SHEEC食管癌细胞中NGAL基因的功能

中性粒细胞明胶酶相关脂质运载蛋白 (neutrophilgelatinase associatedlipocalin ,NGAL)是脂质运载蛋白(lipocalin)家族的一个新成员 ,可能是人类的一种新的癌基因 ,但是在肿瘤中的功能不清楚。以往研究发现NGAL基因在SHEEC食管癌细胞中显著过表达 ,表明该细胞是一种用来揭示NGAL基因在肿瘤中功能的良好模型。采用反义封闭技术 ,同时结合裸鼠成瘤实验等研究了反义封闭NGAL基因转录对SHEEC食管癌细胞的浸润和分裂增殖等行为的影响。结果发现 ,反义封闭NGAL基因转录不但可以有效地降低SHEEC细胞分泌的基质金属蛋白酶 9和基质金属蛋白酶 2的活性 ,而与此同时裸鼠成瘤细胞的浸润行为也相应地受到了明显抑制 ,然而SHEEC细胞端粒的长度、拓扑异构酶II的含量以及细胞增殖指数等未发生明显变化。表明NGAL基因在食管癌细胞SHEEC中的功能可能主要是通过明胶酶在促进肿瘤细胞的浸润中发挥作用 ,而可能与肿瘤细胞分裂增殖的相关性不明显     

Age-Correlated Gene Expression in Normal and Neurodegenerative Human Brain Tissues

Background

Human brain aging has received special attention in part because of the elevated risks of neurodegenerative disorders such as Alzheimer''s disease in seniors. Recent technological advances enable us to investigate whether similar mechanisms underlie aging and neurodegeneration, by quantifying the similarities and differences in their genome-wide gene expression profiles.

Principal Findings

We have developed a computational method for assessing an individual''s “physiological brain age” by comparing global mRNA expression datasets across a range of normal human brain samples. Application of this method to brains samples from select regions in two diseases – Alzheimer''s disease (AD, superior frontal gyrus), frontotemporal lobar degeneration (FTLD, in rostral aspect of frontal cortex ∼BA10) – showed that while control cohorts exhibited no significant difference between physiological and chronological ages, FTLD and AD exhibited prematurely aged expression profiles.

Conclusions

This study establishes a quantitative scale for measuring premature aging in neurodegenerative disease cohorts, and it identifies specific physiological mechanisms common to aging and some forms of neurodegeneration. In addition, accelerated expression profiles associated with AD and FTLD suggest some common mechanisms underlying the risk of developing these diseases.     

Reassessment of CXCR4 Chemokine Receptor Expression in Human Normal and Neoplastic Tissues Using the Novel Rabbit Monoclonal Antibody UMB-2

Background

The CXCR4 chemokine receptor regulates migration and homing of cancer cells to specific metastatic sites. Determination of the CXCR4 receptor status will provide predictive information for disease prognosis and possible therapeutic intervention. However, previous attempts to localize CXCR4 using poorly characterized mouse monoclonal or rabbit polyclonal antibodies have produced predominant nuclear and occasional cytoplasmic staining but did not result in the identification of bona fide cell surface receptors.

Methodology/Principal Findings

In the present study, we extensively characterized the novel rabbit monoclonal anti-CXCR4 antibody (clone UMB-2) using transfected cells and tissues from CXCR4-deficient mice. Specificity of UMB-2 was demonstrated by cell surface staining of CXCR4-transfected cells; translocation of CXCR4 immunostaining after agonist exposure; detection of a broad band migrating at M _r 38,000–43,000 in Western blots of homogenates from CXCR4-expressing cells; selective detection of the receptor in tissues from CXCR4+/+ but not from CXCR4−/− mice; and abolition of tissue immunostaining by preadsorption of UMB-2 with its immunizing peptide. In formalin-fixed, paraffin-embedded human tumor tissues, UMB-2 yielded highly effective plasma membrane staining of a subpopulation of tumor cells, which were often heterogeneously distributed throughout the tumor. A comparative analysis of the mouse monoclonal antibody 12G5 and other frequently used commercially available antibodies revealed that none of these was able to detect CXCR4 under otherwise identical conditions.

