RNA干扰沉默神经轴突导向因子受体4基因增加血肿瘤屏障通透性

目的研究神经轴突导向因子受体(Robo4)对血肿瘤屏障(BTB)通透性的影响。方法建立了体外BTB模型,应用Real-time PCR和Western blot检测Robo4在正常人脑微血管内皮细胞和胶质瘤微血管内皮细胞中的表达变化。设计合成针对Robo4基因的小干扰RNA,转染至人脑微血管内皮h CMEC/D3细胞,下调体外血肿瘤屏障模型内皮细胞中Robo4的表达,跨内皮电阻测量系统和辣根过氧化物酶渗透试验分析血肿瘤屏障通透性变化;Western blot和免疫荧光法检测h CMEC/D3细胞中紧密连接相关蛋白occludin和ZO-1的表达和分布变化。结果和正常人脑微血管内皮细胞相比,Robo4在胶质瘤微血管内皮细胞中的表达显著上调。下调体外血肿瘤屏障模型内皮细胞Robo4的表达后,TEER值显著降低,辣根过氧化物酶透过率显著增高;同时胶质瘤微血管内皮细胞中紧密连接相关蛋白occludin和ZO-1的表达显著降低,在细胞膜上呈不连续分布。结论 RNA干扰沉默Robo4表达能够显著降低紧密连接相关蛋白occludin和ZO-1的表达,增加BTB通透性。     

PLGF通过激活Rho/ROCK信号通路介导人脑微血管内皮细胞间紧密连接开放

目的 探讨胎盘生长因子(PLGF)影响人脑微血管内皮细胞(HBMEC)间通透性的分子机制.方法 利用HBMEC构建体外血脑屏障模型,测定跨内皮细胞电阻抗(TEER)及检测HRP通过率,分析PLGF对血脑屏障完整性的影响;用Western blot法及免疫荧光法检测HBMEC间紧密连接相关蛋白质occludin和ZO-1的表达及分布,用蛋白激酶抑制剂干预和Western blot法检测Rho蛋白活性.结果 25 ng/mL PLGF 30 min显著降低体外血脑屏障模型TEER,使其通透性增高(P＜0.05);增加S-occludin表达的同时降低了IS-occludin的表达,使ZO-1蛋白在细胞周边分布减少;此外,ROCK蛋白激酶抑制剂Y27632抑制了PLGF引起的体外血脑屏障透过性增高(P＜0.05),并激活细胞内Rho激酶活性.结论 PLGF通过激活HBMEC内Rho/ROCK信号通路,影响HBMEC间紧密连接完整性,进而改变血脑屏障的通透性.     

ROCK介导缓激肽开放SD大鼠血肿瘤屏障

目的利用ROCK的特异性抑制剂Y-27632,研究ROCK是否介导缓激肽开放血肿瘤屏障。方法应用ROCK的特异性抑制剂Y-27632预处理大鼠原代脑微血管内皮细胞后,用缓激肽诱导血肿瘤屏障开放,测量跨内皮阻抗值(TEER),辣根过氧化物酶(HRP)渗漏量,分析血肿瘤屏障的通透性的改变;应用Western-blot法检测紧密连接相关蛋白ZO-1的表达;应用免疫荧光方法观察原代大鼠脑微血管内皮细胞紧密连接相关蛋白ZO-1和丝状肌动蛋白结构和分布的改变。结果 Y-27632显著抑制缓激肽诱导TEER值的降低,HRP的升高;Y-27632显著抑制ZO-1的表达;Y-27632抑制ZO-1由内皮细胞的边缘向细胞质转移,抑制丝状肌动蛋白由细胞膜边缘向细胞中央区分布,应力纤维形成明显减少。结论 ROCK介导缓激肽开放血肿瘤屏障。     

