Overexpression of cytoglobin gene inhibits hypoxic injury to SH-SY5Y neuroblastoma cells

A plasmid for cytoglobin expression, pAcGFP1-C1-cytoglobin, was transfected into SH-SY5Y cells. Cobalt chloride was used to establish a model of hypoxia. Western blotting indicated that cytoglobin was overexpressed and there was low expression of hypoxia-inducible factor-1α in SH-SY5Y cells after transfection. Following cobalt chloride-induced hypoxia, cytoglobin and hypoxia-inducible fac-tor-1α expression gradually increased in SH-SY5Y cells. Flow cytometry showed that with increas-ing duration of hypoxia, the proportion of normal cells significantly diminished in the transfected and non-transfected groups. The proportion of cells in the early stages of apoptosis increased. However, the proportion of apoptotic cells was significantly lower in the transfected group compared with the non-transfected group. These results demonstrate that cytoglobin and hypoxia-inducible factor-1α are strongly up-regulated by hypoxia, and that there is a strong relationship between hy-poxia-inducible factor-1α and cytoglobin during hypoxic injury.     

丹参饮合温胆汤加减对稳定型心绞痛患者心肌缺血的保护机制

目的:观察丹参饮合温胆汤加减治疗痰瘀阻滞型稳定型心绞痛(SAP)的疗效及对心肌缺血的保护机制研究。方法:将132例患者随机按数字表法分为对照组和观察组各66例。除去脱落、失访和剔除病例,两组最后分别完成63例。所有患者进行抗心绞痛药物和控制风险因素药物治疗。对照组口服丹蒌片,5片/次,3次/d;观察组给予丹参饮合温胆汤加减,1剂/d;两组疗程均为治疗3个月。每周进行心绞痛发作情况评分;冠心病心肌缺血情况采用心电图平板运动试验评价,中医症状和生活质量分别进行痰瘀阻滞证、西雅图心绞痛量表(SAQ)评分,血液流变学指标、白细胞介素-6(IL-6),肿瘤坏死因子-α(TNF-α),细胞间黏附分子-1(ICAM-1),胱抑素C(CysC),同型半胱氨酸(Hcy),缺血修饰白蛋白(IMA)和巨噬细胞移动抑制因子(MIF)水平,均治疗前后各评价1次;并进行安全性评价。结果:观察组心绞痛发作次数、持续时间、疼痛程度和硝酸甘油用量评分均低于对照组(P<0.01);观察组Duke评分、总运动时间、出现ST段压低1.0 mm的时间、心绞痛出现时间和代谢当量均多于对照组(P<0.01);观察组SAQ评分高于对照组(P<0.01),痰瘀阻滞证积分低于对照组(P<0.01);观察组心绞痛疗效优于对照组(Z=2.091,P<0.05);观察组ICAM-1,CysC,IL-6,TNF-α水平均低于对照组(P<0.01);观察组全血黏度(低切、高切)、全血还原黏度、血浆黏度、血小板聚集率、纤维蛋白原(FIB)均低于对照组(P<0.01);观察组IMA,Hcy和MIF水平均低于对照组(P<0.01)。没有发现服用丹参饮合温胆汤相关不良反应。结论:在西医常规治疗的基础上,丹参饮合温胆汤加减治疗SAP,可控制心绞痛发作,减轻痰瘀阻滞证症状,提高生活质量,有着较好临床疗效,且安全,并能改善患者血液流变性,抑制炎症反应,减轻管腔狭窄或阻塞,从而改善心肌缺血程度。     

