细胞外基质金属蛋白酶诱导分子(CD147)在胰腺癌细胞及胰腺星状细胞中的表达(英文)

目的:探讨细胞外基质金属蛋白酶诱导分子(CD147)在胰腺癌细胞(Panc-1)及胰腺星状细胞(PSCs)的表达。方法:应用QRT-PCR,免疫细胞化学和免疫印迹分析方法检测Panc-1和PSCs细胞中EMMRPIN的表达,应用脱糖基化试剂N-glycosidase F及Endoglycosidase H鉴定CD147糖基化形式。结果:CD147在Panc-1和PSCs细胞质膜及细胞质中高表达,通过脱糖基化法首次鉴定出胰腺癌细胞及胰腺星状细胞中CD147不同的糖基化修饰。结论:CD147的糖基化修饰具有细胞特异性,可能与细胞恶性程度相关。     

N-糖基化修饰在髓性白血病耐药中的作用

目的研究N-糖基化修饰、糖基因表达调控在髓性白血病耐药中的作用,明确N-糖基化修饰、糖基因与白血病耐药的相关性,从而为预测和诊断髓性白血病耐药性,寻求逆转药物提供新策略和靶点。方法通过修饰白血病耐药细胞株的N-糖基化（衣霉素Tunicamycin和PNGase F处理）,Western Blot检测Pgp、CD147糖蛋白的表达水平;MTT法检测N-糖基化修饰前后髓性白血病耐药细胞株的生长情况及对化疗药物的敏感性,观察上述细胞膜型N-糖基化修饰后对化疗药物耐药性的影响;进一步通过RNA干扰技术干预差异表达的糖基因,MTT法检测干扰前后白血病耐药细胞株的生长情况及对化疗药物的敏感性,观测糖基因的表达调控对髓性白血病耐药的影响。结果 NB4/ADR细胞经N-糖基化修饰后,P-gp、CD147糖蛋白的表达水平发生改变,同时该细胞的药物敏感性也增强（P〈0.05）;当通过RNA干扰技术特异性使NB4/ADR细胞中B3GNT8和ST8SIA4表达下调时,该细胞的药物敏感性增强（P〈0.05）。结论髓性白血病细胞株中N-糖基化修饰、糖基因的改变均与白血病多药耐药具有相关性,为预测和诊断髓性白血病耐药性,寻求逆转药物提供新策略和靶点。     

胰腺星状细胞活化相关的信号转导通路

胰腺纤维化是慢性胰腺炎（chronic pancreatitis,CP）和胰腺癌主要的病理学特征,活化的胰腺星状细胞（pancreatic stellate cells,PSCs）是公认的致胰腺纤维化的主要效应细胞。PSCs的活化涉及到几个重要的信号转导通路：有丝分裂原活化蛋白激酶（mitogen-activated protein kinases,MAPK）、磷酯酰肌醇3激酶（phosphatidylinositol 3-kinase,PI3K）=、Smad信号转导蛋白、过氧化物酶体增生物激活受体-γ（PPAR-γ）、Rho-ROCK等细胞内信号途径。探讨这些信号通路在胰腺纤维化中所起的作用对慢性胰腺炎、胰腺癌及糖尿病的治疗有重要意义。现就与PSCs激活有关的信号通路的研究结合最新进展作一综述。     

油红O染色鉴定胰腺星状细胞

目的胰腺星状细胞(pancreatic stellate cells,PSCs)是一类胞浆内含有Vitamin A脂滴的特殊类型的细胞。油红O能够使脂肪组织及细胞内的脂滴着色。本研究的目的是采用油红O染色的方法对分离培养的PSCs进行鉴定。方法采用Histodenz密度梯度离心分离提取PSCs。异丙醇配制油红O染液,培养的PSCs应用油红O染液染色。结果油红O染色后可清晰显示PSCs内的脂滴。结论油红O染色可以特异性地显示脂滴,是鉴别PSCs的有效方法。     

胰腺星状细胞的调控及转归

活化的胰腺星状细胞(pancreatic stellate cells,PSCs)是胰腺炎致胰腺纤维化的主要效应细胞。近年来,学者普遍认为,胰腺纤维化早期阶段是动态可逆的,因此,若在胰腺损伤的早期阶段,抑制PSCs的增殖、迁移,减少损伤部位PSCs的数目,降低细胞外基质(extracellular matrix,ECM)的生成,将可能逆转胰腺纤维化。该文以PSCs为靶点阐述了抗胰腺纤维化的新策略。     