TNF-α激活RhoA/ROCK信号通路增加单核细胞增生性李斯特菌感染内皮细胞的通透性

目的探讨大鼠肉瘤同源基因A/Rho相关螺旋卷曲蛋白激酶(RhoA/ROCK)信号通路在肿瘤坏死因子α(TNF-α)促进单核细胞增生性李斯特(Lm)感染致内皮细胞通透性升高的调控作用。方法培养人脐静脉内皮细胞(HUVEC)分为未感染细胞对照组、 TNF-α处理组(100 ng/mL TNF-α处理2 h)、 Lm感染组(以MOI=10的Lm感染2 h,加入庆大霉素杀菌0.5 h)、 TNF-α处理的Lm感染组(Lm感染组细胞加入100 ng/mL TNF-α处理2 h)、 Y-27632联合TNF-α处理的Lm感染组(细胞用50μmol/L ROCK抑制剂Y-27632处理30 min,再如上进行Lm感染和TNF-α刺激)。Western blot法检测HUVEC的RhoA、闭锁小带蛋白1(ZO-1)、闭合蛋白(occludin)、 ROCK蛋白水平,检测辣根过氧化物酶(HRP)渗漏量分析HUVEC通透性变化,用异硫氰酸荧光素(FITC)标记的鬼笔环肽染色检测纤维型肌动蛋白(F-actin)细胞骨架的改变。结果 TNF-α可降低Lm感染的内皮细胞紧密连接蛋白ZO-1、 occludin的表达,促进其通透性升高、细胞骨架重排,并上调RhoA和ROCK的表达。ROCK抑制剂Y-27632可明显抑制TNF-α所致的HUVEC细胞骨架重排及通透性增高的作用。结论 TNF-α促进感染Lm的血管内皮细胞通透性增高可能通过RhoA/Rock信号通路调控。     

ROCK参与乙醇诱导肠上皮细胞屏障通透性增加

目的探讨Rho相关的卷曲蛋白激酶(ROCK)参与乙醇诱导的肠上皮细胞屏障通透性增加的作用。方法应用ROCK特异性抑制剂Y-27632预处理Caco-2细胞后,测定跨上皮细胞阻抗值(TEER)及荧光黄透过率,分析肠上皮细胞屏障的通透性的变化。应用Western blot的方法检测肌球蛋白轻链(MLC)、p-MLC及紧密连接相关蛋白(occludin)的表达。结果 Y-27632显著抑制乙醇诱导的TEER值降低、荧光黄透过率升高、pMLC/MLC及occludin(S/IS)比值的增加。结论 ROCK参与乙醇诱导肠上皮细胞屏障通透性增加。     

表皮生长因子受体抑制剂AG1478联合Endophilin-1过表达增加血脑屏障通透性

目的研究表皮生长因子受体(EGFR)抑制剂AG1478联合Endophilin-1基因过表达对血脑屏障(BBB)通透性的影响。方法建立了体外BBB模型,实验分为四组:对照组,AG1478组,endophilin-1基因过表达(p IRES2-endophilin-1)转染组;AG1478+p IRES2-endophilin-1转染组。采用跨内皮阻抗值测定和辣根过氧化物酶渗透试验评估血脑屏障通透性变化,Western blot和免疫荧光法检测脑微血管内皮细胞中紧密连接相关蛋白ZO-1和occludin的表达和分布变化。结果与单独应用AG1478或p IRES2-endophilin-1组相比,AG1478+p IRES2-endophilin-1转染组体外血脑屏障模型跨内皮电阻值显著降低,辣根过氧化物酶透过率显著增高(0.01),脑微血管内皮细胞中紧密连接相关蛋白ZO-1和occludin的表达水平显著降低(0.05)。结论 EGFR抑制剂AG1478联合endophilin-1基因过表达增加BBB通透性可能与开放紧密连接相关。     

