慢病毒介导的PRL-3 shRNA对结肠癌细胞增殖、侵袭、凋亡的影响

目的 观察慢病毒介导的 shRNA 沉默肝再生磷酸酶-3（PRL-3）基因对结肠癌 SW480 细胞增殖、侵袭、凋亡的影响。方法 实验分组为空白对照组、阴性对照组、转染组。将携带 PRL-3 shRNA 的慢病毒载体转染结肠癌SW480 细胞,建立稳定沉默 PRL-3 的细胞株,real-time PCR 检测转染后 PRL-3 mRNA 的相对表达水平。采用 MTT法、平板克隆形成实验检测转染后细胞增殖能力;采用 Transwell 侵袭实验、侵袭小室法检测转染后细胞迁移及侵袭能力;采用流式细胞术检测转染后细胞凋亡率变化。结果 稳定沉默 PRL-3 的细胞株构建成功,转染组 PRL-3mRNA 的相对表达水平低于空白对照组、阴性对照组（P＜0.05）,空白对照组、阴性对照组比较差异无统计学意义。PRL-3 shRNA 转染 SW480 细胞 72 h 后,转染组与空白对照组、阴性对照组比较,细胞增殖能力受到抑制,转染 120 h时最明显（P＜0.05）。转染组克隆形成能力较空白对照组、阴性对照组下降（P＜0.05）。转染组与空白对照组、阴性对照组比较,细胞迁移、侵袭能力下降,凋亡率增加（P＜0.05）。结论 结肠癌 SW480 细胞转染 PRL-3 shRNA 可减少 PRL-3 的表达,有效抑制 SW480 细胞增殖,促进其凋亡,PRL-3 可能成为治疗结肠癌的靶基因。     

Killing Effects of IFN R−/− Mouse NK Cells Activated by HN Protein of NDV on Mouse Hepatoma Cells and Possible Mechanism with Syk and NF-κB

The objective of this article is to evaluate whether the tumoricidal activity of mouse IFN R^−/− nature killer (NK) cells is induced by Newcastle disease virus hemagglutinin-neuraminidase (NDV-HN) stimulation, and to investigate what is the mechanism of the HN-stimulated NK cells to kill mouse hepatoma cell line in vitro. The mouse IFN R^−/− NK cells were stimulated for 16 hr with 500 ng/mL NDV-HN in 1640 medium. Quantify the cytotoxic activities of NK cells against mouse hepatoma cells (Hepa1-6) by flow cytometry. Granzymes B (GrB) and Fas/FasL concentrations in the supernatants of IFN R^−/− NK cells medium were determined by specific ELISA assay. The expression of cell surface GrB and Fas was determined by Western blot. NDV-HN stimulation enhanced tumoricidal activity of IFN R^−/− NK cells toward Hepa1-6 in vitro. Treating with anti-HN neutralizing mAb induced significant decline in the cytotoxicity of IFN R^−/− NK cells toward Hepa1-6 cell line (P < 0.05). After treating with anti-HN protein (1 μL/mL), Syk-specific inhibitor Herbimycin A(250 ng/mL) and NF-κB inhibitor PDTC (500 ng/mL) downregulated the tumoricidal activity of HN-stimulated IFN R^−/− NK cells (P < 0.05). Moreover, significant suppressions in the production of GrB and Fas/FasL were observed in HN-stimulated IFN R^−/− NK cells (P < 0.05). Thus, we concluded that killer activation receptors pathway is involved in the IFN-γ-independent GrB and Fas/FasL expression of NDV-HN-stimulated IFN R^−/− NK cells, and these are activated by Syk and NF-κB. Anat Rec, 302:1718–1725, 2019. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association for Anatomy     

Acinetobacter baumannii outer membrane protein A induces HeLa cell autophagy via MAPK/JNK signaling pathway

