尿酸减轻6-OHDA介导PC12细胞SOD活性下降和损伤的实验研究

目的 探讨尿酸对6-羟基多巴(6-OHDA)介导的PC12细胞氧化损伤的作用.方法 采用高分化PC12细胞制作帕金森病细胞模型,实验分为对照组、6-OHDA组、尿酸(Uric acid,UA)组和6-OHDA+UA组.药物作用6 h、12 h和24 h,采用比色法检测细胞超氧化物歧化酶(SOD)的活性和乳酸脱氢酶(LD...     

骨髓间质干细胞与肿瘤细胞中FN1、NME2、TIMP3基因表达检测

目的探讨纤维连接蛋白1(FN1)、肿瘤转移抑制基因2(NME2)、组织金属蛋白酶抑制因子3(TIMP3)基因在胎儿骨髓间质干细胞(FMSCs)、成人骨髓间质干细胞(AMSCs)、F6、SGC及白血病细胞中的差异表达,寻找肿瘤和白血病的分子生物学特征。方法分别提取FMSCs、AMSCs、F6、SGC及白血病细胞的总RNA,逆转录cDNA,以cDNA为模板用PCR和实时PCR技术分别测定FN1、NME2、TIMP3基因表达水平。结果AMSCs组FN1、NME2、TIMP3基因表达水平均高于FMSCs、F6、SGC组(P<0.001);F6细胞以及白血病细胞中未能检出TIMP3基因的表达。结论FN1、NME2、TIMP3基因的下调以及TIMP3基因的缺如,可能是FMSCs突变为F6肿瘤细胞的分子特征之一;TIMP3基因的下调可能是肿瘤细胞的一种分子标志。     

JAK2/STAT3信号转导通路参与调节IL-6介导的肺腺癌细胞株中MMP-10的表达

目的研究Janus激酶2（JAK2）及信号转导和转录激活子3（STAT3）途径对IL-6介导的肺腺癌细胞（A549）中基质金属蛋白酶10（MMP-10）表达调节的作用,探讨MMP-10的调节机制。方法待生长状况良好的A549细胞融合生长至50%～70%时,分别用0,12.5,25,50ng/mlIL-6及AG490（JAK2特异性抑制剂）＋25ng/mlIL-6处理A549细胞24h后,采用实时RT-PCR技术检测JAK2、STAT3和MMP-10mRNA的表达水平;应用Westernblot检测MMP-10蛋白的表达水平。结果25ng/mlIL-6组STAT3mRNA表达水平较0ng/ml组高43.87%（P〈0.05）,AG490＋25ng/mlIL-6组较25ng/mlIL-6组低36.73%（P〈0.01）;AG490＋25ng/mlIL-6组MMP-10mRNA表达水平较25ng/mlIL-6组高43.21%（P〈0.01）;12.5,25,50ng/mlIL-6组的MMP-10蛋白水平显著高于0ng/ml组（P〈0.05）,且峰值出现在25ng/ml组;与25ng/mlIL-6组相比,MMP-10蛋白水平在AG490＋25ng/mlIL-6组明显降低（P〈0.05）。结论JAK2/STAT3信号转导途径可能参与调节IL-6介导的肺癌细胞株A549中MMP-10的表达。     

PAI-1招募中性粒细胞调控IL-6/STAT3信号通路促进脂多糖诱导的肺细胞损伤

目的探讨纤溶酶原激活物抑制物-1(plasminogen activator inhibitor-1,PAI-1)对细菌脂多糖（lipopolysaccharide,LPS）诱导的肺泡Ⅱ型上皮细胞（alveolar epithelial typeⅡcell, AECⅡ）损伤的调控作用及机制。方法 采用免疫印迹法和ELISA检测LPS处理后原代胎鼠AECⅡ细胞内、外PAI-1的表达量。通过CCK8和细胞迁移实验（Transwell）,用PAI-1重组蛋白和PAI-039(PAI-1的抑制剂)检测PAI-1对中性粒细胞活力、趋化的影响。收集Transwell下室的细胞培养液和AECⅡ,采用ELISA检测肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、白细胞介素1β(interleukin-1β,IL-1β)、白细胞介素6(interleukin-6,IL-6)、髓过氧化物酶（myeloperoxidase,MPO）、基质金属蛋白酶-1(matrix metalloproteinase-1,MMP-1)和血小板/内皮黏附因子（platelet/endotheli...     

