槟榔醇提物对H9C2心肌细胞的抗缺氧保护作用

目的 研究槟榔无水醇提物对低氧H9C2心肌细胞的作用及其保护机制。方法 建立H9C2心肌细胞低氧模型,CCK-8法检测槟榔无水醇提物对H9C2细胞存活率的影响;通过检测细胞内丙二醛（MDA）含量,过氧化物歧化酶（SOD）、还原型谷胱甘肽（GSH）活力,研究槟榔无水醇提物对H9C2细胞的保护作用;RT-PCR法检测细胞内Nrf2、Caspase-3 mRNA水平的表达,从分子水平研究槟榔无水醇提物对低氧H9C2细胞的保护机制。结果 与常氧对照组相比,低氧24 h时细胞存活为28.46%（P<0.01）,细胞内MDA含量升高44.33%（P<0.05）,SOD、GSH活力分别下降16.18%、30.64%（P<0.05或P<0.01）。RT-PCR实验结果表明,Nrf2被激活,其mRNA表达上升1.74倍（P<0.05）。与低氧模型组相比,槟榔无水醇提物处理组H9C2细胞存活率明显升高（P<0.01）,且呈现剂量依赖性,槟榔无水醇提物处理组细胞内SOD、GSH活力分别增加14.90%、28.94%（P<0.05）,Nrf2基因mRNA相对表达下降66%（P<0.05）。结论 槟榔无水醇提物对低氧H9C2细胞有显著的保护作用,其保护机制可能与提高细胞内抗氧化物酶活力,减轻细胞氧化应激损伤,提高细胞耐低氧能力有关。     

丁香酚对H2O2诱导H9C2心肌细胞损伤的保护作用

目的 研究丁香酚对H₂O₂诱导H9C2心肌细胞损伤的影响。方法 使用不同浓度H₂O₂建立H9C2心肌细胞氧化损伤模型,将建立好的心肌细胞氧化损伤模型分为对照组、50 μg/mL丁香酚处理组、100 μg/mL丁香酚处理组和200 μg/mL丁香酚处理组,采用MTT法观察吸光度值（OD）变化来评价丁香酚对H9C2心肌细胞活力的影响;采用Hoechst染色法观察细胞核形态变化,评价丁香酚对H9C2心肌细胞凋亡的影响;为进一步研究丁香酚的抗氧化损伤作用,采用比色法检测试剂盒观察丁香酚对H9C2心肌细胞乳酸脱氢酶（LDH）、丙二醛（MDA）及超氧化物歧化酶（SOD）含量的影响。结果 400 μmol/L H₂O₂显著降低H9C2细胞活力,增加细胞凋亡,适宜建立H9C2心肌细胞氧化损伤模型。100 μg/mL和200 μg/mL的丁香酚增加H9C2细胞活力,减少细胞凋亡（P<0.05）;同时,H₂O₂诱导H9C2细胞LDH及MDA含量增加和SOD含量减少的效应也可被100 μg/mL和200 μg/mL的丁香酚抑制。结论 丁香酚对H₂O₂诱导H9C2心肌细胞损伤具有保护作用。     

泽漆抑制三阴乳腺癌MDA-MB-231细胞及其凋亡机制研究

目的 研究泽漆对三阴乳腺癌MDA-MB-231细胞的作用及其作用机制。方法 用MTT法检测细胞活力;用荧光显微镜法测定MDA-MB-231细胞的活性氧（ROS）生成量;用流式细胞仪检测细胞的凋亡率;用TUNEL检测法检测细胞凋亡DNA碎片;用Western blot检测caspase-9、caspase-3、PARP等凋亡相关因子的水平变化。结果 MTT试验显示泽漆提取物对MDA-MB-231细胞具有显著的抑制作用,但作用可被ROS抑制剂NAC及caspase抑制剂Z-VAD-FMK所消除;荧光显微镜检测显示泽漆提取物能显著提高ROS的生成;流式细胞仪检测显示泽漆提取物处理后,PI染色阳性细胞明显增加,但被NAC减弱。Caspase-9、caspase-3在提取物处理后均转为激活形式,PARP被剪切。TUNEL法显示,提取物处理后细胞凋亡碎片明显增多,而提前加入ROS抑制剂NAC和caspase抑制剂Z-VAD-FMK能使泽漆提取物诱导凋亡的DNA碎片明显减少。结论 泽漆乙酸乙酯提取物可以有效抑制MDA-MB-231细胞的生长,诱导其凋亡,其作用机制可能与ROS过量生成所致的线粒体损伤途径有关。     

