慢性低氧对小鼠脑微血管内皮细胞RAGE和LRP-1表达的影响

目的研究慢性低氧状态下血脑屏障上β淀粉样蛋白（amyloidD，Aβ）转运体——晚期糖基化终产物受体（RAGE）及低密度脂蛋白受体相关蛋白-1（LRP-1）表达的变化。方法将鼠脑微血管内皮细胞（BMVECs）分别进行正常气体培养（正常对照组）以及24h、36h及48h的低氧培养，用RT—PCR法和WesternBlot法分别检测细胞RAGE和LRP-1mRNA和蛋白的表达。结果低氧24h组RAGEmRNA和蛋白表达低于正常对照组（P〈0．05），低氧48h组RAGEmRNA和蛋白表达高于正常对照组（P〈O．05），低氧24h、36h及48h组RAGEmRNA和蛋白表达呈时间依赖性增高（P〈0．05）；低氧各组LRP-1mRNA和蛋白表达较正常对照组降低，呈时间依赖性（P〈0．05）；RAGE／LRP，1mRNA和蛋白表达呈时间依赖性增高（P〈0．05）。结论在慢性低氧状态下，血脑屏障BMVECs表达RAGE上调，LRP-1下调，RAGE／LRP-1比值增高，推测可能由此增加了Ap的净入脑量。     

慢性脑缺血海马CA1区微血管LRP-1的表达及其对认知功能的影响

目的探讨慢性脑缺血大鼠海马CA1区微血管LRP—1表达与认知功能改变的相关性。方法应用双侧颈动脉结扎的方法制作SD大鼠慢性缺血模型;Morris水迷宫对大鼠的认知功能进行测试;免疫组化技术测定海马CA1区微血管LRP-1,Ⅷ因子相关抗原及GFAP的表达;放免技术对脑脊液Aβ蛋白的浓度进行测定。采用 MIAS图像分析系统对免疫组化结果进行平均光密度测定及微血管计数。结果术后1个月手术组大鼠认知功能已明显下降,寻找平台所需游走的距离较假手术显著延长。术后6个月手术组大鼠海马CA1区微血管LRP-1的表达较假手术组大鼠显著降低;GFAP的表达显著增强;但VIII因子相关抗原的表达及微血管计数两组之间无显著差异。微血管LRP-1表达与水迷宫成绩呈负相关。手术组大鼠脑脊液Aβ蛋白含量较假手术组显著下降。结论慢性缺血过程中,大鼠认知功能下降与海马CA1区微血管LRP-1的表达下降有显著相关性。     

凝血酶对体外培养脑微血管内皮细胞的影响

目的 :探讨凝血酶 (thrombin ,TM)增加血脑屏障 (blood brainbarrier ,BBB)通透性的机制。 方法 :将脑微血管内皮细胞在体外进行培养 ,在细胞的培养液中加入TM或TM +组织蛋白酶G(cathepsinG ,CATG) ,应用相差显微镜动态观察TM及其受体抑制剂对内皮细胞形态的影响 ,应用免疫组织细胞化学技术检测基质金属蛋白酶 2 (matrixmetalloproteinase 2 ,MMP 2 )表达的变化。结果 :TM加入后 1h内皮细胞开始收缩 ,细胞面积变小 ,细胞间隙增宽 ,细胞收缩程度具有时间依赖性 ,至 12h时细胞面积仅为处理前的 2 0 %。TM使内皮细胞MMP 2的表达水平明显增加 ,与对照组比较 ,存在明显统计学差异 (P <0 .0 1)。TM +CATG加入培养液后 ,细胞形态、MMP 2的表达与对照组比较均无明显统计学差异 (P >0 .0 5 )。 结论 :TM通过激活蛋白酶激活受体 1(proteaseactivatedreceptor 1,PAR 1) ,使内皮细胞发生收缩 ,促进MMP 2表达 ,是TM增加BBB通透性的可能机制。     

脑淀粉样血管病所致脑出血的免疫性血管病理损害初步研究

目的 回顾性分析外科手术治疗的自发性脑出血（intracerebral hemorrhage,ICH）病理标本中脑淀粉样 血管病（cerebral amyloid angiopathy,CAA）及相关免疫-血管因子共表达情况,探讨CAA-ICH的免疫性血 管病理损害可能机制。 方法 收集2010-2011年46例ICH患者的手术病理标本,其中男27例,女19例,平均年龄（56±15）岁。 免疫组化荧光检测β-淀粉样蛋白（amyloid β,Aβ）与晚期糖基化终产物受体（glycosylation end products,RAGE）、低密度脂蛋白受体相关蛋白1（low-density lipoprotein receptor related protein-1,LRP1） 蛋白的共表达情况。 结果 46例ICH患者中8例（男5例,女3例）确诊为CAA,占17.39%,确诊为CAA的8例患者脑组织标 本均有Aβ、RAGE、LRP1蛋白的共表达（阳性率100%）,而在非CAA自发性脑出血患者中均无共表达。 CAA-ICH组的LRP1表达水平较非CAA-ICH组显著降低（P =0.031）,而CAA-ICH组的RAGE表达水平较非 CAA-ICH组显著升高（P =0.015）。 结论 CAA是ICH的重要病因之一,CAA相关的免疫-血管因子RAGE、LRP1蛋白异常表达,在ICH发病机 制中可能有重要作用。     