IL －6／STAT3信号通路在脓毒症早期大鼠下丘脑－垂体－肾上腺轴中的表达及意义

目的：探讨大鼠脓毒症早期出现的HPA轴过度激活与IL－6／STAT3信号通路的内在联系。方法24只健康雄性Wistar大鼠,随机分为正常对照组（ Control组）、假手术组（ Sham组）、模型组（CLP组）三组。采用盲肠结扎穿孔（CLP）法建立脓毒症模型,术后6 h处死,分离出下丘脑、垂体、肾上腺组织。 RT－PCR检测下丘脑组织CRH、IL－6、STAT3、SOCS3 mRNA水平,垂体组织阿片促黑色素原（ POMC）、IL－6、STAT3、SOCS3 mRNA水平,肾上腺组织 IL－6、STAT3、SOCS3 mRNA水平。结果 CLP组较Control组、Sham组下丘脑组织中CRH、IL－6、STAT3、SOCS3 mRNA表达水平明显增加（P＜0.01）,垂体组织中POMC、IL－6、STAT3、SOCS3 mRNA表达水平明显增加（P＜0.01）,肾上腺组织中IL－6、STAT3、SOCS3 mRNA表达水平明显增加（P＜0.01）。Control组与Sham组各指标比较差异无统计学意义。结论脓毒症早期出现的HPA轴过度激活与IL－6／STAT3信号通路有着密切联系。针对IL－6／STAT3信号通路这一靶点进行干预,有望改善脓毒症状态下HPA轴的过度激活,为脓毒症的治疗提供新的思路。     

The m6A methyltransferase METTL14 inhibits the proliferation,migration, and invasion of gastric cancer by regulating the PI3K/AKT/mTOR signaling pathway

Background N6‐methyladenosine (m6A) modification may participate in the regulation of occurrence and development of tumors. However, the m6A level and the potential regulatory mechanism of m6A in gastric cancer (GC) remain uncertain.MethodsRNA m6A quantification assay was conducted to detect the m6A level in GC tissues and cell lines. Methyltransferase‐like 14 (METTL14) expression in GC tissues was explored by bioinformatics and immunohistochemistry. Then, the function of METTL14 in GC cells was examined by CCK‐8, colony formation assay, wound healing assay, and Transwell assay. Besides, Western blotting was conducted to probe the PI3K/AKT/mTOR pathway and the epithelial‐mesenchymal transformation (EMT) pathway‐related gene expression.ResultsThe m6A modification level was decreased in GC and METTL14 was a key regulator resulting in m6A disorder in GC. METTL14 was downregulated in GC by analyzing both clinical samples and bioinformatics. METTL14 overexpression suppressed GC cell proliferation and aggression by deactivating the PI3K/AKT/mTOR pathway and the EMT pathway, respectively.ConclusionsOur findings indicate that METTL14 partakes in the biological process of GC as a tumor suppressor and may be an emerging biomarker in GC.     

Acetylsalicylic acid regulates MMP-2 activity and inhibits colorectal invasion of murine B16F0 melanoma cells in C57BL/6J mice: Effects of prostaglandin F2α

Epidemiological studies indicate that acetylsalicylic acid may reduce the risk of mortality due to colon cancers. Metastasis is the major cause of cancer death. Matrix metalloproteinases (MMPs) play important roles in tumor invasion regulation, and prostaglandin F₂α (PGF₂α) is a key stimulator of MMP production. Thus, we investigated whether acetylsalicylic acid regulated MMP activity and the invasion of cancer cells and whether PGF₂α attenuated acetylsalicylic acid-inhibited invasion of cancer cells. Gelatin-based zymography assays showed that acetylsalicylic acid inhibited the MMP-2 activity of B16F0 melanoma cells. Matrigel-based chemoinvasion assays showed that acetylsalicylic acid inhibited the invasion of B16F0 cells. Acetylsalicylic acid can inhibit PGF₂α synthesis and PGF₂α is a key stimulator of MMP-2 production. Our data showed that PGF₂α treatment attenuated the acetylsalicylic acid-inhibited invasion of B16F0 cells. In animal experiments, acetylsalicylic acid reduced colorectal metastasis of B16F0 cells in C57BL/6J mice by 44%. Our results suggest that PGF₂α is a therapeutic target for metastasis inhibition and acetylsalicylic acid may possess anti-metastasis ability.     

