RHCG基因在非小细胞肺癌中的表达及对细胞增殖的影响

为了探讨Rh type C glycoprotein (RHCG)对非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞增殖的影响及可能的作用机制,本研究使用荧光定量PCR法检测12对NSCLC及癌旁组织样本中RHCG mRNA的表达水平及pcDNA3.1-RHCG质粒对A549细胞RHCG m RNA的表达;采用CCK-8法检测细胞增殖能力;运用PI染色法检测细胞周期;使用免疫印迹法检p-PI3K、PI3K、p-AKT以及AKT蛋白表达水平。本研究发现,与癌旁组织比较,NSCLC中RHCG m RNA表达水平明显降低。RHCG过表达能抑制NSCLC细胞系A549细胞增殖能力。此外,RHCG过表达使A549细胞周期G1/S期转化发生阻滞。本研究还发现,RHCG过表达可下调A549细胞p-PI3K/PI3K和p-AKT/AKT水平。本研究表明,RHCG抑制NSCLC细胞增殖的作用与其抑制PI3K/AKT信号通路有关。     

Cell Surface Cdc37 Participates in Extracellular HSP90 Mediated Cancer Cell Invasion

Cdc37 is a 50 kDa molecular chaperone which targets intrinsically unstable protein kinases to the molecular chaperone HSP90. It is also an over-expressed oncoprotein that mediates carcinogenesis and maintenance of the malignant phenotype by stabilizing the compromised structures of mutant and/or over-expressed oncogenic kinases. Here we report that Cdc37 is not restricted intracellularly but instead it is also present on the surface of MDA-MB-453 and MDA-MB-231 human breast cancer cells, where it is shown to participate in cancer cell motility processes. Furthermore, we demonstrate using an anti-Cdc37 cell impermeable antibody, that similarly to its intracellular counterpart, this surface pool of Cdc37 specifically interacts with HSP90 as well as the kinase receptors HER2 and EGFR on the cell surface, probably acting as a co-factor in HSP90's extracellular chaperoning activities. Finally, we show that functional inhibition of surface HSP90 using mAb 4C5, a cell impermeable monoclonal antibody against this protein, leads not only to disruption of the Cdc37/HSP90 complex but also to inhibition of the Cdc37/ErbB receptors complexes. These results support an essential role for surface Cdc37 in concert with HSP90 on the cell surface during cancer cell invasion processes and strengthen the therapeutic potential of mAb 4C5 for the treatment of cancer.     

温度和病原菌感染对大黄鱼热激蛋白90基因表达的影响

热激蛋白（heat shock protein,HSP）是进化上非常保守的蛋白质家族之一,普遍存在于各种生物体中,在多种生理活动中起到重要作用。该实验克隆了大黄鱼HSP90基因,并分析了温度和病原菌感染对其表达的影响。克隆到的大黄鱼HSP90序列长3 930 nt,含4个外显子和3个内含子,其中编码区2 178 nt,编码725个氨基酸。同源性分析发现,大黄鱼HSP90基因序列和其它鱼类的同源性在90%以上。在不同水温下,HSP90基因在不同组织中的表达量变化不同,心、肠和脑等组织在29 oC时表达量最高,而肌肉、脾和肝等组织在24 oC时表达量最高。用病原菌感染大黄鱼,感染48 h后,鳃、心、脾、肠、肾和脑组织HSP90的表达量明显上升;发病时（感染7天后）,受检的9种组织中HSP90的表达量都比未感染时明显增加。     

高温对原代支持细胞增殖能力和occludin紧密连接蛋白的影响

目的:研究高温对原代大鼠睾丸支持细胞增殖作用和对紧密连接蛋白的损伤机制.方法:体外分离培养雄性Wistar大鼠睾丸支持细胞(Sertoli cell,SC),免疫组化FasL鉴定.实验分为对照组(36℃)和实验组(37℃、38℃、39℃).在相应的温度下培养5d后,CCK-8法检测SC增殖作用,Western印迹法检测OCLN表达水平.结果:体外培养SC的纯度＞90％,CCK-8结果显示,细胞增殖活性在36℃时最强,37～39℃时逐渐降低;Western印迹显示,36℃时OCLN表达量最高;高于36℃时,OCLN表达量随着温度的增加而降低(P＜0.05).结论:高温影响SC增殖活性,破坏了支持细胞紧密连接蛋白的正常表达和血睾屏障的完整性,从而影响正常精子的形成.     

