磷脂酰肌醇蛋白多糖3对胆道闭锁患儿的诊断价值及与肝纤维化的关系

摘要：目的　探讨磷脂酰肌醇蛋白多糖3（GPC3）在胆道闭锁（BA）患儿中表达水平及其与肝纤维化的关系。方法　收集BA患儿41例（BA组）、胆总管囊肿（CC）患儿9例（CC组）、肝功能正常无其他肝胆疾病的体检健康婴儿12例（NC组）。用酶联免疫吸附试验（ELISA）检测3组血清GPC3水平,使用受试者工作特征（ROC）曲线评估GPC3对BA的辅助诊断价值。留取BA组和CC组肝组织样本,通过免疫组织化学染色和实时荧光定量PCR检测GPC3在患儿肝组织中表达水平,分析BA患儿GPC3与肝纤维化分级的关系。结果　（1）BA组GPC3水平高于CC组和NC组（χ2=24.170,P＜0.01）。BA组内日龄≤30 d（21例）及＞30 d的患儿（20例）血清GPC3水平差异无统计学意义,但均高于CC组和NC组（χ2=24.210,P＜0.01）。BA组内Ⅰ~Ⅱ级与Ⅲ~Ⅳ级患儿GPC3水平差异无统计学意义,但均高于CC组和NC组（χ2=24.390,P＜0.01）。GPC3诊断BA的ROC曲线下面积为0.878（95%CI：0.792~0.965,P＜0.01）,敏感度为82.93%,特异度为80.95%,最佳临界值为0.639 μg/L。（2）肝组织免疫组织化学检测结果显示,BA组肝组织中GPC3表达较CC组明显升高（Z=3.565,P＜0.01）;肝组织中GPC3的含量与肝纤维化分级呈正相关（rs=0.619,P＜0.01）。BA组GPC3 mRNA表达较CC组上调（Z=7.361,P＜0.05）。结论　血清GPC3对BA的诊断及鉴别诊断有一定临床价值;BA肝组织中GPC3表达上调,其表达量与胆道闭锁的肝纤维化分级呈正相关。     

Immunohistochemical studies on proteoglycan expression in normal skin and chronic ulcers

BACKGROUND: Proteoglycans (PGs) represent a large family of complex molecules. They are found either as integral membrane components or constituents of the extracellular matrix. Their protein backbones are linked to different glycosaminoglycans, such as dermatan-, chondroitin-, keratan- or heparan sulphate. The molecules have specific functions during developmental processes as well as in diseases, such as cancer and inflammation. OBJECTIVES: The expression patterns of various cell-associated heparan and chondroitin/dermatan-sulphate PGs in human skin and chronic venous ulcers were investigated. METHODS: Tissue sections from 11 patients with chronic venous ulcers were used in this study. Monoclonal antibodies were used for detection of the proteoglycans syndecan-1, -2 and -4, glypican, CD44 and perlecan. RESULTS: The different PGs exhibited individual staining patterns. Syndecan-1 and -4 and glypican expression in chronic ulcers differed from the staining in normal skin. Whereas the expression of syndecan-4 and glypican in intact skin was mostly in the pericellular regions of keratinocytes, the epidermal cells from the wound edge contained mostly intracellular PGs. In the wound edge, syndecan-4 was predominantly expressed by epidermal basal layer cells. Syndecan-1 was less expressed at the epidermal wound margins. PGs bind growth factors, regulate proteolytic activity and act as matrix receptors. CONCLUSIONS: The altered expression patterns of glypican and syndecan-1 and -4 in chronic ulcers reflect their possible roles during inflammation and cell proliferation. Hence, analysis of PG expression should be of interest in future studies on normal as well as defective wound healing.     