miR-19a对血肿瘤屏障通透性的影响

目的研究miR-19a对血肿瘤屏障通透性的影响。方法将miR-19a模拟物转染至人脑微血管内皮细胞hCMEC/D3,应用real-timePCR法检测miR-19a的表达。用过表达miR-19a的hCMEC/D3细胞和人U251胶质瘤细胞建立体外血肿瘤屏障模型,跨内皮电阻测量系统检测血肿瘤屏障跨内皮阻抗值的变化;Western blot和免疫荧光法检测体外血肿瘤屏障hCMEC/D3细胞中,紧密连接相关蛋白ZO-1和occludin的表达。结果经miR-19a模拟物转染后,hCMEC/D3细胞中miR-19a的表达水平显著升高;血肿瘤屏障跨内皮阻抗值显著下降;体外血肿瘤屏障hCMEC/D3细胞中,紧密连接相关蛋白ZO-1和occludin的表达水平显著降低,在细胞膜上呈不连续分布。结论 miR-19a过表达能显著增加血肿瘤屏障的通透性,其机制之一可能与降低紧密连接相关蛋白相关。     

血必净注射液通过降低细胞紧密连接的通透性减轻LPS诱导的脓毒症大鼠肺微血管内皮细胞损伤

目的 探究血必净注射液(XBJ)对脂多糖诱导的脓毒症大鼠肺微血管内皮细胞损伤的作用及其对细胞紧密连接通透性的影响。方法 选择大鼠肺微血管内皮细胞，利用5μg/mL的脂多糖(LPS)处理大鼠肺微血管内皮细胞24 h,构建大鼠脓毒症肺损伤细胞模型，XBJ治疗组采用生药浓度2.5、5、10、25、50 mg/mL的XBJ共同进行干预24 h。CCK-8检测各组细胞活性，筛选最佳XBJ浓度；Hoechst染色检测各组细胞凋亡率；Western blot检测各组细胞凋亡相关蛋白Bax、Bcl-2和cleaved caspase3的表达；Transwell小室检测单层大鼠肺微血管内皮细胞通透性；Western blot检测紧密连接相关蛋白ZO-1、ZO-2和occludin的表达。结果 CCK8实验结果显示，XBJ的最佳浓度为10mg/mL。LPS处理后，大鼠细胞凋亡率明显升高，凋亡相关蛋白Bax/Bcl-2的比值和cleaved caspase3的表达水平增加；而XBJ治疗后，细胞凋亡率下降。LPS处理导致FITC-dextran含量显著增加，ZO-1、ZO-2和Occludin的蛋白表达水平降...     

巨噬细胞炎症因子1α对人脑微血管内皮细胞通透性的影响

目的研究巨噬细胞炎症因子1α(MIP-1α)是否具有开通血脑屏障的作用。方法以重组人MIP-1α直接作用于人脑微血管内皮细胞(HBMEC),免疫荧光方法检测紧密连接蛋白ZO-1的分布变化、跨内皮细胞电阻、HRP穿过HBMEC单层的改变、HBMEC细胞CC趋化因子受体5(CCR5)的表达,以及MIP-1α中和抗体和分泌MIP-1α的模式细胞(6T-CEM)与HBMEC单层共同温育时ZO-1的分布变化。结果MIP-1α作用下,HBMEC单层紧密连接结构被破坏,通透性增加,引起HBMEC细胞CCR5受体的表达,MIP-1α中和抗体阻断6T-CEM细胞对HBMEC单层ZO-1分布的改变。结论MIP-1α可能通过CCR5改变HBMEC单层通透性促进T淋巴细胞穿过血脑屏障。     

腺苷对肺癌脑转移的影响及其可能机制

目的探讨腺苷对肺癌脑转移的影响及其可能机制。方法采用Western blot法检测肺癌细胞内缺氧诱导因子-1(hypoxia inducible factor-1,HIF-1)和血脑屏障脑微血管内皮细胞上的紧密连接蛋白ZO-1的表达水平;应用ELISA法测定肺癌细胞培养液内腺苷的含量。采用荧光分析法检测体外血脑屏障模型的通透性改变;用Hemocytometer计数Transwell下室的A549肺癌细胞数。结果肺癌细胞中HIF-1的表达和肺癌细胞培养液内腺苷含量均于肺癌细胞缺氧12 h时达最高水平。与此同时,血脑屏障ZO-1蛋白的表达最低,血脑屏障通透性最大(7. 11),透过血脑屏障模型的肺癌细胞数最多(84. 6)。腺苷引起血脑屏障通透性增加,其变化趋势与缺氧肺癌细胞培养液的作用一致。结论缺氧可引起肺癌细胞释放腺苷增多,增多的腺苷导致血脑屏障紧密连接蛋白ZO-1表达减少,导致血脑屏障通透性增大,最终引起肺癌脑转移。     