Autophagy is an evolutionary conserved self-balancing process that plays an important role in maintaining cellular homeostasis via the clearance of damaged organelles and misfolded proteins. Infection-triggered autophagy specifically inhibits the invasion of intracellular bacterial replication and hence protects the cells from microbial infections. It has been reported that Acinetobacter baumannii trigger cell autophagy. However, the role of its virulence protein OmpA remains unclear. Therefore, this study aimed to explore the effects of Acinetobacter baumannii OmpA on cell autophagy and its underlying molecular mechanisms. The results showed that OmpA induced autophagy in HeLa and RAW264.7 cells, increased LC3BII expression, and hindered p62 degradation. Moreover, OmpA triggered incomplete autophagy by interfering the fusion of autophagosomes with lysosomes. Besides, OmpA activated MAPK/JNK signaling pathway and enhanced the phosphorylation levels of JNK, p38, and ERK, c-Jun. Inhibition of JNK signaling pathway suppressed OmpA-induced autophagy in HeLa cells. Ab wild-type strains carrying OmpA triggered incomplete autophagy and resulted in a large number of IL-1β production. Ab-△OmpA strain (OmpA gene mutation) restored autophagic flux and reduced the accumulation of p62 and the release of IL-1β in HeLa cells. Rapamycin activated autophagy to inhibit OmpA-induced IL-1β secretion and protect HeLa cells from inflammatory damage. Collectively, these results suggest that OmpA can induce autophagy in HeLa cells through MAPK/JNK signaling pathway. Pre-treatment with Rapamycin activates autophagy and protects against cell death.     

PACAP deficiency aggravates atherosclerosis in ApoE deficient mice

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) plays an important role in cytoprotection, inflammation and cardiovascular regulation. Thus, we studied the involvement of PACAP in atherogenesis. Differentiated human THP-1 macrophages (MΦ) were stimulated with oxidized low-density lipoproteins (oxLDL) and the influence of PACAP38 treatment on lipid content and TNF release was determined. To test the effect of PACAP deficiency (PACAP^?/?) on the development of atherosclerosis under standard chow (SC) or cholesterol-enriched diet (CED) in vivo, PACAP^?/? mice were crossbred with ApoE^?/? to generate PACAP^?/?/ApoE^?/? mice. Blood cholesterol and triglyceride levels were quantified. Lumen stenosis in the brachiocephalic trunk, cellularity and amounts of pro-inflammatory as well as autophagy-, apoptosis- and necroptosis-relevant proteins were analysed in atherosclerotic plaques by quantitative immunohistochemistry. In vitro, PACAP38 inhibited oxLDL-induced intracellular lipid storage as well as TNF release in MФ. In vivo, after SC, but not under CED, PACAP^?/?/ApoE^?/? mice showed an increased lumen stenosis compared to ApoE^?/? mice. In atherosclerotic plaques of PACAP^?/?/ApoE^?/? mice, the immunoreactive areas of TNF⁺, IL-1β⁺, autophagic, apoptotic and necroptotic cells were increased. In contrast, the overall cell density was decreased compared to ApoE^?/? under SC, while no differences were seen under CED. Similar plasma cholesterol levels were observed in PACAP^?/?/ApoE^?/? and ApoE^?/? mice under the respective feeding regime. Thus, PACAP^?/-/ApoE^?/? mice represent a novel mouse model of accelerated atherosclerosis where CED is not required. Our data indicate that PACAP acts as an endogenous atheroprotective neuropeptide. Thus, stable PACAP agonists may have potential as anti-atherosclerotic therapeutics. The specific PACAP receptor(s) mediating atheroprotection remain(s) to be identified.     

Common fragile site instability in normal cells: Lessons and perspectives

Mechanisms and events related to common fragile site (CFS) instability are well known in cancer cells. Here, we argue that normal cells remain an important experimental model to address questions related to CFS instability in the absence of alterations in cell cycle and DNA damage repair pathways, which are common features acquired in cancer. Furthermore, a major gap of knowledge concerns the stability of CFSs during gametogenesis. CFS instability in meiotic or postmeiotic stages of the germ cell line could generate chromosome deletions or large rearrangements. This in turn can lead to the functional loss of the several CFS‐associated genes with tumor suppressor function. Our hypothesis is that such mutations can potentially result in genetic predisposition to develop cancer. Indirect evidence for CFS instability in human germ cells has been provided by genomic investigations in family pedigrees associated with genetic disease. The issue of CFS instability in the germ cell line should represent one of the future efforts, and may take advantage of the existence of sequence and functional conservation of CFSs between rodents and humans.     

复发或难治性非小细胞肺癌的化疗进展

对含铂类药物治疗后复发的非小细胞肺癌,单药泰索帝为标准的二线化疗药物,可明显延长病人的生存时间及改善生活质量.一项泰索帝的Ⅲ期随机对照研究证实,多靶点叶酸抑制剂的抗代谢药Alimta二线化疗治疗非小细胞肺癌,其疗效、中位生存时间及1年生存率与泰索帝相似,可替代泰索帝成为晚期NSCLC的二线化疗药物.泰索帝与健择、卡铂、伊立替康、EGFR-TK抑制剂及抗血管生成剂联合化疗,有一定的疗效.TXT为复发的NSCLC常用的二线标准治疗方案.Alimta为备用的二线治疗方案.Iressa单药对铂类及泰索帝失败后的晚期非小细胞肺癌仍然有效.     