凝血酶对体外培养脑微血管内皮细胞的影响

目的:探讨凝血酶(thrombin,TM)增加血脑屏障(blood-brain barrier,BBB)通透性的机制.方法:将脑微血管内皮细胞在体外进行培养,在细胞的培养液中加入TM或TM+组织蛋白酶G(cathepsin G,CATG),应用相差显微镜动态观察TM及其受体抑制剂对内皮细胞形态的影响,应用免疫组织细胞化学技术检测基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)表达的变化.结果:TM加入后1h内皮细胞开始收缩,细胞面积变小,细胞间隙增宽,细胞收缩程度具有时间依赖性,至12h时细胞面积仅为处理前的20%.TM使内皮细胞MMP-2的表达水平明显增加,与对照组比较,存在明显统计学差异(P＜0.01).TM+CATG加入培养液后,细胞形态、MMP-2的表达与对照组比较均无明显统计学差异(P＞0.05).结论:TM通过激活蛋白酶激活受体-1(protease activated recep-tor-1,PAR-1),使内皮细胞发生收缩,促进MMP-2表达,是TM增加BBB通透性的可能机制.     

大鼠脑微血管内皮细胞的体外培养

目的探讨大鼠脑微血管内皮细胞的培养方法.方法取Wistar大鼠乳鼠脑组织,采用筛网过滤、胶原酶消化、离心等技术获取脑微血管内皮细胞,并进行培养.通过形态学、免疫细胞化学方法进一步鉴定,采用MTT方法测定生长曲线.结果经形态学、免疫细胞化学方法鉴定所培养的细胞为脑微血管内皮细胞,观察到原代脑微血管内皮细胞有3种表型,细胞呈单层生长,并可传代培养.结论建立大鼠脑微血管内皮细胞的培养方法可为体外研究脑血管病提供有益帮助.     

HECTD1调控PI3K/AKT信号通路对低氧诱导的脑微血管内皮细胞损伤的影响

目的 探讨低氧应激下HECT结构域E3泛素连接酶1 (HECTD1)对脑微血管内皮细胞(BMECs)的影响及其功能机制。方法 BMECs细胞在1%O₂的条件下培养以诱导低氧刺激。使用多种细胞生物学功能实验测定来评估BMECs中低氧诱导的损伤。乳酸脱氢酶(LDH)、总抗氧化能力(T-AOC)、丙二醛(MDA)和超氧化物歧化酶(T-SOD)水平的检测以评估低氧诱导的氧化应激水平。Western blot检测PI3K/AKT信号通路相关蛋白的表达。结果 在常氧条件下,过表达HECTD1可以提高BMECs的细胞活力。在低氧条件下,过表达HECTD1显著减轻了低氧诱导的细胞活力抑制作用。此外过表达HECTD1降低了低氧诱导的BMECs细胞凋亡和氧化应激。结论 HECTD1通过阻断PI3K/AKT信号通路来保护BMECs免受低氧应激诱导的损伤,这表明HECTD1在低氧相关的脑疾病中具有治疗价值。     

PAR-1激活剂对体外培养的脑血管内皮细胞的影响

目的探讨蛋白酶激活受体-1(protease activated receptor-1,PAR-1)激活剂SFLLRN对体外培养的脑血管内皮细胞的影响,明确凝血酶增加血脑屏障通透性的可能机制。方法将脑微血管内皮细胞在体外进行培养,在细胞的培养液中加入SFLLRN或凝血酶,应用相差显微镜动态观察SFLLRN或TM对内皮细胞形态的影响,应用免疫组织细胞化学技术检测基质金属蛋白酶2(matrix metalloproteinase-2,MMP-2)表达的变化。结果 SFLLRN加入后1h内皮细胞开始收缩,细胞面积变小,细胞间隙增宽,细胞收缩程度具有时间依赖性,至12h时细胞面积仅为处理前的20%。凝血酶加入培养液后,细胞形态学的变化方式与SFLL-TRN组相似。SFLLTRN孵育后6h,内皮细胞MMP-2的表达水平开始增加,24小时达高峰,与对照组比较,存在明显统计学差异(P0.01),与TM组比较,无明显统计学差异(P0.05)。结论凝血酶通过激活PAR-1,使内皮细胞发生收缩,促进MMP-2表达,是凝血酶增加BBB通透性的可能机制。     