Angiopoietin‐2 mediates blood–brain barrier impairment and colonization of triple‐negative breast cancer cells in brain

Although the incidence of breast cancer metastasis (BCM) in brain has increased significantly in triple‐negative breast cancer (TNBC), the mechanisms remain elusive. Using in vivo mouse models for BCM in brain, we observed that TNBC cells crossed the blood–brain barrier (BBB), lodged in the brain microvasculature and remained adjacent to brain microvascular endothelial cells (BMECs). Breaching of the BBB in vivo by TNBCs resulted in increased BBB permeability and changes in ZO‐1 and claudin‐5 tight junction (TJ) protein structures. Angiopoietin‐2 expression was elevated in BMECs and was correlated with BBB disruption. Secreted Ang‐2 impaired TJ structures and increased BBB permeability. Treatment of mice with the neutralizing Ang‐2 peptibody trebananib prevented changes in the BBB integrity and BMEC destabilization, resulting in inhibition of TNBC colonization in brain. Thus, Ang‐2 is involved in initial steps of brain metastasis cascade, and inhibitors for Ang‐2 may serve as potential therapeutics for brain metastasis. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Stem cell marker expression in small cell lung carcinoma and developing lung tissue

Histopathologic and clinical findings suggest that small cell lung cancer is derived from a multipotent proximal airway epithelial cell. In order to investigate the histogenetic origin of small cell lung cancer, we compared stem cell marker expression in human fetal lung tissue, human adult bronchial tissue, and a cohort of 64 small cell lung cancers. Supporting derivation of a multipotent precursor cell, 87.5% (56/64) of small cell lung cancers showed a dot-like expression of podocalyxin-like protein 1 (PODXL-1), a marker of embryonic and hematopoetic stem cells. Of small cell lung cancers, 98.4% (63/64) ubiquitously expressed Bmi-1, a key player in self-renewal of stem cells. Oct4 and AP2gamma were not expressed. Although podocalyxin-like protein 1 did not correlate with p53 or Wilms tumor suppressor 1, known regulators of podocalyxin-like protein 1, we could demonstrate demethylated CpG islands in the podocalyxin-like protein 1 promoter in small cell lung cancer, indicating epigenetic regulation. During fetal lung development and within adult bronchial mucosa, Bmi-1 was expressed ubiquitously. In contrast, podocalyxin-like protein 1 was detected in few stromal cells during the pseudoglandular phase (n = 7) and, importantly, in clustered epithelial cells within proximal bronchi and the trachea during the canalicular phase (n = 10). Interestingly, podocalyxin-like protein 1 was not expressed in normal or metaplastic adult bronchial epithelium (n = 36) but was expressed in sparse epithelial cells in half of the cases of normal tumor adjacent bronchial mucosa (20/40). Taken together, we show that small cell lung cancers and clustered epithelial cells in developing proximal bronchi share the expression of stem cell markers, suggesting a possible histogenetic link.     

Peripheral T cells overexpress MIP-1alpha to enhance its transendothelial migration in Alzheimer's disease