Conclusions/Significance

Thus, the rabbit monoclonal antibody UMB-2 may prove of great value in the assessment of the CXCR4 receptor status in a variety of human tumors during routine histopathological examination.     

建立NGAL诊断阈值有利于临床判断和发现急性肾功能损伤

为探讨中性粒细胞相关载脂蛋白(neutrophil gelatinase-associated lipocalin, NGAL)检测在肝胆疾病患者继发急性肾功能损伤(acute kidney injury, AKI)中的诊断性能,本研究回顾性收集了476例肝胆疾病患者与225例健康受试者作为实验组,根据血清中性粒细胞相关载脂蛋白(NGAL)、血清肌酐(serum creatinine,s Cr)、尿素(Urea)、半胱氨酸蛋白酶抑制剂C (cystatin C, CysC)、估算肾小球滤过率(estimating glomerular filtration rate, eGFR)水平和尿量,分为急性肾功能损伤(AKI)组和高风险(high-risk, HR)组、低风险(low-risk,LR)组和对照(health control, HC)组,进而建立上述血清指标在急性肾功能损伤(AKI)诊断性能最大时的判断界值;随后,选取145例肝胆疾病患者作为验证组,以评估各观察指标对肝胆疾病继发急性肾功能损伤(AKI)的诊断性能。结果表明:本实验组中各亚组间所观察指标之血清水平均有统计学差异(p<0.05)。中性粒细胞相关载脂蛋白(NGAL)诊断性能最大时的判断界值为205.2μg/L。验证组患者各指标阳性检出率,在低风险(LR)亚组中中性粒细胞相关载脂蛋白(NGAL)均高于其它指标(p<0.05),但在高风险(HR)亚组中中性粒细胞相关载脂蛋白(NGAL)仅高于血清肌酐(sCr)(p<0.05),而与半胱氨酸蛋白酶抑制剂C (CysC)和估算肾小球滤过率(eGFR)无统计学差异(p>0.05);再经分层风险分析各指标预测高风险(HR)亚组患者急性肾功能损伤(AKI)发生的能力,中性粒细胞相关载脂蛋白(NGAL)的优势比(OR=21.0 (2.3, 192.8)),是半胱氨酸蛋白酶抑制剂C(CysC)或估算肾小球滤过率(eGFR)(OR (95%CI)=3.3 (0.7, 15.3))的6.4倍。本研究初步结论表明,肝胆疾病患者诊断急性肾功能损伤(AKI)时应考虑中性粒细胞相关载脂蛋白(NGAL)肝脏合成代谢的作用。因此实验室应建立适宜的中性粒细胞相关载脂蛋白(NGAL)诊断阈值以有利于临床准确判断及早期发现急性肾功能损伤(AKI)。     

MicroRNA-138 Suppresses Neutrophil Gelatinase-Associated Lipocalin Expression and Inhibits Tumorigenicity

The expression of neutrophil gelatinase-associated lipocalin (NGAL) is up-regulated in some cancers; therefore NGAL has potential as a tumor biomarker. Although the regulation mechanism for this is unknown, one study has shown that it is likely to involve a microRNA (miRNA). Here, we investigate the relation between miRNA expression and NGAL expression, and the role of NGAL in tumorigenesis. Using miRNA target–detecting software, we analyze the mRNA sequence of NGAL and identify a target site for microRNA-138 (miR-138) in nucleotides 25–53 of the 3′ UTR. We then analyze NGAL and miR-138 expression in three cancer cell lines originating from breast, endometrial and pancreatic carcinomas (the MCF-7, RL95-2 and AsPC-1 cell lines), respectively, using quantitative (real-time) PCR and western blot analysis. Metastasis is a critical event in cancer progression, in which malignant cell proliferation, migration and invasion increase. To determine whether miR-138-regulated NGAL expression is associated with metastasis, the proliferation and migration of the cell line are examined after miR-138 transfection. Using nude mice, we examine both the tumorigenicity of these cell lines and of miR-138-transfected cancer cells in vivo, as well as the effect of treating tumors with an antibody against NGAL. Our results show that these cancer cell lines down-regulate NGAL when miR-138 is highly expressed. Ectopic transfection of miR-138 suppresses NGAL expression and cell migration in RL95-2 and AsPC-1 cells, demonstrating that miR-138-regulated NGAL expression is associated with cell migration. Additionally, injection of the NGAL antibody diminishes NGAL-mediated tumorigenesis in nude mice, and miR-138 transfection of cancer cells reduces tumor formation. As the cell proliferation data showed that the tumor size should be regulated by NGAL-related cell growth. Taken together, our results indicate that NGAL may be a good target for cancer therapy and suggest that miR-138 acts as a tumor suppressor and may prevent metastasis.     