伊马替尼对K562和K562/AO2细胞细胞色素P4503A亚家族多肽5基因转录、蛋白表达及活性的调控

目的:探讨伊马替尼(商品名:格列卫)对K562及其耐药细胞株K562/AO2细胞色素P4503A亚家族多肽5(CYP3A5)基因转录、蛋白表达及活性的调控。方法:采用实时聚合酶链反应(real-timePCR)、蛋白印迹(Westernblot)和高效液相色谱(HPLC)法检测CYP3A5基因转录、蛋白表达和活性水平。同时将CYP3A5重组质粒稳定转染有CYP3A5基础转录的K562细胞,噻唑蓝(MTT)法测定伊马替尼的半数抑制浓度(IC50)值,观察CYP3A5基因的过表达是否介导白血病细胞对伊马替尼敏感性的改变。结果:K562细胞经伊马替尼作用24h后CYP3A5mRNA转录增强,蛋白表达和活性均增加,与未加药细胞相比,差异有统计学意义(P<0.05、P<0.01、P<0.01、P<0.01、P     

SH2B1沉默对胃癌SGC-7901细胞增殖、凋亡及PI3K/AKT通路的影响

目的研究沉默SH2B1基因表达对胃癌SGC-7901细胞增殖、凋亡及3-磷酸肌醇激酶(PI3K)/蛋白质丝氨酸苏氨酸蛋白激酶(AKT)通路的影响。方法体外培养胃癌SGC-7901细胞,采用SH2B1 siRNA转染SGC-7901细胞作为研究组,采用国际通用的与所有基因均无同源序列的non-target siRNA转染作为阴性对照组(NC组),以未经处理的胃癌SGC-7901细胞作为空白对照组(BC组),48h后收集各组转染成功细胞,采用荧光定量聚合酶链反应(qRT-PCR)检测SH2B1 mRNA表达情况,采用蛋白质印迹法(Western Blot)检测SH2B1蛋白表达情况;采用细胞计数试剂盒(CKK-8)检测细胞增殖情况,采用流式细胞仪检测细胞凋亡,同时检测Ki67、增殖细胞核抗原(PCNA)、Caspase-9、PI3K、AKT、p-AKT蛋白表达情况。结果研究组SGC-7901细胞SH2B1蛋白及mRNA表达量较BC组和NC组明显降低,差异有统计学意义(P<0.05);转染后,siRNA组SGC-7901细胞增殖明显受到抑制、平板克隆形成率较BC组和NC组明显降低,凋亡率明显升高,差异均有统计学意义(P<0.05);与BC和NC组比较,研究组SGC-7901细胞PI3K、p-AKT蛋白、Ki67及PCNA蛋白呈低表达,差异有统计学意义(P<0.05),Caspase-9蛋白呈高表达,AKT蛋白表达差异无统计学意义(P>0.05)。结论沉默SH2B1基因表达可能通过抑制PI3K/ATK信号通路激活,抑制SGC-7901细胞增殖,促进凋亡。     

Targeting MDR-1 gene expression,BAX/BCL2, caspase-3, and Ki-67 by nanoencapsulated imatinib and hesperidin to enhance anticancer activity and ameliorate cardiotoxicity

There is a great demand to introduce new approaches into cancer treatment field due to incidence of increased breast cancer all over the world. The current study was designed to evaluate the role of imatinib mesylate (IM) and/or hesperidin (HES) nanoparticles alone or in combination in enhancing the anticancer activity and to investigate the ability of nanoencapsulation to reduce cardiotoxicity of IM in solid Ehrlich carcinoma (SEC)-bearing mice. IM and HES were loaded into PLGA (poly(lactic-co-glycolic acid) polymer. SEC was induced in female albino mice as a model for experimentally induced breast cancer. Mice were randomly divided into eight groups (n = 10). On day 28 from tumor inoculation, mice were sacrificed and blood samples were collected in heparinized tubes for hematological studies, biochemical determination of lactate dehydrogenase (LDH), and glutamic oxaloacetic transaminase (SGOT) levels. In addition, tumor and cardiac tissues were utilized for histopathological examination as well as determination of MDR-1 gene expression. Immunohistochemical staining of BAX and BCL-2 was done. Nano IM- and/or Nano HES-treated groups showed a significant reduction in tumor volume, weight, hematological, cardiac markers, and tumor MDR-1 gene downregulation compared to free conventional treated groups. In conclusion, the use of HES as an adjuvant therapy with IM could improve its cytotoxic effects and limit its cardiac toxicity. Furthermore, nanoencapsulation of IM and/or HES with PLGA polymer showed a remarkable anticancer activity.     