CD147对白血病细胞U937生长和肿瘤形成的影响

目的：研究CD147对白血病细胞U937生长和肿瘤形成的影响。方法：分别采用脂多糖（LPS）或CD147单克隆抗体处理U937细胞;用RT-PCR和流式细胞术分别在mRNA和蛋白水平检测各组中CD147的表达情况;用流式细胞术检测在LtX3和CD147单克隆抗体作用下U937细胞周期的变化;用MTT法对各组细胞的生长状况进行分析;将细胞经皮下接种于裸鼠体内,对各组间肿瘤生长速度、肿瘤体积及裸鼠存活时间进行统计分析。结果：LPS在体外能够诱导自血病细胞U937表面CD147的表达,同时细胞增殖旺盛,但细胞凋亡数增加;使用CD147抗体阻断CD147后,能够将细胞周期阻断在G0／G1期,细胞活力下降,并诱导细胞凋亡;CD147抗体体外预处理能够抑制U937细胞在裸鼠体内的生长,使小鼠存活时间延长。结论：LPS可诱导U937细胞表面CD147分子表达增加,从而促进U937细胞的生长和肿瘤形成。     

CD真核表达载C 体的构建及在 2KHE39 细胞中的表达

目的：构建含自杀基因胞嘧啶脱氨酶（CD）的真核表达载体pcDNA3．1／HA—myc-His（-）Z—CD,并进行哺乳动物细胞HEK293转染研究。方法：以本实验室保存的含CD基因全长的质粒为模版,用PcR方法扩增CD基因阅读框序列,并定向克隆到带有HAtag的pcDNA3．1／HA—myc-His（-）Z载体上,使目的基因与HAtag在同一阅读框。重组体质粒经EcoRI和BamHI双酶切鉴定,并对插入的CD基因片段进行测序,将鉴定好的阳性重组质粒pcDNA3．1／HA—myc-His（-）Z—CD用脂质体介导转染HEK293,提取细胞蛋白,western blot检测CD基因的表达情况.结果：阳性重组质粒pcDNA3．1／HA—myc-His（-）ZCD经Eco砌和BanHI双酶切后,获得约为5．5kb片段和1．3kb插入片段,序列分析表明插入的片段与GenBank发布的序列一致．western blot检测到CD基因的表达。结论：成功构建了含自杀基因CD的真核表达质粒。     

CD59、CD55在T细胞活化信号转导中的协同作用

目的：研究糖基磷脂酰肌醇（GeD）锚固蛋白CD59、CD55在脂筏介导T细胞信号转导通路中的协同效应。方法：应用siRNA技术,构建特异性针对CD55与CD59基因的重组载体pSUPER—siCD55,pSUPER—siCD59。实验分为未转染的Jurkat细胞组（I组）、转染pSUPER空质粒的Jurkat细胞组（Ⅱ组）、转染pSUPER—siCD59重组质粒的Jurkat细胞组（Ⅲ组）及转染pSUPER—siCD55重组质粒的Jurkat细胞组（Ⅳ组）。RT—PCR检测转染细胞中CD55和CD59基因的表达。噻唑蓝（MTT）比色法和激光共聚焦扫描显微镜分别检测CD55与CD59联合作用对4组Jurkat细胞的增殖效应以及细胞内钙离子的变化。结果：稳定转染后,Ⅲ组细胞CD59分子的表达和Ⅳ组细胞CD55分子的表达被成功抑制。Ⅰ组和Ⅱ组细胞CD55与CD59联合作用后增殖能力和钙离子浓度均明显高于Ⅲ组、Ⅳ组（P〈0．05）,Ⅰ组和Ⅱ组之间无差异。结论：CD59和CD55在T细胞活化信号转导通路中存在协同效应。     

缬沙坦对人肾小球系膜细胞糖基化终产物受体表达的影响

目的：本实验探讨缬沙坦对糖基化终产物诱导的人肾小球系膜细胞氧化应激水平及糖基化终产物受体（RAGE）表达的影响。方法：体外常规培养人肾小球系膜细胞,运用糖基化修饰的牛血清白蛋白（AGE-BSA）和缬沙坦进行干预,流式细胞术检测细胞内活性氧（ROS）,RT-PCR法检测NADPH氧化酶的亚基p47^phox的mRNA表达,RT-PCR和细胞免疫化学法检测RAGE的表达量。结果：缬沙坦干预组人肾小球系膜细胞的ROS产生量、NADPH氧化酶的亚基p47^phox mRNA表达量、RAGE表达量均低于AGE-BSA组（P〈0．05）,且缬沙坦的抑制作用呈浓度和时间依赖性。结论：缬沙坦可能通过降低氧化应激水平来抑制RAGE的表达。     

