Exosomes, as key mediators of intercellular communication, exhibit promising prospects in the biomedical field, particularly in stem cell-based replacement therapy. Current exosome isolation techniques face challenges in achieving batch enrichment and nondestructive release, which severely restrict their applications. Traditional specific enrichment strategies rely primarily on antibodies; however, aptamers can specifically recognize targets and serve as effective alternatives to antibodies, offering advantages such as small molecular size, high structural stability, and low cost, making them more suitable for scalable target isolation. Herein, we established the Graphene Oxide-anchored Releasable Aptamer Scaffold (GRAS) technique targeting CD63, a characteristic surface biomarker of exosomes. Leveraging the high affinity of single-stranded nucleic acids for graphene oxide (GO), along with the specific binding capacity of aptamers, we fabricated aptamer-displayed oligonucleotide scaffolds (Apt-Scaffold) and immobilized them onto GO to prepare Apt-Scaffold@GO. This configuration allows effective presentation of the CD63 aptamer on the GO interface for specific exosome recognition and capture. Different methods were further designed to achieve efficient and controllable exosome release. Our experimental results demonstrated that the GRAS technique outperformed the gold-standard ultracentrifugation (UC) in enriching exosomes from mesenchymal stem cell (MSC) culture medium. This method enables quantitative and specific enrichment of exosomes. With simple operation, low cost, and independence on large sophisticated instruments, it provides a robust technical foundation for advancing exosome-related biomedical applications.

Positive-strand RNA viruses represented by SARS-CoV-2 and DENV spread globally with high infectivity and frequent mutations, posing a severe threat to human health. However, specific and effective antiviral drugs remain limited. RNA-dependent RNA polymerase (RdRp) plays a critical role in the genome replication of RNA viruses and shows high structural conservation. No homologous protein exists in human cells, making RdRp an effective, safe, and broad-spectrum antiviral target. Therefore, establishing an RdRp activity assay for drug development based on its mechanism is of great significance. This paper summarizes the structural characteristics of RdRp and the methods for constructing RdRp activity assay, especially for cell-free activity assay. In addition, several reported RdRp inhibitors and their pharmaceutical progress are also reviewed, aiming to provide a reference for the development of novel antiviral drugs targeting RdRp.

Food safety is a major public health issue of global concern. In recent years, the CRISPR/Cas system has shown promise in the field of molecular detection. The system has been coupled with various nucleic acid amplification methods and combined with different signal output systems to develop a new generation of CRISPR/Cas-based nucleic acid biosensor technology. This review describes the design concept of the CRISPR/Cas-based nucleic acid biosensor and its application in food analysis. A detailed overview of different CRISPR/Cas systems, signal amplification methods, and signal output strategies is provided. CRISPR/Cas-based nucleic acid biosensors have the advantages of high sensitivity, strong specificity, and timeliness, achieving fast analysis of a variety of targets, including bacteria, toxins, metal ions, pesticides, veterinary drugs, and adulteration, promoting the development of rapid food safety detection technology. At the end, we also provide our outlook for the future development of CRISPR/Cas-based nucleic acid biosensors.

基于石墨烯的生物相容性和对单链核酸的吸附性,研究烯壳铁氮磁珠(graphene-shelled ferro-nitride magnetic beads,GFeNMB)运载靶向Survivin的反义寡核苷酸(antisense oligonucleotide,ASO)及其对肿瘤细胞的抑制作用.首先用RNA Draw针对Survivin mRNA的二级结构设计ASO,合成FAM标记和未标记的ASO;基于石墨烯对单链核酸的吸附性和对荧光团的淬灭性,采用酶标仪检测荧光强度方法检测GFeNMB对ASO的吸附能力,并对GFeNMB和GFeNMB-ASO表征;磁极作用下将Survivin ASO磁转染至人非小细胞肺癌细胞A549中,荧光显微镜观察GFeNMB将ASO载入细胞的能力;ASO转染后,采用Western blot检测Survivinr的表达,活性氧(reactive oxygen species,ROS)试剂盒、流式细胞仪检测细胞凋亡,CCK-8和划痕实验检测细胞增殖和迁移能力.结果表明,GFeNMB对ASO具有良好的吸附性,GFeNMB与ASO混合20 min达最大吸附量(0.44 μg·mg-1);FAM-ASO经磁性载入细胞内呈绿色荧光且集中于细胞核,GFeNMB介导的核转染能力显著优于脂质体;ASO经磁转染至细胞后,Survivin蛋白的降低水平优于未处理组和脂质体转染组;磁转染Survivin ASO后,肿瘤细胞凋亡比例增大,细胞内ROS升高,细胞增殖和迁移能力受到抑制.综上,GFeNMB可将Survivin ASO转染至肿瘤细胞中,能够抑制细胞增殖,诱导细胞凋亡.GFeNMB-ASO磁转染细胞后下调靶基因表达的情况表明,GFeNMB有望成为单链寡核苷酸的转染介质.

