多囊肾病基因1和多囊肾病基因2在不同肾组织和肾细胞株中的表达及意义

目的检测多囊肾病基因1（PKD1）和多囊肾病基因2（PKD2）在不同肾组织和肾细胞株中的表达,探讨其表达产物的正常生理功能、相互作用关系及病理意义。方法用免疫沉淀、Westernblotting、原位杂交和免疫组织化学、免疫细胞化学方法检测PKD1和PKD2在不同肾组织和肾细胞株中的表达水平。结果PKD1和PKD2在正常肾组织和常染色体显性多囊肾病（ADPKD1）肾囊肿组织中都有表达。PKD1 mRNA和PKD2mRNA及其表达产物有着共同的组织分布,且表达水平差异无统计学意义。在PKD1突变的多囊肾组织中,二者在囊肿衬里上皮细胞和远端肾小管上皮细胞中都有高水平表达,且明显强于他们在正常肾小管中的表达（P〈0．01）,差异有统计学意义。但在多囊肾临近囊肿壁的小管细胞中,二者表达量明显减低。此外,二者在ADPKD肾囊肿衬里上皮细胞系和猪近端肾小管上皮细胞株（LLC-PK1）中也有表达。多囊蛋白-1（PC-1）的表达位于胞膜和胞质,多囊蛋白-2（PC-2）的表达则局限于胞质。结论PKD1和PKD2几乎共同的表达分布模式及其在正常肾组织和多囊肾组织的表达分布差异提示PC-1和PC-2在功能上的关联可能是以他们分子结构的相互作用为基础的,多囊蛋白可能具有维持肾小管结构稳定性的功能。     

雷帕霉素抑制人多囊肾囊肿衬里上皮细胞增殖及VEGF表达

目的 探讨雷帕霉素对常染色体显性多囊肾病囊肿衬里上皮细胞增殖及血管内皮细胞生长因子（VEGF）表达的抑制作用及其机制。方法MTT法检测WT9-12细胞增殖;流式细胞术检测细胞周期及凋亡;Western Blot检测周期相关蛋白(cyclinD、p21)、凋亡相关蛋白(Bcl2/Bax)及VEGF表达。结果 雷帕霉素可抑制WT9-12细胞的增殖,使细胞周期停滞在G0/G1期并促进细胞凋亡。雷帕霉素可下调WT9-12细胞cyclinD 、上调p21表达,下调Bcl2、上调Bax表达。原代培养的多囊肾囊肿衬里上皮细胞及WT9-12细胞的VEGFmRNA表达明显高于正常肾小管上皮细胞（P<0.05）。雷帕霉素可抑制WT9-12细胞VEGF的表达（P<0.05）。结论 雷帕霉素可通过抑制多囊肾囊肿衬里上皮细胞增殖、促进凋亡及抑制VEGF表达抑制血管形成,从而抑制多囊肾病进展。     

Polycystin-1相关的信号转导通路

已知85%～90%的常染色体显性遗传性多囊肾病是由pkd1突变引起的,因此pkd1编码的多囊蛋白-1的功能受到研究者的关注。多囊蛋白-1是一个具有长的细胞外氨基末端,11个跨膜区,短的细胞内羧基末端的跨膜蛋白。近年来发现多囊蛋白-1与多条信号转导通路有关。文章主要介绍多囊蛋白-1相关的PI3-K/Akt/mTOR、Wnt/β-catenin和JAK-STAT等信号转导通路、各信号通路之间的联系及其在常染色体显性遗传性多囊肾病致病中的作用。     