胶质细胞对共培养的脑微血管内皮细胞增殖及功能的影响

目的 研究体外培养条件下，胶质细胞对脑微血管内皮细胞(BMEC)增殖及功能的影响。方法 模拟血-脑脊液屏障结构及内皮细胞、胶质细胞间相互影响的途径，建立内皮细胞与胶质细胞共培养模型，采用细胞计数、细胞活性检测、酶含量与细胞吞饮量测定对内皮细胞增殖和功能进行研究。结果 共培养和条件培养时，内皮细胞增殖能力减弱，细胞活性以及酶含量增高，细胞吞饮量则无明显变化。结论 胶质细胞可通过两种途径影响内皮细胞的生长。胶质细胞可诱导和维持微血管内皮细胞的脑表型，但并不能促进内皮细胞生长。     

低氧预处理对脑缺血时沙鼠脑红蛋白表达的影响

脑红蛋白（neuroglobin，NGB）是新发现的一种携氧球蛋白，主要存在于人和脊椎动物脑内，在脑组织缺氧时表达增加，起储氧作用。我们观察低氧预处理对沙鼠脑红蛋白表达的影响。     

Levels of Soluble Receptor for Advanced Glycation End Products in Acute Ischemic Stroke without a Source of Cardioembolism

Background and Purpose

Low levels of soluble receptor for advanced glycation end products (sRAGE) are associated with three conventional vascular risk factors (3Fs: diabetes, hypertension, and hypercholesterolemia), nondiabetic coronary artery disease, and Alzheimer''s disease. However, the association between sRAGE and acute ischemic stroke (AS), especially AS without a source of cardioembolism, has not yet been established.

Methods

Patients with AS without a source of cardioembolism (n=259) and age-matched controls (n=300) were grouped according to the presence of 3Fs: AS patients with and without 3Fs (3Fs+ AS and 3Fs- AS, respectively) and controls with and without 3Fs (3Fs+ control and 3Fs- control, respectively). Levels of sRAGE were analyzed among the four groups.

Results

sRAGE was significantly higher in the controls than in the AS patients (855 pg/mL vs. 690 pg/mL, p<0.01). sRAGE was significantly higher in 3Fs- controls (996 pg/mL, p<0.05) than in 3Fs+ controls (721 pg/mL), and in AS group regardless of the 3Fs (629 pg/mL in 3Fs- and 705 pg/mL in 3Fs+). The lowest tertile of sRAGE was associated with an increased risk of AS in the 3Fs- group [adjusted odds ratio (OR) 4.0, 95% confidence interval (CI) 1.6-10.3, p<0.01] but not in the 3Fs+ group. The level of sRAGE was also correlated with neurological severity in the 3Fs- AS group (r=-0.32, p<0.05) but not in the 3Fs+ AS group.

Conclusions

Low plasma levels of sRAGE is a potential biomarker for the risk of AS and may reflect the neurological severity of the condition, especially in subjects without identifiable conventional risk factors.     

High-mobility group box-1 impairs memory in mice through both toll-like receptor 4 and Receptor for Advanced Glycation End Products

High-mobility group box-1 (HMGB1) is a nuclear protein with cytokine-type functions upon its extracellular release. HMGB1 activates inflammatory pathways by stimulating multiple receptors, chiefly toll-like receptor 4 (TLR4) and Receptor for Advanced Glycation End Products (RAGE). TLR4 and RAGE activation has been implicated in memory impairments, although the endogenous ligand subserving these effects is unknown. We examined whether HMGB1 induced memory deficits using novel object recognition test, and which of the two receptor pathways was involved in these effects. Non-spatial long-term memory was examined in wild type, TLR4 knockout, and RAGE knockout mice. Recombinant HMGB1 (10 μg, intracerebroventricularly, i.c.v.) disrupted memory encoding equipotently in wild type, TLR4 knockout and RAGE knockout animals, but affected neither memory consolidation, nor retrieval. Neither TLR4 knockout nor RAGE knockout mice per se, exhibited memory deficits. Blockade of TLR4 in RAGE knockout mice using Rhodobacter sphaeroides lipopolysaccharide (LPS-Rs; 20 μg, i.c.v.) prevented the detrimental effect of HMGB1 on memory. These data show that elevated brain levels of HMGB1 induce memory abnormalities which may be mediated by either TLR4, or RAGE. This mechanism may contribute to memory deficits under various neurological and psychiatric conditions associated with the increased HMGB1 levels, such as epilepsy, Alzheimer's disease and stroke.     