In this study, we prepared a streptavidin magnetic bead based on graphene-coated iron nitride magnetic beads (G@FeN-MB) and tried to use it for the enrichment of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The outer shell of our magnetic bead was wrapped with multiple graphene sheets, and there is no report on the application of graphene to the magnetic-bead-coating material. First, the graphene shell of G@FeN-MB was oxidized by a modified Hummer method so as to generate the carboxyl groups required for the coupling of streptavidin (SA) on the surface of the magnetic beads. X-ray photoelectron spectroscopy (XPS), Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR), and transmission electron microscopy (TEM) were used to characterize the oxidized G@FeN-MB (GO@FeN-MB). Streptavidin was then linked to the surface of the GO@FeN-MB by coupling the amino of the streptavidin with the carboxyl on the magnetic beads by carbodiimide method; thus, the streptavidin magnetic beads (SAMBs) were successfully prepared. To prove the practicality of the SAMBs, biotinylated SARS-CoV-2 S1 antibody was linked with it to respectively capture SARS-CoV-2 Spike-protein-coupled polystyrene beads (S-PS) and pseudovirus with S-protein expressed. Microplate reader and fluorescence microscope results show that the SAMBs can effectively enrich viruses. In conclusion, the preparation of SAMBs with G@FeN-MB is feasible and has potential for application in the field of virus enrichment.

Pioglitazone (PGZ) is utilized as a therapeutic agent in the management of (type 2) diabetes to control blood glucose levels. The existing research work was intended to make and optimize PGZ-containing NLCs (nanostructured lipid carriers). The fabricated nanostructured lipid carrier preparation was optimized by using different concentrations of the surfactants (Tween 80 and Span 80) and solid lipid (Compritol® 888 ATO) and liquid lipid (Labrasol®) while keeping the concentration of drug (PGZ), and co-surfactants (poloxamer 188) the same. The optimized NLC formulation (PGZ-NLCs) was further assessed for physical and chemical characterization, in vitro PGZ release, and stability studies. The optimized PGZ-NLCs have shown an average diameter of 150.4 nm, EE of 92.53%, PDI value of 0.076, and zeta-potential of −29.1 mV, correspondingly. The DSC thermal analysis and XRD diffractograms had not presented the spectrum of PGZ, confirming the comprehensive encapsulation of PGZ in the lipid core. PGZ-NLCs showed significantly extended release (51% in 24 h) compared to the unformulated PGZ. Our study findings confirmed that PGZ-NLCs can be a promising drug delivery system for the treatment of type 2 diabetes.

Objective:The NS5 protein of dengue virus (DENV) mainly acts as the viral RNA-dependent RNA polymerase (RdRp). The aim of the present study was to obtain potent antiviral pro-drugs using the conformational phage display peptide library.Methods:The soluble recombinant DENV-2 NS5 protein was used as the target for screening oligopeptide (OP) with high specificity with the conformational phage display peptide library. Molecular docking was used to verify the interaction between OP and RdRp. DENV infected C6/36 cells were treated with different concentrations of OP. The antiviral effect of OP was assessed by virus titre detecmined by RT-qPCR. Meanwhile, C6/36 cells were culture with exposure to higher concentrations of OP. Cell viability was measured by CCK-8 assay to evaluate the cytotoxicity of OP.Results:The molecular docking indicated that OP bound specifically to the RdRp domain of DENV NS5 protein. Inhibitory effect on DENV-2 replication without cytotoxicity was observed in vitro. In addition, based on the high conservation of the RdRp domain in single-stranded plus RNA (+ssRNA) viruses and the preliminary results of molecular docking, OP may also bind to the RdRp active cavity of a number of +ssRNA viruses. Conclusions:OP may have the potential to inhibit RdRp of certain +ssRNA viruses, and is a potential broad-spectrum antiviral prodrug.

