克酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶p47phoxmRNA表达。结果(1)第12周未DM1组和DM2组质量明显轻于NC组(均P<0.01),血糖明显高于NC组(均P<0.01);DM1组与DM2组间差异无统计学意义。(2)DM1组、DM2组逃避潜伏期较NC组明显延长(P<0.01,P<0.05),DM2组较DM1组明显缩短(P<0.01)。(3)DM1组OH-、MDA水平显著高于DM2组和NC组(均P<0.01),而SOD活性显著低于DM2组和NC组(均P<0.01)。(4)NADPH氧化酶p47phox mRNA的表达量DM1组是NC组的4.89倍,DM2组是NC组的2.67倍,DM2组是DM1组的54.5%;各组间差异有统计学意义(均P<0.01)。结论缬沙坦能提高SOD的活性,降低OH-、MDA的活性和NADPH的表达,抑制机体的氧化应激水平,改善认知功能。     

HIV-1 gp120 proteins alter tight junction protein expression and brain endothelial cell permeability: implications for the pathogenesis of HIV-associated dementia

Breakdown of the blood-brain barrier (BBB) is commonly seen in patients with HIV-associated dementia (HAD) despite the lack of productive infection of the brain endothelium. It is likely that secreted viral products play a major role in BBB damage and the development of HAD. The objective of this study is to determine the effects of gp120 proteins on brain endothelial cell permeability and junctional protein expression. Our results showed that treatment of cultured human brain endothelial cells with gp120 for 24 hours results in increased permeability of the endothelial monolayer. Also, gp120 proteins caused disruption and downregulation of the tight junction proteins ZO-1, ZO-2, and occludin in these cells. Other junctional proteins such as claudin-1 and claudin-5 were unaffected by gp120 treatment. These data demonstrate that HIV gp120 proteins alter both the functional and molecular properties of the BBB, which could increase trafficking of HIV, infected cells, and toxic humoral factors into the central nervous system and contribute to the pathogenesis of HAD.