过表达miR-151a-3p抑制PC-3前列腺癌细胞增殖和迁移

目的研究上调微小RNA-151a-3p(miR-151a-3p)对前列腺癌细胞增殖和迁移的影响和机制。方法采用实时荧光定量PCR检测PC-3M、C4-2B、22RV1、DU-145、PC-3、LNCaP人前列腺癌细胞和RWPE-1人正常前列腺上皮细胞中miR-151a-3p的表达量。以miR-151a-3p表达量最低的PC-3细胞为研究对象,生物信息学预测并采用双荧光素酶报告基因实验检验miR-151a-3p的潜在靶基因。转染miR-151a-3p模拟物或阴性对照微小RNA(miR-NC)至PC-3细胞。实时荧光定量PCR检测miR-151a-3p和潜在靶基因mRNA的表达量,Western blot法检测靶基因及下游信号通路蛋白表达量。采用MTT法检测PC-3细胞的增殖,Transwell~(TM)实验检测PC-3细胞的迁移能力。结果 miR-151a-3p在前列腺癌细胞的表达量明显低于RWPE-1人正常前列腺上皮细胞,其中PC-3细胞中的表达量最低。生物信息学预测及双荧光素酶报告基因实验表明NIMA相关激酶2(NEK2)是miR-151a-3p的潜在靶基因。转染miR-151a-3p模拟物后,PC-3细胞中miR-151a-3p的表达量明显上升,NEK2基因的表达量明显降低,NEK2基因下游磷脂酰肌醇3激酶-蛋白激酶B-哺乳动物雷帕霉素靶蛋白(PI3K-AKT-mTOR)信号通路蛋白水平降低。上调miR-151a-3p明显抑制前列腺癌PC-3细胞的增殖和迁移。结论miR-151a-3p在前列腺癌细胞中表达降低,上调miR-151a-3p的表达可通过降低NEK2基因及下游PI3K-AKT-mTOR信号通路蛋白的表达抑制前列腺癌PC-3细胞的增殖和迁移。     

核转录因子kB／环氧化酶-2参与糖尿病肾病大鼠肾细胞增殖

目的探讨核转录因子-kB（NF-kB）／环氧化酶-2（COX-2）信号与糖尿病肾病大鼠肾细胞增殖的关系。方法单侧肾切除大鼠ip链脲佐菌素诱发糖尿病模型，16周后切除左肾。用免疫组织化学检测肾皮质NF—kB和COX-2蛋白的表达；用正常糖和高糖培养基培养肾小管上皮细胞，24、48和72h后收集细胞，用免疫细胞化学检测PCNA蛋白的表达，流式细胞术检测NF-kB和COX-2蛋白的表达。结果（1）糖尿病模型组肾脏肥大指数增加，肾小球体积增大，系膜基质增多；（2）糖尿病模型组肾组织中活化的NF—kB和COX-2蛋白表达高于对照组，两者呈显著正相关；（3）高糖组肾小管上皮细胞PCNA表达强于正常糖组；（4）高糖能够上调肾小管上皮细胞NF-kB和COX-2蛋白的表达，两者呈显著正相关；同时COX-2蛋白表达与PCNA阳性率呈显著正相关。结论活化NF-kB，上调COX-2蛋白表达从而引起肾脏固有细胞的增殖可能是糖尿病肾脏损伤的机制之一。     

IL-18对肾小管上皮细胞表型转化的影响

目的：研究IL-18对体外培养肾小管上皮细胞表型转化的影响，以明确IL-18在慢性肾脏疾病中的可能作用机制。方法：应用体外细胞培养技术培养人肾小管上皮细胞侏（HK-2）。应用RT-PCR技术检测α-平滑肌肌动蛋白（α-SMA）、转化生长因子-β1（TGF-β1）mRNA水平，用免疫细胞化学方法（ICC）及Western blot技术分别检测IL-18对HK-2细胞表达仪-SMA蛋白的影响。结果：（1）IL-18可促进HK-2细胞表达α-SMA、TGF-β1 mRNA，且两者之间呈正相关（P〈0．05）。（2）IL-18增加α-SMA阳性HK-2细胞百分数（P〈0．05）。（3）IL-18使HK-2细胞α-SMA蛋白表达水平增加。结论：IL-18可剂量和时间依赖性地促进肾小管上皮细胞转分化为肌成纤维细胞，促进肾间质纤维化。     

糖尿病肾病患者尿液sICAM-1浓度及与糖尿病相关指标的关系

糖尿病肾病（Diabetic nephropathy，DN）是糖尿病微血管病变的一部分。近年来的研究认为其形成机制可能与血流动力学障碍、细胞代谢异常等多因素有关，其中黏附分子与糖尿病肾病关系密切。我们探讨了可溶性细胞黏附分子-1（sICAM-1）在DN患者尿液中的浓度及与糖尿病相关指标的关系。     

常显性内脏多囊病三家系报告兼文献复习

多囊性肾脏病（Polycystickidneydisase），包括常显性和常隐性遗传多囊性肾病。以往曾分别称为成人型和儿童型或婴儿型多囊性肾病，现分认为二者的差别在于遗传方式，而非起病年龄差异，因为常显性多囊性肾病（ADPKD）在儿童并不少见，甚至发生在胎内或婴儿[1]。这种科学的分型命名法，末被人所熟知，故本文加以采用，旨在推广应用，ADPKD届世界性疾病，发病率1／200－1／1000，其患病率高，合并症多，惜至今仍未被引起足够重视。ADPKD合并多囊肝、多囊脾、依次为胰、肺、骨、卵巢、子宫等多囊肿。可见是多器官疾病，称为内脏多囊病…     