hSef基因在不同人组织和细胞系的表达分布

将人源Sef蛋白胞内区编码序列与GST融合构建原核表达质粒进行重组蛋白的表达与纯化并制备多抗 .在COS 7细胞中转染表达hSef显示 ,其分子量分别为 80kD ,10 0kD ,比体外翻译的分子量偏大 ,提示可能有糖基化存在 .Northern印迹的结果表明 ,hSefmRNA主要分布在人肾和睾丸组织 .RT PCR检测到hSefmRNA在众多细胞系有广泛存在 .免疫组化的结果显示 ,hSef蛋白在人肾和睾丸及相应癌组织表达水平较高 .     

Expression of a Lipocalin in Human Nasal Mucosa

A 19 kDa soluble protein was purified from human nasal mucus. Its N-terminal amino-acid sequence appeared to be identical to that of a lipocalin synthesised both in lachrymal glands and in von Ebner's glands (VEG) of circumvallate papillae. In order to verify whether this protein was synthesised in the nasal cavity or was the result of tear contamination, we adopted an immunohistochemical approach. Polyclonal antibodies, raised against a primate VEG protein, were used on sections of human nasal mucosa obtained from surgery. The results clearly indicate that the protein is synthesised in sero-mucous glands underlying the respiratory ciliated epithelium. Although ligand-binding experiments with some odorant molecules have given negative results, we cannot exclude a role of odorant solubiliser and carrier for this protein.     

Expression of Ezrin in Human Embryonic, Fetal, and Normal Adult Tissues

Ezrin, which cross-links the cytoskeleton and plasma membrane, was involved in a wide variety of cellular processes. Here, to investigate the distribution of ezrin, tissue microarray technology was employed to perform immunohistochemical experiments on human embryos, fetuses at 4 to 22 weeks’ gestation, and adult tissue specimens. Results showed that ezrin was widely expressed in the gastrointestinal tract throughout the human developmental stages studied. At 6 to 8 weeks’ gestation, ezrin was found in epithelial cells, and this staining pattern was particularly pronounced in the brush border of mature absorptive cells lining the villus in later developmental stages and adult tissues. Throughout neural development, ezrin was only expressed in the neural tube at 4 weeks’ gestation. Ezrin was also detected in the cortex and medulla of the adrenal gland at 8 to 12 weeks’ gestation, whereas its immunoreactivity was increased from the zona glomerulosa through the zona reticularis and was essentially undetectable in the adrenal medulla of adult tissues. Significant expression of ezrin was seen throughout development in the kidney, spleen, lymph nodes, and cells of stratified squamous epithelia. However, ezrin was undetectable in lung, liver, heart, and blood vessels. These results demonstrated that the expression pattern of ezrin was highly time specific and tissue specific.     