新型化合物FK106诱导白血病细胞凋亡的作用研究

目的 研究新型化合物FK106对白血病细胞增殖的抑制作用及其可能的机制。方法 采用噻唑蓝（MTT）法检测FK106对白血病MV4-11细胞存活率的影响,采用PI染色流式细胞仪检测FK106对细胞周期的影响,通过Annexin V-FITC/PI 双染流式细胞仪检测FK106对细胞凋亡的影响,采用Western blot检测凋亡相关蛋白表达的影响。结果 FK106对白血病细胞MV4-11的增殖具有抑制作用,且在0.1~1.0 mmol·L^-1浓度范围内,对白血病MV4-11的抑制率具有浓度、时间依赖关系;FK106能阻滞 MV4-11细胞G₀/G₁期;随着FK106浓度的增加,MV4-11细胞凋亡率呈现增高的趋势,并呈剂量依赖性;FK106可以抑制 Bcl-2和Fas mRNA的表达,同时Bax和Caspase-3 mRNA表达明显增加,以上均呈依赖性。结论 FK106能抑制人白血病细胞MV4-11细胞的增殖,并诱导其发生凋亡,可能通过Bcl-2和Fas途径诱导MV4-11细胞凋亡,具有较好的临床应用前景。     

丁苯酞对神经干细胞向神经细胞分化的促进作用

目的 研究丁苯酞通过PI3K-AKT信号通路促进神经干细胞向神经细胞分化的作用。方法 分离培养SD大鼠胚胎脑皮质层的神经干细胞,采用分化专用完全培养基培养后,通过光镜下观察和Nestin蛋白免疫荧光法进行鉴定。设置对照组和实验组,其中实验组添加丁苯酞辅助分化,分为丁苯酞低剂量组（1 μmol·L^-1）、中剂量组（5 μmol·L^-1）和高剂量组（10 μmol·L^-1）,作用24 h后,CCK-8法检测神经干细胞的细胞活力,碱性磷酸酶染色法检测神经干细胞的分化能力,免疫荧光法检测神经细胞分化程度,RT-qPCR法检测转录因子SOX2、PAX6和NeuN的mRNA表达水平,Western-blot及RT-qPCR检测PI3K-AKT信号通路的表达。结果 丁苯酞干预后神经干细胞活力提高,分化能力提高,碱性磷酸酶试验呈强阳性,转录因子SOX2、PAX6和NeuN的mRNA表达水平增高,PI3K-AKT信号通路蛋白和mRNA表达水平均增高,且呈现剂量依赖性。结论 丁苯酞可以促进神经干细胞向神经细胞分化,其作用机制可能与PI3K-AKT的激活有关。     

羽扇豆醇抑制人肝癌Huh-7细胞增殖机制研究

摘 要 目的：研究羽扇豆醇（lupeol）对人肝癌Huh-7细胞增殖的影响。方法: 运用CCK-8法检测lupeol（10~150 μmol·L^-1）对T24细胞活力的影响;运用caspase抑制剂确证凋亡相关蛋白caspase是否与lupeol对细胞增殖抑制作用有关;运用western blot检测lupeol对c-Jun蛋白表达的影响;运用real time PCR评价lupeol对miR-21、MMP-2、MMP-9基因mRNA表达的影响;运用pGL3 Basic荧光素酶报告系统研究lupeol对MMP-9启动子活性的影响。结果: Lupeol在72 h能够抑制Huh-7细胞增殖,该抑制作用具有剂量依赖性;lupeol处理细胞72 h后半抑制浓度（IC50）为80 μmol·L^-1;与对照组相比,运用lupeol能够使Huh-7细胞中c-Jun蛋白表达下调;lupeol能够降低miR-21表达水平;与对照组相比,lupeol处理Huh-7细胞降低MMP-9的mRNA表达,并使MMP-9基因启动子活性下调40%;以上结果与对照组比较差异均有统计学意义（P<0.05）。结论: Lupeol能够抑制人肝癌Huh-7细胞增殖,该作用与抑制miR-21/c-Jun/MMP-9信号通路有关。     