显性失活IκBα质粒对胰腺癌PC-3细胞株核因子-κB和环氧合酶-2表达的影响

目的：观察显性失活IκBα质粒转染胰腺癌PC-3细胞株后，对细胞核因子-κB（NF-κB）和环氧合酶-2（COX-2）表达的影响。 方法： 免疫组织化学证实NF-κB和COX-2在胰腺癌PC-3细胞株中的表达，逆转录-聚合酶链反应（RT-PCR）和蛋白免疫印迹（Western blotting）检测PC-3细胞转染显性失活IκBα质粒后，细胞中NF-κB和COX-2表达的变化。 结果： 胰腺癌PC-3细胞株中存在NF-κB和COX-2的表达，转染显性失活IκBα质粒后，细胞中NF-κB和COX-2表达均下调，且体现出一定的时间依赖性关系。 结论： 胰腺癌PC-3细胞株中存在NF-κB和COX-2的阳性表达。显性失活的IκBα质粒可抑制细胞中NF-κB和COX-2的表达。     

重组人β2糖蛋白1第一结构域对抗磷脂抗体综合征患者血清诱导人脐静脉内皮细胞产生ICAM-1、MCP-1的影响

目的：探讨重组人β2糖蛋白1第一结构域（hrβ2GPⅠDⅠ）对抗磷脂抗体综合征（APS）患者血清诱导人脐静脉内皮细胞产生细胞间黏附分子-1（ICAM-1）、单核细胞趋化蛋白-1（MCP-1）的影响。方法：应用hrβ2GPⅠDⅠ二聚体处理前后的APS患者血清分别与人脐静脉内皮细胞（HUVEC）共孵育，细胞EL／SA和RT-PCR方法分析处理前后HUVEC表达ICAM-1、MCP-1的变化。结果：hrβ2GPⅠDⅠ二聚体处理后的APS患者血清较处理前相比，与其孵育的HUVEC表达ICAM-1、MCP-1在蛋白和mRNA水平均明显降低。结论：邮：GPⅠDⅠ可中和APS患者血清中的大部分致病性抗β2GPⅠ抗体（anti-β2 GPⅠ）。     

Polycystin-1 expression in fetal, adult and autosomal dominant polycystic kidney

The mutation of the PKD1 gene causes autosomal dominant polycystic kidney disease (ADPKD), and the PKD1 gene encodes polycystin-1 (PC-1). PC-1 is thought to be a cell-cell/matrix adhesion receptor molecule at the cell surface that is widely expressed in the kidney. However, there are controversies about the role of PC-1 protein and its expression when using different antibodies to detect it. We used two PC-1 antibodies; C-20 (Santa Cruz, sc-10372) as the C-terminal antibody, and P-15 (Santa Cruz, sc-10307) as the N-terminal antibody. We evaluated the PC-1 expression by performing immunoblotting on the human embryonic kidney (HEK) 293 cells and the renal proximal tubular epithelial cell (RPTEC) lysates. We characterized the expression of PC-1 in the fetal, adult and polycystic kidneys tissues by performing immunohistochemistry. We confirmed the PC-1 expression in the HEK 293 cells and the RPTEC lysates, but the expression was very low. The PC-1 proteins were diffusely expressed in the tubular epithelial cells cytoplasm in the fetal and adult kidneys, and the PC-1 expression was more prominent in the proximal tubules of the fetal kidney. In the ADPKD kidney, the PC-1 proteins were heterogenously and weakly expressed in the tubular or cyst lining epithelial cells. Our data suggests that the development of the kidney may regulate the expression of PC-1, and an altered PC-1 expression may contribute to cyst formation in ADPKD.     

ERBIN associates with p0071, an armadillo protein,at cell-cell junctions of epithelial cells