It is unclear how circulating T cells cross the blood-brain barrier (BBB) and participate in the inflammation process in Alzheimer's disease (AD). Here we showed significantly higher macrophage inflammatory protein-1alpha (MIP-1alpha) expression in peripheral T lymphocytes of AD patients than age-matched controls. T cells crossing of the human brain microvascular endothelial cells (HBMECs) which constitute the BBB, were almost completely abrogated by anti-MIP-1alpha antibody. MIP-1alpha induced the expression of CCR5, a potential MIP-1alpha receptor, on HBMECs. HBMECs tranfected with CCR5 resulted in increased T cells transendothelial migration. CCR5 antagonist (2D7 mAb) blocked the T cells transmigration. The MIP-1alpha-CCR5 interaction promoted T cells transendothelial migration via ROCK (Rho kinase). Furthermore, Abeta injection into rats' hippocampus induced MIP-1alpha overexpression accompanied with increased T lymphocytes occurrence in the brain cortex and this enhanced T cells entry was effectively blocked by anti-MIP-1alpha antibody. These data are the first to suggest that the interaction between MIP-1alpha overexpressed by T cells and CCR5 on HBMECs is involved in AD patients' T cells migrating from blood to brain.     

Expression and modulation of IFN-gamma-inducible chemokines (IP-10, Mig, and I-TAC) in human brain endothelium and astrocytes: possible relevance for the immune invasion of the central nervous system and the pathogenesis of multiple sclerosis.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS) mediated by blood-derived immune cells invading the CNS. This invasion could be determined by chemokines, and their role within the MS-affected brain is still poorly defined. We investigated the expression by RT-PCR and protein release by ELISA of the interferon-gamma (IFN-gamma)-inducible chemokines in human brain microvascular endothelial cells (HBMECs) and astrocytes. The monokine induced by IFN-gamma (Mig) behaves as a homing chemokine constitutively expressed in HBMECs and astrocytes, whereas the IFN-gamma-inducible 10-kDa protein (IP-10) and IFN-inducible T cell alpha-chemoattractant (I-TAC) are induced only after inflammatory stimuli. The biologic activity of IFN-gamma-inducible chemokines from an endothelial source was analyzed, and the transendothelial migration of activated lymphocytes was partly antagonized by specific antibodies, especially anti-Mig antibody. Our data highlight the capability of cells of the CNS to activate the chemoattractant machinery in a proinflammatory environment and in MS.     

Expression of the Alpha Subunit of Human Chorionic Gonadotropin in Normal and Neoplastic Neuroendocrine Cells

The alpha subunit of human chorionic gonadotropin (HCG) was localized Immunohistochemically in paraffin sections of normal human tissues and neuroendocrine tumors. A small subset of dispersed neuroendocrine cells was positive in normal adult tissues, including gastric antrum, urachal remnant, anal glands and prostate. Positive cells were consistently present in perinatal lung but rare in adult lung. In contrast, the beta subunit was absent from these cells. Seventy two of 151 extrapituitary neuroendocrine tumors (48%) were alpha subunit positive. Thirty three of 37 bronchial carcinoids (92%) were immunore-active, with a high percentage of the tumors (54%) containing moderate to large numbers of positive cells. The alpha subunit was further demonstrated in 9 of 45 small cell lung carcinomas (20%), 19 of 35 extrapulmonary carcinoids (54%), 3 of 11 islet cell tumors (27%) and 8 of 13 medullary thyroid carcinomas (62%). Two of three malignant islet cell tumors were positive. Positive cells were usually few in number, except for two small cell lung carcinomas, two rectal carcinoids, one thymic carcinoid and one malignant islet cell tumor. Pheochromocytomas (n=10) were negative. Eleven of 19 pulmonary tumorlets (58%) were alpha subunit immunoreactive. A few beta subunit positive cells were detected in only 6 lung lesions. The physiological significance of the imbalance of expression of HCG subunits by certain neuroendocrine cells and their tumors remains unknown. Acta Pathol Jpn 39: 413 419, 1989.     