白藜芦醇通过抑制Akt/mTOR信号通路活性促进肺癌细胞自噬发挥抗肿瘤效应的研究

目的 探讨白藜芦醇对肺癌细胞生长活性的抑制效应及可能的机制。方法 稳定培养肺癌细胞系H292,分别加入含10、20、40 μmol·L^-1白藜芦醇的培养基进行干预,采用CCK-8、流式细胞术、细胞集落形成实验检测白芦藜醇对H292细胞生长活性的影响,采用免疫荧光、Western blotting检测白藜芦醇对细胞Akt/mTOR信号通路相关蛋白的影响。结果 （1） CCK-8检测结果显示,白藜芦醇对H292细胞增殖有明显抑制作用（P<0.05）,且呈时间和剂量依赖性,同时对H292细胞周期有明显影响,能阻滞细胞周期于G₂/M期。（2）白藜芦醇可明显抑制H292细胞集落形成（P<0.05）,并呈剂量依赖性。（3） Western blotting检测结果显示,白藜芦醇以剂量依赖的方式诱导H292细胞中LC3-II的累积。免疫荧光检测发现,白藜芦醇可诱导细胞核和线粒体中GFP-LC3显著增加。（4）自噬抑制剂3-MA能有效减弱白藜芦醇对H292细胞活力的抑制作用,溶酶体酸化抑制剂Bafilomycin A1则可促进白藜芦醇导致的LC3-II累积。（5）白藜芦醇可下调H292细胞中p-Akt、p-P70S6K、p-mTOR蛋白的表达（P<0.05）,而对Akt、P70S6K、mTOR蛋白的表达无明显影响（P>0.05）。结论 白藜芦醇可通过抑制Akt/mTOR信号通路抑制肺癌细胞的增殖。     

颗粒状角膜营养不良Ⅱ型家系的TGFBI基因筛查

目的 对颗粒状角膜营养不良II型（GCD2）的家系进行TGFBI基因筛查，寻找患病原因。方法 对GCD2家系患者TGFBI基因的所有外显子进行突变检测，以GCD2家系中的健康成员和100例无亲缘关系的正常志愿者DNA样本作为对照。结果 GCD2家系所有患者TGFBI基因4号外显子的第370位碱基发生纯合或杂合的G>A突变，导致TGFBI基因编码的蛋白质的第124位氨基酸R变为H。结论 错义纯合和杂合R124H突变是导致GCD2家系角膜营养不良不同临床表型的主要病因。     

蛇床子素联合肿瘤坏死因子诱导凋亡配体对乳腺癌干细胞的杀伤效应

目的 探讨蛇床子素联合肿瘤坏死因子诱导凋亡配体[tumor necrosis factor （TNF）-related apoptosis inducing ligand,TRAIL]对乳腺癌干细胞的杀伤效应及机制。方法 用TRAIL及蛇床子素体外处理MCF-7乳腺癌非干细胞及MCF-7乳腺癌干细胞,MTT法检测乳腺癌细胞的细胞活力;流式细胞术检测乳腺癌细胞的凋亡;Western blot试验检测caspase-9和caspase-3的活化,凋亡酶激活因子（apoptotic protease activating facter-1,Apaf-1）的表达水平,细胞色素C的释放;免疫共沉淀法检测Apaf-1和caspase-9前体的相互作用。结果 MCF-7肿瘤干细胞对TRAIL的敏感性显著低于MCF-7非干细胞。在MCF-7肿瘤干细胞的培养体系中加入蛇床子素能显著提高TRAIL对MCF-7肿瘤干细胞的细胞活力抑制率。Western blot实验结果表明,蛇床子素能显著上调MCF-7肿瘤干细胞中Apaf-1的表达水平,但不影响细胞色素C的释放。在MCF-7肿瘤干细胞中转染Apaf-1 siRNA后,蛇床子素联合TRAIL对MCF-7肿瘤干细胞的协同杀伤活性受到显著抑制。另外,免疫共沉淀实验结果表明,蛇床子素联合TRAIL能显著诱导MCF-7肿瘤干细胞中Apaf-1与caspase-9前体的相互作用,并使之发生活化。结论 蛇床子素通过上调Apaf-1的表达促进TRAIL对乳腺癌干细胞caspase-9的活化,从而增强TRAIL对乳腺癌干细胞的凋亡诱导效应。     