课程思政是新时期高校教育新模式.如何充分发掘医学基础专业基础课程蕴含的课程思政元素,将其融入教学实践过程中,对实现三全育人,引导学生建立正确的人生观、价值观及职业观,具有重要的现实意义.通过对医学基础的课程思政元素和教案设计进行探索与实践,设计医学基础课程思政的切入点、思路.通过教学实践与教学反思,准确地把握思想政治教育的时机与方法.主要探索课程思政与医学基础课程的有效融合教学范式,发挥立德树人和三全育人的重要作用.

To investigate the effect of 1800 MHz electromagnetic radiation (EMR) on apoptosis, we exposed NIH/3T3 cells at 1800 MHz with a specific absorption rate (SAR) of 2 W/kg intermittently for 12, 24, 36, and 48 h. After exposure, Cell Counting Kit-8 (CCK-8) and flow cytometry were used to detect cell viability and apoptosis; the expression of p53, a molecule with the key role in apoptosis, was measured by real-time qPCR, western blot, and immunofluorescence; and images of the structure of the mitochondria, directly reflecting apoptosis, were captured by electron microscopy. The results showed that the viability of cells in the 12, 36, and 48 h exposure groups significantly decreased compared with the sham groups; after 48 h of exposure, the percentage of late apoptotic cells in the exposure group was significantly higher. Real-time qPCR results showed that p53 mRNA in the 48 h exposure group was 1.4-fold of that in the sham group; significant differences of p53 protein fluorescence expression were observed between the exposure groups and the sham groups after 24 h and 48 h. The mitochondrial swelling and vesicular morphology were found in the electron microscopy images after 48 h exposure. These findings demonstrated 1800 MHz, SAR 2 W/kg EMR for 48 h may cause apoptosis in NIH/3T3 cells and that this apoptosis might be attributed to mitochondrial damage and upregulation of p53 expression.

目的:利用CRISPR/Cas9技术对K562细胞系JAK2基因进行编辑,构建JAK2基因敲除的K562细胞系.方法:使用CRISPR在线设计工具,针对JAK2基因设计sgRNA,构建Cas9-sgRNA共表达质粒.使用第二代慢病毒包装系统包装慢病毒并感染K562细胞,提取细胞基因组DNA,Sanger测序和TA克隆检测基因编辑活性.无限稀释法将编辑阳性的细胞接种于96孔板并扩培得到单克隆细胞株,提取基因组DNA,Sanger测序和TA克隆分析敲除JAK2单克隆细胞的基因型.结果:成功构建靶向敲除JAK2基因的lentiCRISPRv2-sgRNA3-1质粒.优化方案得到低细胞毒性高转染效率的感染K562细胞慢病毒量.CRISPR/Cas9系统成功在JAK2基因sgRNA3-1识别位点发挥基因组编辑活性,获得纯合敲除JAK2基因细胞株K562-JAK2-/-(两个等位分别发生移码突变,预期编码没有功能的JAK2蛋白).结论:CRIAPR/Cas9系统通过慢病毒感染方式获得JAK2基因纯合敲除的K562细胞株,该细胞模型可用于研究在慢性髓系白血病中JAK2基因的作用,为构建K562敲除其他基因细胞系提供实验依据,为探究造血分化机制的研究奠定实验基础.

Dengue fever is a mosquito-borne viral disease with dramatically increasing morbidity rate worldwide in decades. Since there is no specific treatment to date, early diagnosis is important for providing proper timely medical care to minimize mortality, and for the prompt initiation of public health control measures. NS5 is a potential biomarker for dengue virus infection due to its highly conserved and immunogenic properties. In this study, the DENV 2 NS5 full-length and the DENV 2 NS5 C-terminus RNA-dependent RNA polymerase domain fragment (NS5-C70) expression plasmids were constructed, and the 104 kDa full-length NS5 and the 70 kDa NS5-C70 were respectively expressed in Escherichia coli. These two purified recombinant products were found to react with the sera of patients infected with dengue virus when analyzed by an enzyme-linked immunosorbent assay (ELISA), which resulted in significantly higher absorption values than those of control sera. The recombinant DENV 2 NS5 exhibited strong reactivity to each of the four types of sera, whereas the NS5-C70 showed strong reactivity only to DENV 2 and 4. In comparison, the positive agreement value of recombinant NS5-based assay with either MyBioSource or Panbio assay was higher than that of the two commercially available IgG indirect ELISA kits. These results suggest that the recombinant DENV 2 NS5 be an effective antigen for detection of dengue virus infection. The recombinant NS5-C70 may also be used as an auxiliary antigen for diagnostic purposes.