Apoptosis Versus Cell Differentiation: Role of Heat Shock Proteins HSP90, HSP70 and HSP27

Heat shock proteins HSP27, HSP70 and HSP90 are molecular chaperones whose expression is increased after many different types of stress. They have a protective function helping the cell to cope with lethal conditions. The cytoprotective function of HSPs is largely explained by their anti-apoptotic function. HSPs have been shown to interact with different key apoptotic proteins. As a result, HSPs can block essentially all apoptotic pathways, most of them involving the activation of cystein proteases called caspases. Apoptosis and differentiation are physiological processes that share many common features, for instance, chromatin condensation and the activation of caspases are frequently observed. It is, therefore, not surprising that many recent reports imply HSPs in the differentiation process. This review will comment on the role of HSP90, HSP70 and HSP27 in apoptosis and cell differentiation. HSPs may determine de fate of the cells by orchestrating the decision of apoptosis versus differentiation.Key Words: apoptosis, differentiation, heat shock proteins, chaperones, cancer cells, anticancer drugs     

华蟾素注射液对前列腺癌PC3细胞株增殖及VEGF及其受体KDR表达的影响

目的:探讨华蟾素注射液对前列腺癌PC3细胞的生长抑制作用及其对VEGF及KDR表达水平的影响。方法:体外培养前列腺癌PC3细胞株,采用MTT(四甲基偶氮唑蓝)法检测不同浓度华蟾素注射液对前列腺癌PC3细胞增殖的影响;采用FCM法分析该药对PC3细胞周期分布的影响;蛋白印迹法检测药物处理PC3细胞后,细胞中VEGF及KDR蛋白表达的变化。结果:不同浓度浓度的华蟾素注射液对前列腺癌PC3细胞具有抑制作用(P<0.05),并呈一定的时间剂量依赖性;流式细胞仪检测显示,华蟾素可阻滞细胞进程于S期(P<0.05);蛋白印迹法检测结果显示,华蟾素注射液能使VEGF及KDR蛋白表达下降,并呈浓度依赖性。结论:华蟾素注射液能抑制PC3细胞的增殖作用,其机制可能与下调VEGF及KDR表达有关。     

HSP90 Inhibition Enhances Antimitotic Drug-Induced Mitotic Arrest and Cell Death in Preclinical Models of Non-Small Cell Lung Cancer

HSP90 inhibitors are currently undergoing clinical evaluation in combination with antimitotic drugs in non-small cell lung cancer (NSCLC), but little is known about the cellular effects of this novel drug combination. Therefore, we investigated the molecular mechanism of action of IPI-504 (retaspimycin HCl), a potent and selective inhibitor of HSP90, in combination with the microtubule targeting agent (MTA) docetaxel, in preclinical models of NSCLC. We identified a subset of NSCLC cell lines in which these drugs act in synergy to enhance cell death. Xenograft models of NSCLC demonstrated tumor growth inhibition, and in some cases, regression in response to combination treatment. Treatment with IPI-504 enhanced the antimitotic effects of docetaxel leading to the hypothesis that the mitotic checkpoint is required for the response to drug combination. Supporting this hypothesis, overriding the checkpoint with an Aurora kinase inhibitor diminished the cell death synergy of IPI-504 and docetaxel. To investigate the molecular basis of synergy, an unbiased stable isotope labeling by amino acids in cell culture (SILAC) proteomic approach was employed. Several mitotic regulators, including components of the ubiquitin ligase, anaphase promoting complex (APC/C), were specifically down-regulated in response to combination treatment. Loss of APC/C by RNAi sensitized cells to docetaxel and enhanced its antimitotic effects. Treatment with a PLK1 inhibitor (BI2536) also sensitized cells to IPI-504, indicating that combination effects may be broadly applicable to other classes of mitotic inhibitors. Our data provide a preclinical rationale for testing the combination of IPI-504 and docetaxel in NSCLC.     