干预磷脂酰肌醇蛋白多糖3基因转录联合抗肿瘤药物抑制肝癌细胞增殖的协同作用

目的探讨干预磷脂酰肌醇蛋白多糖(GPC)3基因转录和联合抗肿瘤药物对肝癌细胞增殖的抑制效果。方法构建4种GPC-3-shRNA质粒转染肝癌HepG2细胞,以realtime-PCR、Western Blot法观察GPC-3基因及蛋白表达水平,分析其与肝癌细胞增殖、凋亡的关系。计量资料两组间比较采用独立样本t检验,多组间比较采用单因素方差分析。结果 4种转染质粒中shRNA1转染HepG2细胞效率85%,在mRNA水平的沉默效率为89.3%,GPC-3蛋白表达显著抑制(P0.01)。shRNA1干扰组72 h HepG2细胞抑制率71.1%,与阴性组比较差异有统计学意义(t=18.092,P0.001);shRNA1干扰组肝癌细胞发生生物学特性改变,迁移抑制率为89.1%,明显慢于阴性组,差异有统计学意义(t=8.326,P0.001);HepG2细胞运动抑制率为53.6%、侵袭抑制率60.1%,与阴性组比较差异均有统计学意义(t值分别为52.400、48.245,P值均0.001)。shRNA1干扰组β-catenin mRNA抑制率为46.9%,Gli1 mRNA上调率为7.4%,与对照组比较差异均有统计学意义(t值分别为30.108、-3.551,P值分别为0.001、0.009)。在24 h时,10μmol/L索拉非尼与shRNA1联合,对肝癌细胞抑制率为52.6%,100μmol/L索拉非尼与shRNA1联合,对肝癌细胞的抑制率为79.5%,与对照组比较差异有统计学意义(t值分别为23.314、50.352,P值均0.001)。索拉非尼对HepG2细胞的半数抑制浓度(IC_(50))值为(4.67±1.20)μmol/L、雷帕霉素为(7.85±2.00)nmol/L、厄洛替尼为(18.36±0.56)μmol/L,与shRNA1联合使用后HepG2细胞抑制率达95.11%。结论特异性shRNA干预GPC-3转录可抑制肝癌细胞增殖、迁移运动和侵袭能力,并诱导肝癌细胞凋亡,与抗肿瘤药物协同抑制癌细胞增殖,提示GPC-3可能是肝癌治疗有效靶点,联合靶向治疗将为肝癌提供更佳治疗策略。     

Glypican-3过表达对A549细胞增殖、凋亡和迁移的影响

目的探讨glypican-3对人肺癌A549细胞增殖、凋亡和迁移的影响。方法采用glypican-3 cDNA瞬时转染A549细胞,Western blot方法检测转染前后glypican-3在A549细胞中的表达水平,分别采用MTT、流式和Transwell小室研究glypican-3 cDNA对A549细胞增殖、凋亡和迁移的作用。结果转染后glypican-3在A549细胞中表达上调,细胞增殖和迁移能力增强,细胞凋亡率降低（均P〈0.05）。结论 A549细胞中glypican-3过表达促进细胞增殖和迁移,抑制细胞凋亡,可能对A549细胞的生长和存活具有重要作用。     

The tumor suppressor genes dachsous and fat modulate different signalling pathways by regulating dally and dally-like

The activity of different signaling pathways must be precisely regulated during development to define the final size and pattern of an organ. The Drosophila tumor suppressor genes dachsous (ds) and fat (ft) modulate organ size and pattern formation during imaginal disc development. Recent studies have proposed that Fat acts through the conserved Hippo signaling pathway to repress the expression of cycE, bantam, and diap-1. However, the combined ectopic expression of all of these target genes does not account for the hyperplasic phenotypes and patterning defects displayed by Hippo pathway mutants. Here, we identify the glypicans dally and dally-like as two target genes for both ft and ds acting via the Hippo pathway. Dally and Dally-like modulate organ growth and patterning by regulating the diffusion and efficiency of signaling of several morphogens such as Decapentaplegic, Hedgehog, and Wingless. Our findings therefore provide significant insights into the mechanisms by which mutations in the Hippo pathway genes can simultaneously alter the activity of several signaling pathways, compromising the control of growth and pattern formation.     

Novel insights into Notum and glypicans regulation in colorectal cancer

The connection between colorectal cancer (CRC) and Wnt signaling pathway activation is well known, but full elucidation of the underlying regulation of the Wnt/β-catenin pathway and its biological functions in CRC pathogenesis is still needed. Here, the azoxymethane/dextran sulfate sodium salt (AOM/DSS) murine model has been used as an experimental platform able to mimic human sporadic CRC development with predictable timing. We performed genome-wide expression profiling of AOM/DSS-induced tumors and normal colon mucosa to identify potential novel CRC biomarkers. Remarkably, the enhanced expression of Notum, a conserved feedback antagonist of Wnt, was observed in tumors along with alterations in Glypican-1 and Glypican-3 levels. These findings were confirmed in a set of human CRC samples. Here, we provide the first demonstration of significant changes in Notum and glypicans gene expression during CRC development and present evidence to suggest them as potential new biomarkers of CRC pathogenesis.     