通过"我国公民的素养因素之一——多维健康观""专业教育与通识教育是相互促进的统一体"以及"'健康生活方式与健康传播'"是现代高等教育不可或缺的重要内容"几个方面,对"健康生活方式与健康传播"在高等教育中的价值进行初步分析和讨论,旨在依托通识教育载体使大学生摒弃陋习,养成健康生活习惯,倡导科学生活方式,形成知晓健康信息、认同健康信念、建立健康态度、自觉采纳健康行为的四个层次的健康新理念.

In this study,we aimed to establish an immortal neural-like cell strain that could be used to study the effects of long-term exposure to environmental factors.After preconditioning with bFGF,human umbilical cord-derived mesenchymal stem cells (MSCs) were then induced with BHA,DMSO,and β-mercaptoethanol.Changes in cell morphology were observed under microscope and the expression levels ofNestin,NSE,GFAP,and MAP2 were determined by immunofluorescence and real-time PCR to estimate nerve cell specificity.The successfully induced cells were serially passaged,and single-cell clones were isolated.A cell clone that was subcultured more than 50 times was selected.Based on Nestin and MAP2 expression levels,we obtained a cell line with nerve-like properties.In addition,the induced cells showed surface projections,and the expression ofNestin,NSE,and GFAP indicated that the MSCs had differentiated into neuron-like cells.Nestin expression increased 1.32-fold at the 11th subculture and decreased 8.85-fold at the 50th subculture.Specifically,MAP2 expression increased 2.12-and 7.29-fold at the 11th and 50th subculture,respectively,confirming that the neural stem cells differentiated into neuron-like cells that exhibited the neuron-like cell properties.The monoclonal neural-like cells could be serially passaged,and they maintained their neuron-like properties.Thus,these cells can be used to study the chronic effects of long-term environmental exposures on the nervous system.

Objective To observe the proliferation ability of NIH/3T3 cells after a long-term exposure to 1800 MHz electromagnetic radiation.Methods Cells were exposed to 1800 MHz electromagnetic radiation,at SAR of 2 W/kg,intermittently exposure mode (5 min on/10 min off),5 h/d.Then plate clone forming test and soft agar clone forming test of the exposed and sham exposed cells were cultured respectively after 60-day and 138-day exposure.Telomerase expressions of each group were detected by ELISA.Results In plate clone experiments,plate clone forming ratio was significantly lowcr than that of the sham group (P＜O.05) after 60-day exposure,however,there was no significant difference between the exposed cells and the sham after 138-day exposure.In soft agar clone experiments,soft agar clone forming was no significantly different from sham group after 60-day exposure,however,138-day exposure group formation was significantly higher than that of the sham group (P＜0.05).In telomerase experiments,after 60 days of exposure,the expression of telomerase was no significantly different from sham group,whereas 138-day exposure group was significantly higher than sham group (P ＜ 0.05).Conclusions 1800 MHz electromagnetic radiation could damage the proliferation of NIH/3T3 cells exposed for 60 days,whereas the ability of cell transformation and proliferation increased significantly after more long-term exposure (138 days).

登革热(dengue fever,DF)是一种主要在热带、亚热带地区流行的蚊媒传染疾病,近年来疫情愈发严重.登革病毒(dengue virus,DENV)非结构蛋白(non-structural protein,NS) NS5是所有黄病毒属蛋白中最大也是最保守的.DENV NS5具有三种酶活性:甲基转移酶(methyltransferase,MTase)、核苷酸末端转移酶(terminal nueleotide transferase,TNTase)和RNA依赖的RNA聚合酶(RNAdependent RNApolymerase,RdRp).DENV NS5的MTase和RdRp活性在结构和功能上具有鲜明特点,在病毒基因组复制过程起着核心作用,因此NS5蛋白具有成为阻断或抑制病毒增殖靶标的潜力.本文综述了近年来DENVNS5的研究进展.