成球培养法富集胰腺癌肿瘤干细胞的研究

目的 研究成球培养法培养胰腺癌肿瘤干细胞的可行性。方法 通过细胞成球培养体系富集胰腺癌肿瘤干细胞并进行培养,流式细胞术检测分析干细胞的表型特征。结果 光镜下,成球培养细胞形态发生明显变化,细胞由贴壁时的多边形变成圆形,并形成肿瘤细胞微球。流式细胞仪检测发现,与普通细胞培养相比,成球培养体系富集的胰腺癌肿瘤干细胞干性相关基因CD24、CD44共表达水平明显升高。结论 利用成球培养法能够富集得到CD24、CD44高表达的胰腺癌肿瘤干细胞。     

A zwitterionic polymer-inspired material mediated efficient CRISPR-Cas9 gene editing

The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR/Cas9) adaptive immune system is a cutting-edge genome-editing toolbox. However, its applications are still limited by its inefficient transduction. Herein, we present a novel gene vector, the zwitterionic polymer-inspired material with branched structure (ZEBRA) for efficient CRISPR/Cas9 delivery. Polo-like kinase 1 (PLK1) acts as a master regulator of mitosis and overexpresses in multiple tumor cells. The Cas9 and single guide sgRNA (sgRNA)-encoded plasmid was transduced to knockout Plk1 gene, which was expected to inhibit the expression of PLK1. Our studies demonstrated that ZEBRA enabled to transduce the CRISPR/Cas9 system with large size into the cells efficiently. The transduction with ZEBRA was cell line dependent, which showed ～10-fold higher in CD44-positive cancer cell lines compared with CD44-negative ones. Furthermore, ZEBRA induced high-level expression of Cas9 proteins by the delivery of CRISPR/Cas9 and efficient gene editing of Plk1 gene, and inhibited the tumor cell growth significantly. This zwitterionic polymer-inspired material is an effective and targeted gene delivery vector and further studies are required to explore its potential in gene delivery applications.     

Delivery approaches for CRISPR/Cas9 therapeutics in vivo: advances and challenges

Introduction: Therapeutic gene editing is becoming a viable biomedical tool with the emergence of the CRISPR/Cas9 system. CRISPR-based technologies have promise as a therapeutic platform for many human genetic diseases previously considered untreatable, providing a flexible approach to high-fidelity gene editing. For many diseases, such as sickle-cell disease and beta thalassemia, curative therapy may already be on the horizon, with CRISPR-based clinical trials slated for the next few years. Translation of CRISPR-based therapy to in vivo application however, is no small feat, and major hurdles remain for efficacious use of the CRISPR/Cas9 system in clinical contexts.

Areas covered: In this topical review, we highlight recent advances to in vivo delivery of the CRISPR/Cas9 system using various packaging formats, including viral, mRNA, plasmid, and protein-based approaches. We also discuss some of the barriers which have yet to be overcome for successful translation of this technology.

Expert opinion: This review focuses on the challenges to efficacy for various delivery formats, with specific emphasis on overcoming these challenges through the development of carrier vehicles for transient approaches to CRISPR/Cas9 delivery in vivo.     

Cas9 Ribonucleoprotein Complex Delivery: Methods and Applications for Neuroinflammation

The CRISPR/Cas9 system is a revolutionary gene editing technology that combines simplicity of use and efficiency of mutagenesis. As this technology progresses toward human therapies, valid concerns including off-target mutations and immunogenicity must be addressed. One approach to address these issues is to minimize the presence of the CRISPR/Cas9 components by maintaining a tighter temporal control of Cas9 endonuclease and reducing the time period of activity. This has been achieved to some degree by delivering the CRISPR/Cas9 system via pre-formed Cas9 + gRNA ribonucleoprotein (RNP) complexes. In this review, we first discuss the molecular modifications that can be made using CRISPR/Cas9 and provide an overview of current methods for delivering Cas9 RNP complexes both in vitro and in vivo. We conclude with examples of how Cas9 RNP delivery may be used to target neuroinflammatory processes, namely in regard to viral infections of the central nervous system and neurodegenerative diseases. We propose that Cas9 RNP delivery is a viable approach when considering the CRISPR/Cas9 system for both experimentation and the treatment of disease.