LRP16对乳腺癌MCF-7细胞增殖的影响

用Northern印迹方法检测雌二醇 (17β E2 )对LRP16mRNA表达的时间及剂量依赖性调控作用 .构建LRP16基因启动子序列调控的萤光素酶报告子 (pS0 ) ,并与雌激素受体α和 β(ERα和ERβ)表达载体共转染COS 7和MCF 7细胞后测定萤光素酶活性 .将LRP16基因的表达载体转染MCF 7细胞 ,测定过表达LRP16对细胞的生长特性的影响 .17β E2 使MCF 7细胞中LRP16mRNA表达水平增加 ,增加幅度未显示出 17β E2 培养时间和剂量的依赖性 .pS0 与ERα表达载体共转染细胞的相对萤光素酶活性较非共转染组 (对照组 )及pS0 ERβ表载体共转染组升高 5～ 10倍 .LRP16基因过表达促进MCF 7细胞的增殖 .研究表明 ,雌激素可能通过ERα上调乳腺癌MCF 7细胞LRP16基因的表达并促进细胞增殖     

RTN4对于结肠癌细胞增殖的调控作用

2018年全球癌症统计调查显示,结直肠癌约占患癌新病例的12.1%。因此,寻找新的结肠癌发生有关的基因,发现新的治疗靶点显得尤为迫切。通过数据库分析发现,RTN4基因的表达水平与结肠癌患者生存率的相关性具有统计学意义。针对RTN4基因构建其干扰质粒,将慢病毒作为载体转染结肠癌HCT116细胞中构建敲低RTN4的结肠癌细胞系,最后检测了低表达后RTN4基因的细胞增殖。结果发现,敲低RTN4基因后显著促进了结肠癌细胞HCT116的增殖,研究通过Western blot观察敲低RTN4后HCT116细胞自噬通路相关蛋白p62和LC3的表达情况,发现与对照组相比较,敲低RTN4组LC3转化量（LC3-II/LC3-I）增多,而p62蛋白减少。研究分析了RTN4的潜在抑癌作用,发现敲低RTN4基因会显著增强结肠癌细胞的增殖能力,并且诱导自噬,说明RTN4可能与激活LC3/p62自噬途径有关。     

截短型rBTI的表达、纯化及其对EC9706细胞生长的抑制作用

依据先前获得的重组荞麦胰蛋白酶抑制剂（rBTI）氨基酸序列及三维分子构像,分别构建了rBTI C末端缺失VVM、TPVVM或VDTPVVM的截短型pExsecI BTI t1,pExsecI BTI t2和pExsecI BTI t3重组质粒.转入大肠杆菌BL21中进行表达,并通过Resource Q阴离子交换层析分离.实验结果显示,3个工程菌均以可溶方式表达,目的蛋白在SDS PAGE图谱中显示单一条带,其纯度达98％以上. 理化性质分析表明,截短型rBTI与野生型rBTI具有相似的胰蛋白酶抑制活性,并具有很好的热稳定性及酸碱稳定性. 将野生型和截短型rBTI分别作用于人食管癌EC9706细胞.MTT检测发现,C末端截短不同数目的氨基酸后,与野生型rBTI相比,在相同浓度下截短型rBTI仍具有一定的抑制肿瘤细胞生长作用,其抑制作用范围是截短前的50%左右. 这些结果提示, rBTI的 C末端氨基酸残基缺失未引起活性区域或功能部位的较大改变,从而保留了其对胰蛋白酶的抑制作用和部分生物学功能.     

新基因BCAPP2Ac在膀胱癌中的表达 及其对膀胱癌细胞增殖的影响

为探讨膀胱癌相关蛋白磷酸酶2A催化亚单位（BCAPP2Ac）新基因在膀胱癌组织中的表达及其对膀胱癌细胞增殖的影响,通过合成抗原多肽免疫家兔获得抗BCAPP2Ac多克隆抗体及通过慢病毒感染方式获得稳定表达BCAPP2Ac的膀胱癌细胞.采用实时PCR及免疫组化染色方法检测BCAPP2Ac mRNA和蛋白在膀胱癌组织、癌旁组织及其它多种肿瘤组织中的表达;用细胞增殖实验检测BCAPP2Ac对膀胱癌细胞增殖的影响.实时PCR及免疫组化结果显示,BCAPP2Ac mRNA及蛋白在膀胱癌组织较癌旁组织表达明显下调;过表达BCAPP2Ac能抑制膀胱癌EJ、T24细胞的增殖, 蛋白磷酸酶2A催化结构域缺失（△PP2Ac）能逆转BCAPP2Ac的增殖抑制作用.研究结果提示,新基因BCAPP2Ac能抑制膀胱癌细胞的增殖,PP2Ac结构域在其抑制细胞增殖作用中发挥重要作用.     