Respiratory syncytial virus infection influences tight junction integrity

Respiratory syncytial virus (RSV) is an important risk factor of asthma development and is responsible for severe respiratory tract infections. However, the influence of RSV infection on barrier function of bronchial epithelial cells in vitro and in vivo is still unclear. The aim of this study was to analyse the role of RSV in tight junction (TJ) regulation and to compare epithelial integrity between asthmatic and healthy individuals upon RSV infection. Healthy and asthmatic human bronchial epithelial cells (HBECs) were differentiated at air–liquid interface (ALI) and infected with RSV and ultraviolet (UV)‐irradiated RSV. TJ expression and their integrity were analysed by quantitative polymerase chain reaction (qPCR), transepithelial resistance (TER) and paracellular flux. To determine the effect in vivo, BALB/c mice were infected intranasally with RSV or UV‐irradiated RSV A2. Bronchoalveolar lavage and TJ integrity were analysed on days 1, 2, 4 and 6 post‐infection by qPCR, bioplex and confocal microscopy. RSV increased barrier integrity in ALI cultures of HBEC from healthy subjects, but no effect was found in HBECs from asthmatics. This was not associated with an increase in TJ mRNA expression. In vivo, RSV induced lung inflammation in mice and down‐regulated claudin‐1 and occludin mRNA expression in whole lungs. Surprisingly, RSV infection was not observed in bronchial epithelial cells, but was found in the lung parenchyma. Decreased expression of occludin upon RSV infection was visible in mouse bronchial epithelial cells in confocal microscopy. However, there was no regulation of claudin‐1 and claudin‐7 at protein level.     

Cloning and expression of the Escherichia coli K1 outer membrane protein A receptor,a gp96 homologue

Escherichia coli is one of the most common gram-negative bacteria that cause meningitis in neonates. Our previous studies have shown that outer membrane protein A (OmpA) of E. coli interacts with a 95-kDa human brain microvascular endothelial cell (HBMEC) glycoprotein, Ecgp, for invasion. Here, we report the identification of a gene that encodes Ecgp by screening of an HBMEC cDNA expression library as well as by 5' rapid amplification of cDNA ends. The sequence of the Ecgp gene shows that it is highly similar to gp96, a tumor rejection antigen-1, and contains an endoplasmic reticulum retention signal, KDEL. Overexpression of either Ecgp or gp96 in both HBMECs and CHO cells increases E. coli binding and invasion. We further show that Ecgp gene-transfected HBMECs express Ecgp on the cell surface despite the presence of the KDEL motif. Northern blot analysis of total RNA from various eukaryotic cells indicates that Ecgp is significantly expressed in HBMECs. Recombinant His-tagged Ecgp blocked E. coli invasion efficiently by binding directly to the bacteria. These results suggest that OmpA of E. coli K1 interacts with a gp96-like molecule on HBMECs for invasion.     

Neurotrophins and neurotrophin receptors in human lung cancer.

The expression of neurotrophins (NTs) and related high- and low-affinity receptors was studied in surgical samples of histologically diagnosed human tumors of the lower respiratory tract. The experiment was conducted with 30 non-small cell lung cancer specimens and in eight small cell lung cancer specimens by Western blot analysis and immunohistochemistry to assess expression and distribution of NT and NT receptor proteins in tissues examined. Immunoblots of homogenates from human tumors displayed binding of anti-nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and NT-3 antibodies as well as of anti-tyrosine-specific protein kinase (Trk) A, TrkB, and TrkC receptor antibodies, with similar migration characteristics than those displayed by human beta-NGF and proteins from rat brain. A specific immunoreactivity for NTs and NT receptors was demonstrated in vessel walls, stromal fibroblasts, immune cells, and sometimes within neoplastic cell bodies. Approximately 33% of bronchioloalveolar carcinomas exhibited a strong membrane NGF and TrkA immunoreactivity, whereas 46% adenocarcinomas expressed an intense TrkA immunoreactivity but a weak immunostaining for NGF within tumor cells. Moreover, squamous cell carcinomas developed an intense TrkA immunoreactivity only within stroma surrounding neoplastic cells. A faint BDNF and TrkB immunoreactivity was documented in adenocarcinomas, squamous cell carcinomas, and small cell lung cancers. NT-3 and its corresponding TrkC receptor were found in a small number of squamous cell carcinomas within large-size tumor cells. No expression of low-affinity p75 receptor protein was found in tumor cells. The detection of NTs and NT receptor proteins in tumors of the lower respiratory tract suggests that NTs may be involved in controlling growth and differentiation of human lung cancer and/or influencing tumor behavior.