BACKGROUND: ERBIN, an ErbB2 receptor-interacting protein, belongs to a recently described family of proteins termed the LAP [leucine-rich repeats and PSD-95/dLg-A/ZO-1 (PDZ) domains] family which has essential roles in establishment of cell polarity. RESULTS: To identify new ERBIN-binding proteins, we screened a yeast two-hybrid library, using the carboxyl-terminal fragment of ERBIN containing PDZ domain as the bait, and we isolated p0071 (also called plakophilin-4) as an ERBIN-interacting protein. p0071 is a member of the p120 catenin family, which are defined as proteins with 10 armadillo repeats, and localizes along the cell-cell border. The ERBIN PDZ domain binds the COOH-terminus of p0071 containing the PDZ domain-binding sequence. Endogenous ERBIN was co-immunoprecipitated with p0071. In fully polarized Madin-Darby canine kidney (MDCK) cells, ERBIN co-localized largely with beta-catenin and partly with desmoplakin along the lateral plasma membrane domain. At these cell-cell contact regions, ERBIN co-localizes with p0071. Over-expression of the dominant active forms of Cdc42, Rac1 or RhoA, Rho family small GTPases, resulted in a marked accumulation of ERBIN at the cell-cell contacts of MDCK and HeLa cells. CONCLUSION: These results show that ERBIN interacts in vivo with p0071 and that it may be involved in the organization of adherens junctions and the desmosomes of epithelia. In addition, we demonstrated that the subcellular localization of ERBIN might be regulated by Rho family small GTPases.     

Expression of podoplanin in the mouse tooth germ and apical bud cells

This study was designed to investigate the distribution of cells expressing podoplanin in the mouse tooth bud. Podoplanin expression was detected in enamel epithelia of the cervical loop at cell-cell contacts strongly, and weakly on the loosely aggregated stellate reticulum in the center and the neighboring stratum intermedium. Odontoblasts exhibited intense podoplanin expression at the junction with predentin while no expression was detected in the enamel organ containing ameloblasts. These results suggest that proliferating inner and outer enamel epithelia express podoplanin but that the expression is suppressed in the differentiated epithelia containing ameloblasts. On the other hand the podoplanin expression occurs in the differentiating odontoblasts and the expression is sustained in differentiated odontoblasts, indicating that odontoblasts have the strong ability to express podoplanin. In cultured apical bud cells podoplanin was detected at cell-cell contacts. In real-time PCR analysis the amount of podoplanin mRNA of the apical buds was 2-fold compared with the amount of kidney used as a positive control. These findings indicate that apical bud cells have the strong ability to express the podoplanin gene. Podoplanin is a mucin-type glycoprotein negatively charged by extensive O-glycosylation and a high content of sialic acid, which expresses the adhesive property. The podoplanin may contribute to form odontoblastic fiber or function as the anchorage to the tooth development and in proliferating epithelial cells of cervical loop and apical bud.     

Serum amyloid A in the mouse. Sites of uptake and mRNA expression.

Murine serum amyloid A1 (SAA1) and serum amyloid A2 (SAA2) are circulating, acute phase, high density apolipoproteins of unknown function. To pursue issues relating to their possible function their uptake and formation were studied. Kinetics of SAA protein distribution and gene expression after acute phase stimulation by casein or lipopolysaccharide were examined using immunocytochemistry for protein and RNA blot and in situ hybridization with probes for SAA1 and SAA2 mRNA. After casein injection, interstitial cells of testes, cells of adrenal cortex, kidney proximal convoluted tubule epithelia, and some parafollicular cells of spleen took up SAA in a time pattern related to plasma SAA levels. Extrahepatic SAA1 and SAA2 mRNA were induced by lipopolysaccharide in kidney proximal and distal convoluted tubule epithelia, and SAA1 mRNA was induced in epithelial lining the mucosa of the ileum and large intestine, indicating that there may be more than one function for the apoSAA gene family related to site of and stimulus for expression.     

The PKD1 gene product, "polycystin-1," is a tyrosine-phosphorylated protein that colocalizes with alpha2beta1-integrin in focal clusters in adherent renal epithelia.

Mutations in the PKD1 gene are responsible for autosomal dominant polycystic kidney disease (ADPKD). Although PKD1 has been cloned and shown to be expressed at high levels in the fetal ureteric bud and ADPKD cystic epithelia in the human kidney, the function of its encoded protein, "polycystin-1" is unknown. In this study we used primary and immortalized human renal epithelial cell lines derived from normal fetal, adult, and ADPKD kidneys, that endogenously express PKD1, to study the biologic function of the polycystin-1 protein. ADPKD renal epithelial cells expressed high levels of polycystin-1 protein and showed increased adhesion to type I collagen by comparison with normal adult human renal epithelia that expressed little polycystin. Adherent ADPKD cells also expressed high levels of alpha2beta1-integrin and their attachment was inhibited by a functional monoclonal antibody to alpha2-integrin. Double labeling and confocal microscopy as well as coimmunoprecipitation analysis showed overlapping colocalization of polycystin-1 with alpha2beta1-integrin as well as with the focal adhesion proteins vinculin and paxillin in multiprotein clusters localized to focal areas of cell membrane contact with type I collagen matrix after short periods of attachment. Immunoprecipitation and Western immunoblot studies also showed that polycystin-1 was posttranslationally modified by tyrosine phosphorylation. These studies suggest that the PKD1-encoded protein is part of a large multiprotein complex in epithelial cells that functions in the regulation of extracellular matrix interactions with the plasma membrane and cell cytoskeleton.     