目的 研究1 800 MHz电磁辐射对细胞的氧化损伤作用.方法 使用1 800 MHz,比吸收率为2、3、4W/kg电磁辐射辐照小鼠皮肤细胞(JB6),细胞分为辐照组与对照组.辐照结束后,采用流式细胞术,DCFH-DA荧光探针检测细胞内活性氧(ROS)水平,再加入ROS中和剂富氢水,以观察其对ROS的中和作用.通过硫代巴比妥酸比色法检测丙二醛含量,来评价电磁辐射后细胞氧化损伤程度.结果 比吸收率为2、3 W/kg时,暴露2h内细胞内ROS水平变化差异无统计学意义;4 W/kg暴露20 min、80 min、120 min后,暴露组细胞与假暴露组比较ROS水平均显著升高(P＜0.05);加入富氢水后,暴露组与普通培养基暴露组比较,降低了50.79％;4 W/kg暴露120 min后丙二醛含量显著升高(P＜0.05).结论 4 W/kg 1 800 MHz电磁辐射会引发细胞内ROS升高并造成细胞氧化损伤.

我们对本校生物技术专业的人才培养模式及专业建设进行了的新的探索和研究,以多维度谋求创新人才培养新突破的对策为出发点,将凝练特色,凸显大学生实践创新能力培养为目标,以深化改革,推动人才培养模式改革创新为目的,搭建基于能力培养的创新人才培养体系,构建科学、合理的实验教学体系.以“注重能力,自主创新,协调发展”的实验教学理念为先导,紧紧抓住人才培养这条主线,从优化整合实践教学体系入手,将搭建教学科研互动基地、产学研互动平台为切入点,形成了“理论教学模块和实践教学平台”的人才培养方案.

目的 探讨1 800 MHz电磁辐射对NIH/3T3细胞周期和凋亡的影响.方法 将NIH/3T3细胞假暴露或间断暴露(5 min开,10 min关)在比吸收率(specific absorption rate,SAR)为2 W/kg,模拟全球移动通信系统(global system for mobile communications,GSM)通话状态信号的1 800 MHz电磁场中.每天暴露5h,连续60 d,期间每3～4 d传代一次.暴露完毕后,用Cell Counting Kit-8(CCK-8)检测细胞活性;染色后用流式细胞仪检测细胞凋亡、细胞周期的变化;用Caspase-3活性检测试剂盒检测细胞凋亡因子Caspase-3的活性变化.结果 与假暴露组相比,暴露组细胞活力下降且培养96 h后明显下降(t=30.78,P=0.048);暴露组细胞处于S期(t=3.267,P=0.022)和G2期(t=3.844,P=0.022 9)的百分数较假暴露组有明显增加;细胞凋亡率明显增加(t=3.349,P=0.047),Caspase-3活性增高(t=13.593,P=0.013).结论 NIH/3T3细胞经60d 1 800 MHz模拟手机电磁辐射后,细胞活性下降,S期和G2期阻滞,同时细胞凋亡率增加.

[目的]研究900MHz、最大功率密度为10.644μW/cm2的手机射频辐射对体外细胞DNA损伤和病毒癌基因EB病毒早期抗原(EBV-EA)的激活作用. [方法] (1)将NIH/3T3细胞分别暴露在手机辐射下0、1、2、4、6、8、12h,并分别设定相应的正常对照组,用彗星电泳检测细胞DNA受损程度.(2)将Raji细胞分为A辐照(-)TPA(-)、B辐照(-)TPA(+)、C辐照(+)TPA(-)、D辐照(+)TPA(+)4组,接受手机辐照,强度为4h/d,实验持续4周;每周结束时,B和D两组细胞分别使用促癌剂12-0-十四烷酰佛波醇-13-乙酸酯(TPA)1ng/mL处理48h.用免疫细胞化学检测各组细胞中EBV-EA的阳性表达情况. [结果]当NIH/3T3细胞辐照2h后,与对照组相比细胞Olive尾距有明显差异(P＜0.05),但在随后的2～12h内的各个检测时间点上,Olive尾距并没有随着时间的增加而增加(P＞0.05); Raji细胞在实验4周后,辐照C、D组细胞的EBV-EA阳性表达率较A组明显增加(P＜0.05),并且在加入促癌剂TPA的D组,阳性表达率更加明显(P＜0.01). [结论] 900 MHz手机射频辐射会对细胞DNA产生一定的损伤,并且能够激活EBV-EA的表达.