Spexin Expression in Normal Rat Tissues

Spexin is a highly conserved peptide which was recently identified through the bioinformatics approach. Immunohistochemical analysis of its expression has not yet been performed. Thus, in this study, we examined spexin location in a wide range of rat organs by both RT-PCR and IHC. RT-PCR identified spexin mRNA in all tissues examined. Spexin immunoreaction was mainly cytoplasmic. Spexin was immunohistochemically detected, although with different staining intensities, in epithelia and glands of skin and respiratory, digestive, urinary, and reproductive systems. Smooth muscle cells showed weak immunostaining, and connective tissue was negative. In the central nervous system, neuronal groups showed different intensities for reaction product. Immunoreaction was also found in ganglionic cells of both trigeminal and superior cervical ganglia and in photoreceptor, inner nuclear, and ganglionic layers of the retina. In the endocrine system, spexin immunoreaction was detected in the hypothalamic paraventricular and supraoptic nuclei; adenohypophysis, thyroid, and parathyroid glands; adrenal cortex and medulla (mainly ganglionic cells); Leydig cells; and thecal, luteal, and interstitial cells of the ovary. Because of its widespread expression, spexin is probably involved in many different physiological functions; in particular, location of spexin in neurons and endocrine cells suggests its roles as neurotransmitter/neuromodulator and endocrine factor. (J Histochem Cytochem 58:825–837, 2010)     

组织专一性表达自杀基因治疗人结肠癌的研究

构建了以癌胚抗原(CEA)基因启动子控制的HSV-TK和ECCD的表达质粒PCEA-TK和pCEA-CD. 将它们分别与pSV2-neo共转染人结肠癌细胞株LoVo和人宫颈癌细胞株HeLa. G418筛选得到细胞克隆LoVo/CEA-TK、toVo/CEA-CD、HeLa/CEA-TK和HeLa/CEA-CD. 与野生型LoVo细胞相比, LoVo/CEA-TK和LoVo/CEA-CD形态无明显改变, 生长曲线也相似, 但对GCV或5-FC的细胞毒的敏感性分别提高了2000倍或700倍.而HeLa/CEA-TK(或HeLa/CEA-CD)仍对低浓度GCV(或5-FC)不敏感. 以上结果显示了应用组织专一性表达的自杀基因治疗人结肠癌的可能性.     

Enhanced Expression of CD13 in Vessels of Inflammatory and Neoplastic Tissues

Aminopeptidase-N (CD13) is an important target of tumor vasculature-targeting drugs. The authors investigated its expression by immunohistochemistry with three anti-CD13 monoclonal antibodies (WM15, 3D8, and BF10) in normal and pathological human tissues, including 58 normal, 32 inflammatory, and 149 tumor tissue specimens. The three antibodies stained vessels in most neoplastic tissues, interestingly with different patterns. As a matter of fact, WM15 stained almost all intratumor and peritumor capillaries and only partially large vessels, whereas BF10 and 3D8 reacted with arteries and venules and to a lesser extent with capillaries. These antibodies also stained the stroma in about half of neoplastic tissues. In inflammatory lesions, the three antibodies stained vessels and stroma, whereas in normal tissues, they stained a small percentage of blood vessels. Finally, the three antibodies failed to stain endothelial cells of normal colon, whereas they reacted with activated human umbilical vein endothelial cells and with endothelial cells of colon adenocarcinoma vessels. Overall, WM15 was the most specific antibody for angiogenic tumor vessels, suggesting that it may be a good tool for detecting the CD13 form associated with the tumor vasculature. This finding may be relevant for CD13-mediated vascular targeting therapies.     

Matrix Metalloproteinases of Normal Human Tissues

This review considers biochemical properties of the family of matrix metalloproteinases (MMPs) of normal human tissues and the involvement of these enzymes in morphogenesis. Four main MMP subfamilies are characterized, and a group of other MMPs is described. Data on mechanisms of activation and inhibition of MMPs in certain tissues during various physiological processes (embryogenesis, angiogenesis, tissue growth and involution) are considered. Information about tissue inhibitors of MMP is presented, and the ability of these inhibitors to regulate the activity of MMPs is analyzed.     