N-Cadherin Expression in Human Prostate Carcinoma Cell Lines : An Epithelial-Mesenchymal Transformation Mediating Adhesion with Stromal Cells

In human prostate adenocarcinoma, an association between loss of E-cadherin, increased Gleason score, and extracapsular dissemination has been observed. Further characterization of the E-cadherin/catenin phenotype of human prostate carcinoma cell lines showed loss of E-cadherin and expression of N-cadherin in poorly differentiated prostate carcinoma cell lines (PC-3N derived from PC-3, PC-3, and JCA1). We showed that N-cadherin is concentrated at sites of cell-cell contact in PC-3N cellular extensions. N-cadherin was also expressed in prostate stromal fibroblasts both in vitro and in prostate tissue. Co-cultures of prostate stromal fibroblasts and PC-3N cells showed the immunolocalization of N-cadherin in intercellular contacts. In addition, the isoform expression of the cadherin binding protein p120(ctn) differed in relation to the expression of E- versus N-cadherin by the prostate carcinoma cell lines. The p100 isoform was more highly expressed in E-cadherin-positive carcinoma cell lines, whereas p120 was predominantly expressed only in N-cadherin-positive prostate carcinoma cell lines and prostate stromal fibroblasts. The N-cadherin-positive carcinoma cell line, PC-3N, displayed aggressive invasion into the surface of the diaphragm muscle after intraperitoneal injection of SCID mice. The gain of N-cadherin and loss of E-cadherin by invasive prostate carcinoma cell lines suggests a progression from an epithelial to a mesenchymal phenotype, which may allow for their interaction with surrounding stromal fibroblasts and facilitate metastasis.     

AMPKα2 reduces renal epithelial transdifferentiation and inflammation after injury through interaction with CK2β

TGFβ1/Smad, Wnt/β‐catenin and snail1 are preferentially activated in renal tubular epithelia after injury, leading to epithelial–mesenchymal transition (EMT). The stress response is coupled to EMT and kidney injury; however, the underlying mechanism of the stress response in EMT remains elusive. AMP‐activated protein kinase (AMPK) signalling is responsive to stress and regulates cell energy balance and differentiation. We found that knockdown of AMPKα, especially AMPKα2, enhanced EMT by up‐regulating β‐catenin and Smad3 in vitro. AMPKα2 deficiency enhanced EMT and fibrosis in a murine unilateral ureteral obstruction (UUO) model. AMPKα2 deficiency also increased the expression of chemokines KC and MCP‐1, along with enhanced infiltration of inflammatory cells into the kidney after UUO. CK2β interacted physically with AMPKα and enhanced AMPKα Thr172 phosphorylation and its catalytic activity. Thus, activated AMPKα signalling suppresses EMT and secretion of chemokines in renal tubular epithelia through interaction with CK2β to attenuate renal injury. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

利多卡因抑制前列腺癌PC-3细胞恶性生物学行为和ERK/JNK的磷酸化

目的 研究利多卡因对前列腺癌PC-3细胞恶性生物学行为和细胞外调节蛋白激酶/c-Jun氨基末端激酶(ERK/JNK)磷酸化的影响.方法 以PC-3细胞为模型进行研究.分组:对照组、利多卡因处理组(低25μmol/L、中50μmol/L、高100μmol/L).采用克隆实验检测细胞增殖;流式检测凋亡和线粒体膜电位;Transwell实验检测细胞侵袭能力;Western印迹检测血管内皮生长因子(vascular endothelial growth factor,VEGF),E-cadherin和Vimentin的蛋白表达和ERK1/2和JNK1/2的磷酸化情况.结果 与对照组比较,利多卡因处理可抑制PC-3细胞增殖、侵袭和迁移,促进其线粒体膜电位的变化和凋亡.Western印迹结果显示,利多卡因可抑制PC-3细胞中ERK1/2和JNK1/2的磷酸化水平.结论 利多卡因处理可有效地抑制前列腺癌PC-3细胞恶性生物学行为,其潜在机制或与利多卡因对ERK/JNK的磷酸化的抑制有关.