The Effect of Manganese-induced Cytotoxicity on mRNA Expressions of HSP27, HSP40, HSP60, HSP70 and HSP90 in Chicken Spleen Lymphocytes in Vitro

The purpose of this study was to investigate the effect of manganese (Mn)-induced cytotoxicity on heat shock proteins in chicken spleen lymphocytes. Lymphocytes were cultured in medium in the absence and presence of MnCl₂ (2?×?10^?4, 4?×?10^?4, 6?×?10^?4, 8?×?10^?4, 10?×?10^?4, and 12?×?10^?4 mmol/L) for 12, 24, 36, and 48 h in vitro. Then, the mRNA levels of HSP27, HSP40, HSP60, HSP70, and HSP90 were examined by real-time quantitative PCR. The results showed that the mRNA levels of HSP27, HSP40, HSP60, HSP70, and HSP90 in all treatment groups at all time points, except mRNA levels of HSP27 at 48 h, had the same tendency. As manganese concentration increased, the mRNA expression of the heat shock proteins first increased and then decreased. In other words, we demonstrated that the mRNA expression of the heat shock proteins was induced at lower concentrations of manganese and was inhibited at higher concentrations. Mn had a dosage-dependent effect on HSP27, HSP40, HSP60, HSP70, and HSP90 mRNA expression in chicken spleen lymphocytes in vitro.     

Ndrg2基因表达对胃癌细胞增殖调控及其机理的研究

为研究Ndrg2基因在人类肿瘤发生发展中的作用,以不表达Ndrg2基因的胃癌细胞系HGC-27和表达Ndrg2基因的胃癌细胞系SGC-7901作为对比材料,以Ndrg2基因转染HGC-27胃癌细胞系,以及用Ndrg2的反义寡核苷酸封闭SGC-7901胃癌细胞系中Ndrg2基因的表达.发现Ndrg2可以抑制HGC-27胃癌细胞的软琼脂集落形成,有一定诱导细胞凋亡的作用,对细胞周期蛋白E的表达有明显下调作用.当封闭了SGC-7901胃癌细胞中Ndrg2基因表达的软琼脂集落形成受到抑制,流式细胞仪检测发现此时的SGC-7901细胞周期被阻滞在G1期,细胞周期蛋白D1和E表达下调.Ndrg2基因对两种肿瘤细胞中的细胞外信号调节激酶(ERK)和P38的表达也有不同的影响.     

Cellular Expression of Cyclooxygenase,Aromatase, Adipokines,Inflammation and Cell Proliferation Markers in Breast Cancer Specimen

Current evidences suggest that expression of Ki67, cyclooxygenase (COX), aromatase, adipokines, prostaglandins, free radicals, β-catenin and α-SMA might be involved in breast cancer pathogenesis. The main objective of this study was to compare expression/localization of these potential compounds in breast cancer tissues with tissues collected adjacent to the tumor using immunohistochemistry and correlated with clinical pathology. The breast cancer specimens were collected from 30 women aged between 49 and 89 years who underwent breast surgery following cancer diagnosis. Expression levels of molecules by different stainings were graded as a score on a scale based upon staining intensity and proportion of positive cells/area or individually. AdipoR1, adiponectin, Ob-R, leptin, COX-1, COX-2, aromatase, PGF_2α, F₂-isoprostanes and α-SMA were localised on higher levels in the breast tissues adjacent to the tumor compared to tumor specimens when considering either score or staining area whereas COX-2 and AdipoR2 were found to be higher considering staining intensity and Ki67 on score level in the tumor tissue. There was no significant difference observed on β-catenin either on score nor on staining area and intensity between tissues adjacent to the tumor and tumor tissues. A positive correlation was found between COX-1 and COX-2 in the tumor tissues. In conclusion, these suggest that Ki67, COXs, aromatase, prostaglandin, free radicals, adipokines, β-catenin and α-SMA are involved in breast cancer. These further focus the need of examination of tissues adjacent to tumor, tumor itself and compare them with normal or benign breast tissues for a better understanding of breast cancer pathology and future evaluation of therapeutic benefit.     