高糖高胰岛素促进小鼠胰腺星状细胞活化和半乳凝素-3表达

该研究观察了高糖高胰岛素对小鼠胰腺星状细胞(pancreatic stellate cells,PSCs)活化、增殖、细胞外基质(extracellular matrix,ECM)合成和半乳凝素-3(galectin-3,Gal-3)表达的影响。分离PSCs并培养至3~5代后进行实验。PSCs干预分为低糖对照组(5 mmol/L葡萄糖)、高糖组(25 mmol/L葡萄糖)、高胰岛素组(5 mmol/L葡萄糖+100 nmol/L胰岛素)、高糖高胰岛素组(25 mmol/L葡萄糖+100 nmol/L胰岛素)。细胞免疫荧光检测胰岛素受体(insulin receptor,IR)和胰岛素样生长因子-1型受体(insulin like growth factor-1 receptor,IGF-1R)在PSCs的表达;MTT法检测PSCs增殖;RT-PCR和Western blot测定平α-滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、I型胶原(type I collagen,Col I)、纤连蛋白(fi bronectin,Fn)和Gal-3的m RNA和蛋白质水平。结果发现,PSCs细胞表达IR和IGF-1R;与低糖对照组相比,高糖组、高胰岛素组、高糖高胰岛素组均诱导PSCs活化、增殖并促进Col I、Fn生成和Gal-3表达,其中以高糖高胰岛素组最为显著。以上结果说明,2型糖尿病高糖、高胰岛素微环境可能促进PSCs活化、增殖、ECM生成和Gal-3表达,在一定程度上可导致胰腺纤维化。     

结缔组织生长因子在胰腺组织纤维化中的作用及机制探讨

目的 观察胰腺纤维化后结缔组织生长因子(Connective tissue growth factor,CTGF)在胰腺组织内的表达;进一步研究参与CTGF作用于胰腺星状细胞(pancreatic stellate cells,PSCs)的分子信号调控通路.方法 建立大鼠胰腺纤维化动物模型,HE染色、天狼猩红染色和免疫组织化学染色等方法观察胰腺纤维化后PSCs的活化情况及CTGF在胰腺组织的表达.Real-time RT PCR检测CTGF的基因表达.Western Blot检测PSCa内α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)及胶原蛋白Ⅰ(CollagenⅠ)水平.结果 胰腺组织纤维化后,PSCs大量活化,并显著表达CTGF.CTGF作用后,PSCs内CTGF mRNA、α-SMA和Collagen Ⅰ的合成均有显著增加,在给予不同的细胞信号通路阻断剂后,PSCs内α-SMA的合成有显著下降,而Collagen Ⅰ的降低没有表现出统计学差异.结论 CTGF参与了胰腺纤维化的调控,MAPK和PI3-K信号通路均参与了CTGF的调控作用.     

MRP在胰腺癌组织中的表达及其临床意义

目的：了解MRP蛋白在胰腺癌组织中的表达情况,并探讨其临床意义。方法：应用免疫组织化学法检测72例胰腺癌组织,10例正常胰腺组织中MRP蛋白的表达,并分析其与临床病理学特征及预后的关系。结果：MRP蛋白在胰腺癌组织中的阳性表达率为91．7％,明显高于其在正常胰腺组织中的表达（p〈0．05）。胰腺癌组织中的MRP蛋白高表达与病理学分级低显著相关（p=0．031）,亦与年龄有关（p〈0．05）,而与肿瘤生长部位、肿瘤大小、淋巴结有无转移、临床分期及神经有无浸润无显著相关性（p〉0．05）。MRP蛋白高表达者的中位生存期明显长于低表达者（21．7个月vs11．1个月,p=0．040）。结论：胰腺癌组织中MRP蛋白的高表达与胰腺癌术后的预后好相关,检测胰腺癌中MRP蛋白的表达具有重要的临床意义。     

CD271+ Subpopulation of Pancreatic Stellate Cells Correlates with Prognosis of Pancreatic Cancer and Is Regulated by Interaction with Cancer Cells