Effects of ginkgo biloba extract EGb761 on expression of RAGE and LRP-1 in cerebral microvascular endothelial cells under chronic hypoxia and hypoglycemia

Alzheimer’s disease (AD), characterized by accumulation of amyloid-beta protein (Aβ) in brain parenchyma, is closely associated with brain ischemia. Decreased clearance of Aβ from brain is the main cause of Aβ accumulation in sporadic AD. However, the mechanisms underlying ischemia-mediated AD pathogenesis remain unclear. The receptor for advanced end glycation products (RAGE) and low-density lipoprotein receptor-related protein-1 (LRP-1) expressed at blood brain barrier (BBB) are actively involved in Aβ clearance. RAGE is thought to be a primary transporter of Aβ across BBB into the brain from the systemic circulation, while LRP-1 mediates the transport of Aβ out of the brain. Ginkgo biloba extract EGb761, a traditional Chinese medicine, has been widely used in the treatment of AD. To investigate the effects of EGb761 on the expression of RAGE and LRP-1 in endothelial cells in response to ischemic injury, we cultured bEnd.3 cells, an immortalized mouse cerebral microvessel endothelial cell line, under a chronic hypoxic and hypoglycemic condition (CHH) to mimic ischemic injury of BBB, and then treated with EGb 761. We found that EGb 761 markedly ameliorated the damage (evaluated by MTT assay) from CHH. Moreover, we demonstrated that CHH led to a significant increase in the expression of RAGE both at the mRNA and protein levels at all times (24, 36, and 48 h), conversely; CHH induced a dramatic decrease in LRP-1 mRNA and protein expression at both 36 and 48 h. The results indicated that CHH has differential effects on the expression of RAGE and LRP-1. Furthermore, EGb761 significantly reversed CHH-induced upregulation of RAGE expression and downregulation of LRP-1 expression. Our findings suggest that EGb761 favor clearance of Aβ via regulating the expression of RAGE and LRP-1 during brain ischemia. This may provide a new insight into a possible molecular mechanism underlying brain ischemia-mediated AD pathogenesis, and potential therapeutic application of EGb 761 in treatment of AD.     

亚低温对局灶性脑缺血大鼠白细胞介素-6和细胞间黏附分子-1表达的影响

目的 探讨亚低温对局灶性脑缺血大鼠缺血局部细胞间黏附分子-1（ICAM-1）表达和血清白细胞介素-6（IL-6）含量的影响。方法 将30只体重在250～300&#8197;g的雌性SD大鼠随机分为实验组（n=15）和对照组（n=15），采用线栓法阻断大鼠一侧大脑中动脉制作局灶性脑缺血模型.制模成功后，实验组和对照组大鼠分别给予亚低温、常温处理，使其肛温保持在（33±1）℃和（37±0.5）℃。12&#8197;h后，自左室取血，断头取脑，检测缺血区ICAM-1阳性血管数目（免疫组化方法）和血清IL-6含量（免疫放射测定法）。结果 实验组缺血局部ICAM-1的表达明显低于对照组[（2.45±1.56）vs（18.27±2.45）个/HP，P<0.01］；对照组血清IL-6含量、神经功能缺陷评分明显高于实验组[（184±38）vs（124±41）ng/L），P<0.01；（2.13±0.35）vs（1.48±0.25），P<0.01]。结论 亚低温对脑缺血性损害的神经保护作用可能与降低IL-6含量和减少ICAM-1的表达有关。     

Orexin-1受体拮抗剂对慢性点燃癫大鼠学习记忆的影响及海马神经细胞的增殖变化

目的:研究orexin-1受体(OX1R)拮抗剂(SB334867,SB)对戊四氮(PTZ)慢性点燃癫大鼠空间学习记忆能力及海马齿状回神经细胞增殖的影响。方法:Wistar大鼠随机分为①对照组[腹腔和侧脑室均注射生理盐水(NS)];②PTZ组(腹腔注射PTZ+侧脑室注射NS);③PTZ+orexin-A(OXA)组(腹腔注射PTZ+侧脑室注射OXA);④PTZ+SB组(腹腔注射PTZ+侧脑室注射SB);⑤PTZ+SB+OXA组(腹腔注射PTZ+侧脑室注射SB和OXA)。观察各组大鼠的空间学习记忆能力及海马齿状回区BrdU+和BrdU+/NeuN+细胞的表达。结果:与PTZ+OXA组比较,PTZ+SB+OXA组大鼠逃避潜伏期延长、穿越平台象限的次数减少(P<0.05)。免疫荧光显示,PTZ+OXA组大鼠齿状回区BrdU+和BrdU+/NeuN+细胞表达增多(P<0.01),而PTZ+SB+OXA组大鼠齿状回区BrdU+/NeuN+细胞表达比PTZ+OXA组减少(P<0.01)。结论:OXA通过OX1R能改善癫大鼠的空间学习记忆能力,可能与OX1R介导的海马齿状回神经细胞增殖与分化作用有关。