The interaction between β1 integrins and ERK1/2 in osteogenic differentiation of human mesenchymal stem cells under fluid shear stress modelled by a perfusion system

Fluid shear stress (FSS) is an important biomechanical factor regulating the osteogenic differentiation of human mesenchymal stem cells (hMSCs) and is therefore widely used in bone tissue engineering. However, the mechanotransduction of FSS in hMSCs remains largely unknown. As β1 integrins are considered to be important mechanoreceptors in other cells, we suspect that β1 integrins should also be important for hMSCs to sense the stimulation of FSS. We used a perfusion culture system to produce FSS loading on hMSCs seeded in PLGA three‐dimensional (3D) scaffolds and investigated the roles of β1 integrins, FAK and ERK1/2 in FSS‐induced osteogenic differentiation of hMSCs. Our results showed that FSS not only markedly increased ALP activity and the expression of ALP, OCN, Runx2 and COLIα genes but also significantly enhanced the phosphorylation of ERK1/2, Runx2 and FAK. FSS‐induced activation of ERK1/2 and FAK was inhibited by blockade of the connection between β1 integrins and ECM with RGDS peptide and integrins β1 monoclonal antibody. Our study also found that FSS could upregulate the expression level of β1 integrins and that this upregulation could be abolished by PD98059. Further investigation indicated that FSS‐activated ERK1/2 led to the phosphorylation of IκBα and NFκB p65. The activation of NFκB p65 resulted in the upregulation of β1 integrin expression. Therefore, it could be inferred that β1 integrins should sense the stimulation of FSS and thus activate ERK1/2 through activating of FAK, and FSS‐activated ERK1/2 feedback to upregulate the expression of β1 integrins through activating NFκB. Copyright © 2012 John Wiley & Sons, Ltd.     

Pharmacological control of the human gastric histamine H2 receptor by famotidine: comparison with H1, H2 and H3 receptor agonists and antagonists

Histamine 0.1 microM-0.1 mM increased adenylate cyclase activity five- to ten-fold in human fundic membranes, with a potency Ka = 3 microM. The histamine dose-response curve was mimicked by the H3 receptor agonist (R) alpha-MeHA, but at 100 times lower potency, Ka = 0.3 mM. Histamine-induced adenylate cyclase activation was abolished by H2, H1 and H3 receptor antagonists, according to the following order of potency IC50: famotidine (0.3 microM) greater than triprolidine (0.1 mM) thioperamide (2 mM), respectively. Famotidine has no action on membrane components activating the adenylate cyclase system, including the Gs subunit of the enzyme stimulated by forskolin and cell surface receptors sensitive to isoproterenol (beta 2-type), PGE2 and VIP. The Schild plot was linear for famotidine (P less than 0.01) with a regression coefficient r = 0.678. The slope of the regression line was 0.64 and differs from unity. Accordingly, famotidine showed a slow onset of inhibition and dissociation from the H2 receptor in human cancerous HGT-1 cells. The results demonstrate that famotidine is a potent and selective H2 receptor antagonist with uncompetitive actions in human gastric mucosa. Consequently, famotidine might be a suitable drug with long-lasting actions in the treatment of Zollinger-Ellison syndrome. The results also confirm and extend the previous observations that (R) alpha-MeHA and thioperamide are two selective ligands at histamine H3 receptor sites. In the human gastric mucosa, these drugs are respectively 330 and 6700 times less potent than histamine and famotidine on the adenylate cyclase system. The possible involvement of histamine H3 receptors in the regulation of gastric secretion is proposed.