AFP的亚细胞定位及对肿瘤细胞生长的影响

目的观察甲胎蛋白（AFP）在不同肿瘤细胞中的亚细胞定位及对肿瘤细胞生长的影响。方法运用免疫荧光的方法观察内源性AFP在HeI。a细胞、QGY-7703细胞、MCF-7细胞中的亚细胞定位。将构建的表达AFP的质粒pcDNA3-AFP及AFP腺病毒siRNA干涉载体Adv—AFPsiRNA作用于QGY-7703细胞,MCF-7细胞,运用M1Tr,集落形成实验检测细胞增殖状况。结果免疫荧光显示,内源性的AFP在HeLa细胞、QGY-7703细胞、MCF-7细胞均只在细胞质中表达。pcDNA3-AFP使QGY-7703的细胞活性增加了2l％（P〈0．05）及集落形成能力增加了32％（P〈0．01）,MCF-7实验组比对照组细胞活性降低了30％（P〈0．01）．克隆形成能力降低82％（P〈0．01）。Adv—AFPsiRNA使QGY-7703的细胞活性降低了22％（P〈0．05）,平均克隆形成能力降低52％（P〈0．01）,MCF-7细胞活性提高了24．5％（P〈0．05）,克隆形成能力提高了89％（P〈O．01）。结论内源性的AFP只在细胞质中表达。AFP能促进QGY-7703细胞的增殖及克隆形成能力,而在MCF-7细胞中发挥相反的作用。腺病毒介导的内源性的AFP表达的下调能降低QGY-7703的增殖,却增加了MCF-7的细胞活性及克隆形成能力。     

黄芪甲苷对人骨髓间充质干细胞体外增殖及细胞因子表达的影响

目的：探讨黄芪甲苷对人骨髓间充质干细胞体外增殖及细胞因子表达的影响。方法：采用Percoll密度离心法和贴壁法分离纯化hBMSC;流式细胞术检测hBMSC表面标志;MTT法检测不同浓度黄芪甲苷干预72h后hBMSC的增殖情况;Real-timePCR法检测经黄芪甲苷干预后hBMSC对SCF、VEGF、SDF-1、GM-CSFmRNA的表达水平。结果：成功分离培养出laBMSC;不同浓度（20、40、80、160、320mg／mL）的黄芪甲苷可促进hBMSC增殖（P〈0．05）,其中160mg／mL组促增殖最明显;黄芪甲苷可促进hBMSC对SCF、VEGF、SDF-1mRNA的表达,而GM—CSFmRNA表达无明显变化。结论：黄芪甲苷可促进hBMSC体外增殖,可能与其促进hBMSC对SCF、VEGF、SDF-1mRNA表达有关。     

穿心莲内酯对人肺癌细胞增殖以及侵袭相关分子表达的影响

目的:观察穿心莲内酯(Andrographolide,AD)对人非小细胞肺癌细胞系H3255细胞的生长抑制作用,并研究其对肿瘤生长相关标记物血管内皮生长因子(vascular endothelial growth factor,VEGF)、转化生长因子(transforming growth factor,TGF)β1的表达以及蛋白激酶C(protein kinase C,PKC)的活性有无影响。方法:体外培养H3255细胞,分别用1.0、2.5、5.0μmol/L的AD在处理细胞24h。MTT法检测细胞的增殖,比色法检测乳酸脱氢酶(lactate dehydrogenase,LDH)的释放情况。ELISA检测DNA片段化情况以及VEGF和TGF-β1的产生;无机磷法检测Na+-K+-ATP酶活性以及磷基转移法测得PKC活性。结果:AD处理能呈剂量依赖性方式降低H3255细胞的活性以及Na+-K+-ATP酶活性(P<0.05),同时也能促进LDH的释放和DNA片段的形成,并减少肺癌细胞VEGF和TGFβ1的水平和PKC的磷酸化。结论:AD对肺癌细胞具有一定的抑制作用,有望成为一种潜在的肿瘤治疗药物。     

URI基因稳定表达对肝癌SMMC-7721细胞增殖能力的影响

目的:瞬转以及筛选出能够稳定表达URI(RPB5-mediating protein)基因的SMMC-7721细胞株,以其为模型研究URI基因对人肝癌SMMC-7721细胞增殖的影响.方法:首先,抽提URI的重组质粒并转染到SMMC-7721细胞中,在G418药物的筛选下选出能够稳定表达URI基因的细胞株,RT-PCR法和酶切检测该稳定细胞中URI基因的表达效率以确认URI是否稳定表达,MTT法和克隆形成实验检测URI基因对SMMC-7721细胞增殖的影响.结果:成功建立URI基因过表达的稳定细胞株.与SMMC-7721细胞对照组比较,其URI mRNA表达水平显著上调,能稳定表达URI细胞株的增殖,克隆形成率明显升高.结论:pFLAG-CMV-4-URI重组质粒能使URI在肝癌SMMC-7721细胞内稳定表达,过表达URI基因有可能帮助细胞通过G2/M期检验点来提高肝癌细胞SMMC-7721的增殖能力.