NRAGE影响人食管癌细胞放射抗性的作用及其机制研究

背景与目的：食管癌是全球威胁人类生命和健康的常见恶性肿瘤之一，中国每年食管癌发病人数占全球发病总人数的一半以上，以食管鳞癌最为常见。放疗是食管癌三大治疗手段之一，而放射抗性是导致其治疗失败的主要原因。神经营养因子受体相互作用MAGE类药物（neurotrophin receptor-interacting MAGE homolog，NRAGE）在放射抗性细胞株TE13R120中表达量明显高于亲本TE13细胞，且NRAGE亚细胞定位变化可能参与食管癌细胞放射抗性的形成。通过基因转染构建NRAGE稳定表达的食管癌细胞系，以进一步明确NRAGE基因与食管鳞癌细胞放射抗性的关系，分析该基因影响放射抗性的具体机制。方法：通过基因转染构建NRAGE稳定表达的食管癌细胞系。采用细胞克隆形成实验检测细胞放射敏感性，采用流式细胞术检测细胞周期及凋亡；细胞划痕、Transwell侵袭实验检测细胞迁移、侵袭能力，采用实时荧光定量聚合酶链反应（real-time fluorescence quantitive polymerase chain reaction，RTFQ-PCR）和蛋白质印迹法（Western blot）检测细胞中β-catenin表达情况。组间差异采用t检验或方差分析。结果：实验分为转染过表达组（Eca109/NRAGE组）和空白对照组（Eca109组）。Eca109/NRAGE细胞中NRAGE的表达量明显高于Eca109（t=29.65，P<0.05）。照射后Eca109/NRAGE细胞的放射抗性显著高于Eca109。流式细胞术检测结果显示，Eca109/NRAGE细胞中对射线抵抗性最强的S期细胞比例增加，对射线最敏感的G ₂ /M期减少。Eca109/NRAGE细胞凋亡率较Eca109细胞降低（t=3.268，P<0.05）。Eca109/NRAGE细胞迁移和侵袭能力均高于Eca109。RTFQ-PCR和Western blot检测结果显示，β-catenin的mRNA表达及蛋白水平在Eca109/NRAGE细胞中明显高于Eca109（t=15.87，P<0.05）。结论：NRAGE参与Eca109细胞放射抗性的形成，改变细胞的细胞周期分布和凋亡情况，影响细胞迁移及侵袭能力并可能影响食管癌细胞的放射敏感性，该作用可能与激活Wnt/β-catenin信号转导通路有关。     

Targeting the PI3K/AKT/mTOR pathway in biliary tract cancers: A review of current evidences and future perspectives

Biliary tract cancers (BTCs) are a group of invasive neoplasms, with increasing incidence and dismal prognosis. In advanced disease, the standard of care is represented by first-line chemotherapy with cisplatin and gemcitabine. In subsequent lines, no clear recommendations are currently available, highlighting the need for novel therapeutic approaches.The PI3K/AKT/mTOR pathway is a core regulator of cell metabolism, growth and survival, and is involved in BTCs carcinogenesis and progression. Mutations, gene copy number alterations and aberrant protein phosphorylation of PI3K, AKT, mTOR and PTEN have been thoroughly described in BTCs and correlate with poor survival outcomes.Several pre-clinical evidences state the efficacy of PI3K/AKT/mTOR pathway inhibitors in BTCs, both in vitro and in vivo. In the clinical setting, initial studies with rapamycin analogs have shown interesting activity with an acceptable toxicity profile. Novel strategies evaluating AKT and PI3K inhibitors have risen serious safety concerns, pointing out the need for improved patient selection and increased target specificity for the clinical development of these agents, both alone and in combination with chemotherapy.This review extensively describes the role of the PI3K/AKT/mTOR pathway in BTCs and examines the rationale of its targeting in these tumors, with particular focus on clinical activity, toxicities and perspectives on further development of PI3K/AKT/mTOR pathway inhibitors.     

Coronary Artery Disease in Patients ≥80 Years of Age

Coronary artery disease (CAD) is a major cause of morbidity and mortality in patients ≥80 years of age. Nonetheless, older patients have typically been under-represented in cardiovascular clinical trials. Understanding the pathophysiology, epidemiology, and optimal means of diagnosis and treatment of CAD in older adults is crucial to improving outcomes in this high-risk population. A patient-centered approach, taking into account health status, functional ability and frailty, cognitive skills, and patient preferences is essential when caring for older adults with CAD. The present systematic review focuses on the current knowledge base, gaps in understanding, and directions for future investigation pertaining to CAD in patients ≥80 years of age.     

Bacterial ghosts as a novel advanced targeting system for drug and DNA delivery

Although there are powerful drugs against infectious diseases and cancer on the market, delivery systems are needed to decrease serious toxic and noncurative side effects. In order to enhance compliance, several delivery systems such as polymeric micro- and nanoparticles, liposomal systems and erythrocyte ghosts have been developed. Bacterial ghosts representing novel advanced delivery and targeting vehicles suitable for the delivery of hydrophobic or water-soluble drugs, are the main focus of this review. They are useful nonliving carriers, as they can carry different active substances in more than one cellular location separately and simultaneously. Bacterial ghosts combine excellent natural or engineered adhesion properties with versatile carrier functions for drugs, proteins and DNA plasmids or DNA minicircles. The simplicity of both bacterial ghost production and packaging of drugs and/or DNA makes them particularly suitable for the use as a delivery system. Further advantages of bacterial ghost delivery vehicles include high bioavailability and a long shelf life without the need of cold-chain storage due to the possibility to freeze-dry the material.     