Pancreatic stellate cells (PSCs) play a crucial role in the aggressive behavior of pancreatic cancer. Although heterogeneity of PSCs has been identified, the functional differences remain unclear. We characterized CD271⁺ PSCs in human pancreatic cancer. Immunohistochemistry for CD271 was performed for 31 normal pancreatic tissues and 105 pancreatic ductal adenocarcinomas (PDACs). We performed flow cytometry and quantitative RT-PCR, and assessed CD271 expression in PSCs isolated from pancreatic tissues and the changes in CD271 expression in PSCs cocultured with cancer cells. We also investigated the pattern of CD271 expression in a SCID mouse xenograft model. In the immunohistochemical analyses, the CD271-high staining rates in pancreatic stroma in normal pancreatic tissues and PDACs were 2/31 (6.5%) and 29/105 (27.6%), respectively (p = 0.0069). In PDACs, CD271⁺ stromal cells were frequently observed on the edge rather than the center of the tumors. Stromal CD271 high expression was associated with a good prognosis (p = 0.0040). Flow cytometric analyses demonstrated CD271-positive rates in PSCs were 0–2.1%. Quantitative RT-PCR analyses revealed that CD271 mRNA expression was increased in PSCs after coculture with pancreatic cancer cells. However, the level of CD271 mRNA expression subsequently decreased after the transient increase. Furthermore, CD271 mRNA expression was decreased in PSCs migrating toward pancreatic cancer cells through Matrigel. In the xenograft model, CD271⁺ PSCs were present at tumor margins/periphery and were absent in the tumor core. In conclusion, CD271 was expressed in PSCs around pancreatic tumors, but not in the center of the tumors, and expression decreased after long coculture with pancreatic cancer cells or after movement toward pancreatic cancer cells. These findings suggest that CD271⁺ PSCs appear at the early stage of pancreatic carcinogenesis and that CD271 expression is significantly correlated with a better prognosis in patients with PDAC.     

缺氧诱导因子1α、血管内皮生长因子和成纤维细胞生长因子在胰腺癌中的表达及意义

目的：研究胰腺癌组织中缺氧诱导因子1alpha（Hypoxia-inducible factor-1alpha,HIF-1α）、血管内皮生长因子（vascular endothelial growth factor,VEGF）和成纤维细胞生长因子（fibroblast growth factor,FGF）的表达并探讨其意义。方法：Western blot法检测22例胰腺癌及癌旁组织中HIF-1α、VEGF和FGF蛋白的表达,分析HIF-1α与VEGF、FGF之间的相关性以及与性别、年龄、肿瘤大小、淋巴结转移和TNM分期之间的关系。结果：HIF-1α、VEGF和FGF在胰腺癌组织中的蛋白表达水平明显高于胰腺癌周组织（P〈0.01）,HIF-1α与VEGF、FGF之间的表达具有显著相关性（P〈0.01）。HIF-1α的表达与胰腺癌的TNM分期、肿瘤大小和淋巴结转移有关（P〈0.01）,VEGF和FGF的表达与胰腺癌的肿瘤大小和淋巴结转移有关（P〈0.05）。结论：HIF-1α可以上调VEGF和FGF的表达,在胰腺癌的发生、发展中起着重要作用。     

基质金属蛋白酶-9表达与胰腺癌生物学行为及预后的相关性研究

目的：探讨基质金属蛋白酶-9（matrix metalloproteinase-9,MMP-9）的表达与胰腺癌生物学特性及对患者预后的影响。方法：采用免疫组织化学法检测63例原发性胰腺癌及11例癌旁正常组织中MMP-9的表达.比较MMP-9的表达与胰腺癌患者生物学特性及预后的关系。结果：MMP-9阳性表达率为68.3%（43/63）,而11例癌旁正常组织无MMP-9表达。MMP-9的表达与肿瘤大小（P=0.014）、分化程度（P=0.039）、TNM分期（P=0.001）、远处转移（P=0.011）及淋巴结转移（P=0.009）显著相关。MMP-9表达阳性者生存时间明显低于阴性表达者（Log-rank检验,x2=32.70,P=0.000）。Cox回归分析表明：分化程度（P=0.000）、TNM分期（P=0.006）、淋巴结转移（P=0.035）及MMP-9过表达（P=0.000）是胰腺癌预后的独立影响因素。结论：MMP-9过表达在胰腺癌的浸润转移中发挥重要作用,过表达提示预后不良.对MMP-9的检测可有效的评估胰腺癌患者的预后。     

Pancreatic tumor cells influence the composition of the extracellular matrix

The malignant behavior of cancers depends on the microenvironmental context. We investigated compositional alterations of the extracellular matrix (ECM) in pancreatic cancer, with special emphasis on the proteoglycans decorin, lumican, and versican. Compared with normal controls (n=18), marked overexpression of these proteoglycans was observed in pancreatic cancer tissues (n=30) by quantitative RT-PCR (p<0.0001). Immunohistochemistry revealed abundance of proteoglycans in the ECM of pancreatic cancer specimens, whereas tumor cells themselves were devoid of either decorin, lumican or versican. RT-PCR confirmed pancreatic stellate cells (PSCs) as the major source of these proteins. Interestingly, TGFbeta1 and conditioned medium derived from pancreatic cancer cell lines synergistically suppressed the expression of known anti-tumor factors decorin and lumican, but stimulated the expression of pro-metastatic factor versican in cultured PSCs. These findings indicate that malignant cells can actively influence the composition of the ECM through TGFbeta1 and other soluble factors, altering their microenvironment in a tumor-favorable way.     