Glypican-1 在脑胶质瘤中的表达及与 临床病理特征和预后的关系

目的 探究Glypican-1 在脑胶质瘤中的表达、分布,以及与临床病理特征和预后的关系。方法 通 过免疫组织化学法检测Glypican-1 在171 例脑胶质瘤组织芯片中的表达及分布,分析其与脑胶质瘤患者临床病 理特征及总生存期的关系。结果 Glypican-1 在脑胶质瘤细胞质、细胞膜中均有表达。不同年龄患者脑胶质 瘤细胞质Glypican-1 高表达率比较,差异有统计学意义（P <0.05）;不同性别、肿瘤分级患者脑胶质瘤细胞质 Glypican-1高表达率比较,差异无统计学意义（P >0.05）。肿瘤分级Ⅲ、Ⅳ级患者脑胶质瘤细胞膜Glypican-1 表达水平高于Ⅰ、Ⅱ级患者（P <0.05）;而不同性别、年龄患者脑胶质瘤细胞膜Glypican-1 高表达率比较,差 异无统计学意义（P >0.05）。不同脑胶质瘤细胞质Glypican-1 表达水平患者的总生存期比较,差异无统计学意义 （P >0.05）;而脑胶质瘤细胞膜Glypican-1 表达水平越高,患者总生存期越短（P <0.05）。年龄>50 岁[Hl ^ R=0.325 （95% CI ：0.185,0.570）]、肿瘤分级Ⅲ 和Ⅳ 级[Hl ^ R=14.658（95% CI ：6.835,31.432）]、Glypican-1 在脑胶 质瘤细胞膜中高表达[Hl ^ R=2.351（95% CI ：1.081,5.114）] 是患者预后的影响因素。结论 脑胶质瘤细胞膜 Glypican-1 表达水平与患者肿瘤分级有关,且脑胶质瘤细胞膜Glypican-1 表达水平越高,总生存期越短。     

抗人GPC3单克隆抗体在肝脏肿瘤组织学检测中的应用

目的应用抗人GPC3单克隆抗体检测肝脏良、恶性肿瘤组织中GPC3蛋白的表达。方法采用自制鼠抗人GPC3单克隆抗体及免疫组织化学技术检测19例正常肝组织、94例肝细胞癌(HCC)组织、20例胆管细胞癌组织、42例肝脏良性肿瘤组织中GPC3蛋白的表达,并分析GPC3蛋白表达水平与HCC患者临床病理参数的关系。结果 GPC3蛋白在肝细胞癌组织中阳性率达到79.8%,而血管瘤旁正常肝组织、胆管细胞癌组织和肝脏良性肿瘤组织中均不表达,差异具有统计学意义(P<0.001)。HCC组织中GPC3蛋白的表达与血清AFP水平(P=0.020)、是否伴有门静脉癌栓(P=0.050)、有无包膜等因素相关(P=0.043)。结论抗人GPC3单克隆抗体可特异性检出肝细胞癌组织GPC3蛋白的表达,值得进一步研究以应用于临床诊治。     

磷脂酰肌醇蛋白聚糖-3和甲胎蛋白联合检测对原发性肝癌的诊断价值

目的通过联合检测磷脂酰肌醇蛋白聚糖-3（GPC3）、甲胎蛋白（AFP）在原发性肝癌患者血清及组织中的表达情况,探讨对原发性肝癌的诊断价值。方法分别采用ELISA和免疫组织化学法检测57例肝癌、74例肝炎后肝硬化患者和47例正常血清和肝组织GPC3、AFP表达水平,根据不同临床病理指标进行分组比较。结果 （1）肝癌患者、肝炎后肝硬化及正常对照者血清中GPC3水平分别为（212.6±137.5）、（60.9±27.8）、（39.5±18.7）ng/ml;肝癌患者血清GPC3浓度显著高于正常及肝炎后肝硬化患者（t=4.503,P〈0.05;t=6.045,P〈0.05）;血清GPC3、AFP联合检测原发性肝癌的敏感性和特异性为84.2%和95.7%,均显著高于任一单项检测（t=4.132,P〈0.05;t=6.514,P〈0.05）;（2）GPC3在肝癌组织表达高于癌旁和正常肝组织（t=3.724,P〈0.05;t=15.799,P〈0.05）;GPC3与肿瘤大小、肿瘤数目、HBsAg及AFP水平无明显相关,而与病理分级和临床分期有关;（3）AFP阳性肝癌血清GPC3阳性率为91.4%,而在AFP阴性肝癌中GPC3阳性率为59.1%。结论 GPC3联合AFP检测有助于提高原发性肝癌的确诊率,检测GPC3有助于提高AFP阴性患者肝癌的确诊率。