121例二线抗结核药品不良反应分析

目的探讨二线抗结核药品药品不良反应(ADR)发生的特点。方法从某院2017年度ADR报告汇总表中选取二线抗结核药品所致ADR患者的病历,对不同性别、年龄的患者ADR发生情况,ADR类型以及患者各种ADR所涉及的器官系统及其主要临床表现、转归数据信息进行统计分析。结果报告的121例二线抗结核药品ADR患者中,男性占比较高(72例,59. 50%),年龄 60岁者居多(70例,57. 85%)。引发ADR的药品剂型主要是注射剂型(80例,66. 12%),主要药品是莫西沙星(38例,31. 40%)和左氧氟沙星(29例,23. 96%)。ADR累及的器官系统以全身性为主(69/128,53. 91%),其次是中枢及外周神经系统(22/128,17. 19%)。严重的ADR患者9例(7. 44%),痊愈和好转的ADR患者118例(97. 52%)。结论使用二线抗结核药品ADR多发生于老年患者,尤其以喹诺酮类药品的全身性损害和中枢及外周神经系统损害为主。在诊治过程中,应及时对高危人群和主要治疗药品进行ADR监测和主动干预,并作出应对处理。     

温针灸对类风湿性关节炎大鼠血清免疫球蛋白及滑膜组织中VCAM-1的影响

目的:观察温针灸对类风湿性关节炎(Rheumatoid arthritis,RA)大鼠血清免疫球蛋白及滑膜组织中血管细胞黏附分子-1(vascular cell adhesion molecule-1,VCAM-1)的影响,探讨温针灸改善类风湿性关节炎的作用机制。方法:选取清洁级3月龄SD健康大鼠60只,雌雄各半,体质量(208±4.5)g,先适应性喂养1周,然后按照数字表法随机抽取15只为正常组进行常规喂养,其余大鼠进行模型复制,并随机分为模型组、针刺组和温针灸组,每组15只。采用皮内注射牛Ⅱ型胶原接种诱发制备大鼠类风湿性关节炎模型。温针灸组于造模后第1天取"足三里""肾俞"和"悬钟"温针灸治疗,采用手针,刺激量以大鼠耐受为度,日1次,共治疗21 d。针刺组针刺方法同温针灸组,不用艾灸。模型组不给予任何处理。疗程结束后滑膜取材,采用MD-100自动生化分析仪检测血清中IgG、IgM和IgA的含量,以免疫组化法检测VCAM-1在滑膜组织中的表达。结果:与正常组比较,模型组大鼠血清IgG、IgM、IgA含量和滑膜组织中VCAM-1表达水平显著升高(P0.05);与模型组比较,温针灸组和针刺组大鼠血清IgG、IgM、IgA含量和滑膜组织中VCAM-1表达水平显著降低(P0.05);与针刺组比较,温针灸组大鼠血清IgG、IgM、IgA含量和滑膜组织中VCAM-1表达水平降低幅度更明显(P0.05)。结论:温针灸可升高RA大鼠血清IgG、IgM和IgA的含量,抑制VCAM-1在滑膜组织中的表达,可能是温针灸通过调节RA大鼠慢性炎症以改善关节损伤的调节机制之一。     

质量源于设计在银杏叶片制粒工艺中的应用(Ⅲ):基于设计空间的过程控制策略

该文在质量源于设计(QbD)理念的指导下,建立基于设计空间的银杏叶片高速剪切湿法制粒工艺控制策略,提高过程质量可控性和产品质量一致性。以颗粒中间体的中值粒径(D_(50))和松装密度(D_a)为关键质量属性(CQAs),采用失败模式和效应分析(FMEA)辨识潜在关键工艺参数(pCPPs)。采用Plackett-Burmann设计对潜在关键工艺参数进行筛选,确定黏合剂用量、湿混时间和湿混搅拌桨转速为关键工艺参数(CPPs)。在关键工艺参数范围内,采用Box-Behnken设计和二次多项式回归模型开发工艺设计空间。ANOVA分析显示回归模型的P0.05,且失拟值0.1,表明其可较好地定量描述CQAs和CPPs之间的关系。设计空间内任一CPPs组合均能分别将颗粒D_(50)和D_a控制在170~500μm和0.30~0.44 g·cm~(-3),进而满足银杏叶片机械性质要求。