血浆中性粒细胞明胶酶相关脂质运载蛋白(NGAL)的变化与婴幼儿先心病术后急性肺损伤的关系

目的:明确血浆中性粒细胞明胶酶相关脂质运载蛋白(NGAL)的变化能否预测婴幼儿先心病术后急性肺损伤(ALI)的发生。方法:纳入2013年10月至2014年5月年龄≤36月的先心病合并肺动脉高压的婴幼儿患者的相关资料,检测体外循环(CPB)术后2 h、12 h、24 h、48 h和72 h的血浆NGAL水平,分析NGAL与术后发生ALI之间的关系。结果:本研究共纳入患者61例,术后发生ALI患者9例(14.8%)。所有患者CPB术后5个时间点的变化趋势为:CPB术后2 h NGAL浓度开始升高,均值为27.28μg/L,至12 h达到最高峰,均值为30.61μg/L,24 h开始下降;其中12 h、24 h和72 h与前一时间点比较均具有显著性差异(P0.05)。进一步分组显示,NGAL浓度变化趋势主要在ALI组:ALI组患者12 h、24 h、48 h和72 h的NGAL浓度与前一个时间点比较,变化均具有显著差异(P0.05),NGAL峰值浓度出现在12 h,其均值为40.82μg/L;而无ALI组患者NGAL浓度只在24 h和12 h之间比较有显著差异(P0.05);ALI组患者5个时间点的NGAL浓度均比无ALI组患者高,具有显著性差异(P0.05)。结论:血浆NGAL在早期预测婴幼儿体外循环术后发生ALI可能具有重要作用。     

Identification of Normal or Neoplastic Murine Mesenchymal Cells in Chimeric Tissues or Heterotransplants

An improved method for identifying murine mesenchymal cells in chimeric tissues or heterotransplants using Hoechst dye 33258 is described. Following fixation in formalin-saline, tissues are embedded in JB-4 plastic Sections 3 μm thick are then stained in a 10 μg/ml solution of Hoechst 33258 in Hanks' balanced salt solution for 5-10 min at 4 C. After rinsing, the sections are coverslipped using a modified polyvinyl alcohol mounting medium. This approach offers several advantages over existing techniques: 1) uniform section thickness is more easily obtained than with paraffin or cryostat microtomy, thereby allowing improved resolution and more reliable identification of mesenchymal cells with small nuclei such as skeletal muscle myocytes or fibroblasts, 2) the preparations are stable over long periods and can be repeatedly viewed or photographed, and 3) calcified tissues can be examined without prior decalcification. An example is shown of species identification using rat chondrosarcoma cells grown in nude mice.     

Isoenzymes of Acid Phosphatase and Non-specific Esterases in Cultures of Neoplastic and Normal Tobacco Tissues

Axenic cultures of normal, habituated and crown gall teratoma tissues were grown under varying conditions to examine the effects of environment on the expression of neoplastic character. Acid phosphatase patterns on polyacrylamide gels did not vary greatly among tissues although there were differences in acid phosphatase activity between various strains of Agrobacterium tumefaciens , the bacteria which cause crown gall. Certain esterase isoenzymes were found only in tissues grown on specific media, while others were tissue-specific but independent of the nature of the medium. Comparisons of liquid and solid grown cultures revealed that culture conditions also influence esterase expression. Both sunflower and tobacco crown gall tissue contained an esterase not found in habituated or normal tissues, and similar in electrophoretic mobility to an esterase found in extracts of the bacteria that had induced the tumors. The basic difference between the three tissue types studied is the manner in which they respond to a given environment.     

Immunohistochemical Study of Expression of Sohlh1 and Sohlh2 in Normal Adult Human Tissues