E-cadherin在胰腺癌中的表达及其对预后的影响

目的：探讨钙粘蛋白E-cadherin的表达与胰腺导管腺癌临床病理特征及预后的关系。方法：利用组织芯片,采用免疫组织化学En Vision二步法检测106例胰腺导管腺癌组织和12例正常胰腺组织E-cadherin的表达情况。结果：E-cadherin在胰腺导管腺癌组织中表达部分缺失率为53.8%（57/106）,完全缺失率11.3%（12/106）,正常组织中的缺失率为0%,差异有统计学意义（P〈0.01）;E-cadherin的表达与患者性别、年龄,临床分期、神经浸润和淋巴结转移无关,与肿瘤分化程度有关。106例胰腺导管腺癌患者E-cadherin表达完全缺失、部分缺失和完整表达的中位总生存期分别为7个月、19个月和25个月,两者差异有统计学意义（P〈0.01）。单因素分析显示,肿瘤分化程度、淋巴结转移、切缘情况和E-cadherin表达缺失水平与预后相关;多因素分析显示,切缘情况和E-cadherin表达缺失水平与预后相关。结论：E-cadherin可以作为胰腺导管腺癌患者预后的独立指标。     

ADAMl0在胰腺癌患者血清中表达及其临床意义

目的：探讨ADAM10在胰腺癌患者外周血中的表达及其临床意义。方法：应用酶联免疫吸附试验法（ELISA）检测40例胰腺癌患者和20例健康体检者的外周血ADAM10的表达水平,分析其与临床病理特征的关系。结果：胰腺癌患者血清ADAM10水平显著高于正常对照组（P〈0.01）;胰腺癌患者血清中ADAM10的表达水平与胰腺癌淋巴结转移、远处转移及TNM分期密切相关（P〈0.05）,且行根治性手术切除的胰腺癌患者ADAM10表达水平低于姑息性手术切除的患者（P〈0.05）;ADAM10对胰腺癌诊断的敏感度、特异性分别为51.7%、76.9%,联合检测CA19-9有助于提高胰腺癌诊断敏感度,但特异性有所下降;根治性切除后胰腺癌患者血清中ADAM10水平明显下降。结论：胰腺癌癌患者血清中ADAM10水平明显增高,检测血清ADAM10有助于胰腺癌的诊断和治疗。     

Pancreatic stellate cells promote epithelial-mesenchymal transition in pancreatic cancer cells

The interaction between pancreatic cancer cells and pancreatic stellate cells (PSCs), a major profibrogenic cell type in the pancreas, is receiving increasing attention. There is accumulating evidence that PSCs promote the progression of pancreatic cancer by increasing cancer cell proliferation and invasion as well as by protecting them from radiation- and gemcitabine-induced apoptosis. Because epithelial-mesenchymal transition (EMT) plays a critical role in the progression of pancreatic cancer, we hypothesized that PSCs promote EMT in pancreatic cancer cells. Panc-1 and SUIT-2 pancreatic cancer cells were indirectly co-cultured with human PSCs isolated from patients undergoing operation for pancreatic cancer. The expression of epithelial and mesenchymal markers was examined by real-time PCR and immunofluorescent staining. The migration of pancreatic cancer cells was examined by scratch and two-chamber assays. Pancreatic cancer cells co-cultured with PSCs showed loose cell contacts and a scattered, fibroblast-like appearance. The expression of E-cadherin, cytokeratin 19, and membrane-associated β-catenin was decreased, whereas vimentin and Snail (Snai-1) expression was increased more in cancer cells co-cultured with PSCs than in mono-cultured cells. The migration of pancreatic cancer cells was increased by co-culture with PSCs. The PSC-induced decrease of E-cadherin expression was not altered by treatment with anti-TGF-β-neutralizing antibody, excluding a central role of TGF-β in this process. In conclusion, PSCs promoted EMT in pancreatic cancer cells suggesting a novel mechanism by which PSCs contribute to the aggressive behavior of pancreatic cancer cells.