Graphical Abstract

     

Effective PEI-mediated delivery of CRISPR-Cas9 complex for targeted gene therapy

The-state-of-art CRISPR/Cas9 is one of the most powerful among the approaches being developed to rescue fundamental causes of gene-based inheritable diseases. Several strategies for delivering such genome editing materials have been developed, but the safety, efficacy over time, cost of production, and gene size limitations are still under debate and must be addressed to further improve applications. In this study, we evaluated branched forms of the polyethylenimine (PEI) – branched PEI 25 kDa (BPEI-25K) – and found that it could efficiently deliver CRISPR/Cas9 plasmids. Plasmid DNA expressing both guide RNA and Cas9 to target the Slc26a4 locus was successfully delivered into Neuro2a cells and meditated genome editing within the targeted locus. Our results demonstrated that BPEI-25K is a promising non-viral vector to deliver the CRISPR/Cas9 system in vitro to mediate targeted gene therapy, and these findings contribute to an understanding of CRISPR/Cas9 delivery that may enable development of successful in vivo techniques.     

The development of high-content screening (HCS) technology and its importance to drug discovery

ABSTRACT

Introduction: High-content screening (HCS) was introduced about twenty years ago as a promising analytical approach to facilitate some critical aspects of drug discovery. Its application has spread progressively within the pharmaceutical industry and academia to the point that it today represents a fundamental tool in supporting drug discovery and development.

Areas covered: Here, the authors review some of significant progress in the HCS field in terms of biological models and assay readouts. They highlight the importance of high-content screening in drug discovery, as testified by its numerous applications in a variety of therapeutic areas: oncology, infective diseases, cardiovascular and neurodegenerative diseases. They also dissect the role of HCS technology in different phases of the drug discovery pipeline: target identification, primary compound screening, secondary assays, mechanism of action studies and in vitro toxicology.

Expert opinion: Recent advances in cellular assay technologies, such as the introduction of three-dimensional (3D) cultures, induced pluripotent stem cells (iPSCs) and genome editing technologies (e.g., CRISPR/Cas9), have tremendously expanded the potential of high-content assays to contribute to the drug discovery process. Increasingly predictive cellular models and readouts, together with the development of more sophisticated and affordable HCS readers, will further consolidate the role of HCS technology in drug discovery.     

烟酰胺磷酸核糖转移酶抑制剂的设计合成及抗肿瘤细胞增殖活性

目的寻找新的烟酰胺磷酸核糖转移酶（Nampt）抑制剂,为进一步药物设计提供依据。方法以进入临床研究的APO866为先导化合物,设计合成一系列Nampt抑制剂。以哌嗪为原料,经取代、SN2反应、Gabriel反应、成胍、环合等多步反应得到关键中间体（7a7,b）,该中间体再与酰氯衍生物反应,最终合成目标化合物。结果与结论合成了8个全新的Nampt抑制剂,化合物的结构经质谱和核磁共振氢谱确证;初步体外抗肿瘤实验结果表明,其中3个化合物（H1、H5、H7）具有一定的抗肿瘤活性,其活性与阳性对照APO866相当。     

Applications of CRISPR genome editing technology in drug target identification and validation

Introduction: The analysis of pharmaceutical industry data indicates that the major reason for drug candidates failing in late stage clinical development is lack of efficacy, with a high proportion of these due to erroneous hypotheses about target to disease linkage. More than ever, there is a requirement to better understand potential new drug targets and their role in disease biology in order to reduce attrition in drug development. Genome editing technology enables precise modification of individual protein coding genes, as well as noncoding regulatory sequences, enabling the elucidation of functional effects in human disease relevant cellular systems.

Areas covered: This article outlines applications of CRISPR genome editing technology in target identification and target validation studies.

Expert opinion: Applications of CRISPR technology in target validation studies are in evidence and gaining momentum. Whilst technical challenges remain, we are on the cusp of CRISPR being applied in complex cell systems such as iPS derived differentiated cells and stem cell derived organoids. In the meantime, our experience to date suggests that precise genome editing of putative targets in primary cell systems is possible, offering more human disease relevant systems than conventional cell lines.