The expression pattern of Sohlh1 (spermatogenesis and oogenesis specific basic helix-loop-helix 1) and Sohlh2 in mice has been reported in previous studies. Sohlh1 and Sohlh2 are specifically expressed in spermatogonia, prespermatogonia in male mice and oocytes of primordial and primary follicles in female mice. In this report, we studied the expression pattern of Sohlh1 and Sohlh2 in human adult tissues. Immunohistochemical staining of Sohlh1 and Sohlh2 was performed in 5 samples of normal ovaries and testes, respectively. The results revealed that Sohlh genes are not only expressed in oocytes and spermatogonia, but also in granular cells, theca cells, Sertoli cells and Leydig cells, and in smooth muscles of blood vessel walls. To further investigate the expression of Sohlh genes in other adult human tissues, we collected representative normal adult tissues developed from three embryonic germ layers. Compared with the expression in mice, Sohlhs exhibited a much more extensive expression pattern in human tissues. Sohlhs were detected in testis, ovary and epithelia developed from embryonic endoderm, ectoderm and tissues developed from embryonic mesoderm. Sohlh signals were found in spermatogonia, Sertoli cells and also Leydig cells in testis, while in ovary, the expression was mainly in oocytes of primordial and primary follicles, granular cells and theca cells of secondary follicles. Compared with Sohlh2, the expression of Sohlh1 was stronger and more extensive. Our study explored the expression of Sohlh genes in human tissues and might provide insights for functional studies of Sohlh genes.     

嗜中性粒细胞是人抵抗素表达的主要细胞

抵抗素(resistin)是小鼠白色脂肪组织大量表达的富含半胱氨酸的 分泌型蛋白.近年研究发现,人与啮齿类动物的抵抗素组织表达分布存在很 大差异．小鼠抵抗素主要在白色脂肪组织表达,而人抵抗素主要在单核细 胞/巨噬细胞表达,且在骨髓组织中大量表达,但目前骨髓中的细胞定位还 不清楚．本研究的目的是明确成人骨髓及外周血白细胞中抵抗素表达细胞 的类型．免疫荧光法检测骨髓中抵抗素表达细胞,结果显示,抵抗素主要表 达在细胞核呈杆状和分叶核状的成熟粒细胞中,其中杆状核粒细胞表达较 高,分叶核粒细胞表达减弱．Anti-hresistin IgG-Biotin-PE单色荧光流 式细胞术分选外周血白细胞中抵抗素表达细胞后经瑞氏化学染色,结果显 示,抵抗素表达细胞主要为杆状和分叶核状的嗜中性粒细胞,还有少量嗜酸 性粒细胞,且抵抗素蛋白分布在细胞质中. RT-qPCR结果在RNA水平上证明, 人抵抗素在嗜中性粒细胞中大量表达．Anti-hresistin IgG-FITC和anti- HNL IgG-Biotin-PE 双色荧光流式细胞术进一步证明,抵抗素的主要表达细 胞为成熟的嗜中性粒细胞．嗜中性粒细胞在机体免疫防御中起重要作用, 人骨髓及外周血中抵抗素主要在成熟嗜中性粒细胞中表达,这一研究结论 为人抵抗素与炎症反应的关联性及其功能的进一步研究奠定了基础.     

MUC16 对子宫内膜容受性的影响及其意义

目的:多囊卵巢综合征(Polycystic Ovary Syndrome,PCOS)系由于体内复杂的内分泌和代谢环境影响了子宫内膜稳态,导致子宫内膜容受性下降,造成患者生育力减弱和不良妊娠结局.通过测定多囊卵巢综合征患者子宫内膜与正常生育力妇女增生期、分泌早、中、晚期子宫内膜中MUC16的相对表达量,本文探讨MUC16与PCOS患者子宫内膜容受性下降的关系,为临床上改善PCOS患者子宫内膜容受性,提高PCOS患者的妊娠率,降低流产率提供一条新的可能途径.方法:选择PCOS患者子宫内膜、正常生育力妇女增生期、分泌早、中、晚期子宫内膜各20例,用免疫组化SP法检测MUC16在各组的表达情况.结果:(1)MUC16在月经周期各期均有表达,在分泌中期表达最强.(2)PCOS组MUC16的表达较分泌中期组弱,差异有显著性(P＜0.05).结论:(1)在子宫内膜中的表达呈周期性变化.(2)PCOS患者子宫内膜中MUC16表达异常可能使子宫内膜容受性下降,推测其与胚胎不能正常着床或着床后发育不良、导致流产有关.