Antagomir-30d对大鼠骨髓基质干细胞成骨分化的影响

目的：探讨miR-30d拮抗剂（antagomir-30d）正性调控大鼠骨髓基质干细胞（BMSCs）的成骨分化作用并确认转染的最佳浓度。方法：BMSCs培养并分组，分为不同浓度的antagomir-30d（antagomir-30d组）、阴性对照（NC组）和未做转染的BMSCs（空白组）；将不同浓度的antagomir-30d及其NC转染至BMSCs后进行成骨诱导。通过RT-PCR技术进行比较，测定成骨基因碱性磷酸酶（ALP）、骨钙素（OC）、Runt相关转录因子2（RUNX2）的mRNA的表达情况，并确定最佳转染浓度及其转染效率。通过ALP染色验证antagomir-30d对成骨分化的调控作用。结果：RT-PCR结果表明，antagomir-30d体外可以促进BMSCs的成骨分化。不同浓度antagomir-30d组的成骨基因表达量随浓度变化而改变，当antagomir-30d转染浓度为150 nmol/L时，ALP、OC、RUNX2的mRNA水平均显著高于空白组和NC组（P＜0.05）。Antagomir-30d的转染最佳浓度为150 nmol/L，此时ALP染色结果显示其活性最高，成骨分化作用好。结论：Antagomir-30d体外正性调控BMSCs的成骨分化，其转染至BMSCs的最佳浓度为150 nmol/L。     

Accelerated mass production of influenza virus seed stocks in HEK-293 suspension cell cultures by reverse genetics

Despite major advances in developing capacities and alternative technologies to egg-based production of influenza vaccines, responsiveness to an influenza pandemic threat is limited by the time it takes to generate a Candidate Vaccine Virus (CVV) as reported by the 2015 WHO Informal Consultation report titled “Influenza Vaccine Response during the Start of a Pandemic”.In previous work, we have shown that HEK-293 cell culture in suspension and serum free medium is an efficient production platform for cell culture manufacturing of influenza candidate vaccines. This report, took advantage of, recombinant DNA technology using Reverse Genetics of influenza strains, and advances in the large-scale transfection of suspension cultured HEK-293 cells. We demonstrate the efficient generation of H1N1 with the PR8 backbone reassortant under controlled bioreactor conditions in two sequential steps (transfection/rescue and infection/production). This approach could deliver a CVV for influenza vaccine manufacturing within two-weeks, starting from HA and NA pandemic sequences. Furthermore, the scalability of the transfection technology combined with the HEK-293 platform has been extensively demonstrated at >100 L scale for several biologics, including recombinant viruses.Thus, this innovative approach is better suited to rationally engineer and mass produce influenza CVV within significantly shorter timelines to enable an effective global response in pandemic situations.     

恶性肿瘤治疗中维和策略的实施与思考

健康是一种和谐的状态,是一些成对的相反因素之间的平衡,中医学谓之"阴平阳秘,精神乃治"。人体内外环境失和,脏腑阴阳气血失调,正气亏虚,加之外邪、情志、饮食等多种因素作用于人体,就会出现虚、痰、瘀、毒等病理改变,导致肿瘤的发生发展。晚期肿瘤不能手术,不能耐受放化疗,但并非意味着"不治",可通过一定的方法和手段,维持人体内环境的相对和谐稳定,使肿瘤失去生长的环境,调动人体自身的抗病能力达到抑制肿瘤生长的目的,使患者长期带瘤生存。常用的治法有补法、消法、和法等,一般而言,发病早中期正盛邪实,正气尚盛,消法为主以祛邪;中后期正虚邪稽,正气衰败,注重补法以扶正;放化疗期则以和法为主。临床表现复杂多样,具体治法运用必须知常达变,消中有补,补中有消,和中有消,消中有和,从而获得预期效果。