Study on the regulatory effect of liver X receptor in HEK293 cells by six main diterpene esters in Semen Euphorbiae

Background:To study the effects of the main diterpene esters in Euphorbia factor L_(1),L_(2),L_(3),L_(7a),L_(7b)and L_(8)on the transcriptional activity and protein expression of liver X receptor(LXR).Methods:The effect of the main diterpene ester components in Semen Euphorbiae on the viability of HEK293 cells were studied by MTT assay.The LXR-Luc plasmid vector was transfected into HEK293 cells and treated with Euphorbia factor L_(1),L_(2),L_(3),L_(7a),L_(7b)and L_(8)for 24 h.The effect of the main diterpene ester components of Semen Euphorbiae on LXR-Luc luciferase activity was investigated by dual luciferase reporter gene system,and the expression of LXRαprotein was detected by Western Blot.Results:Euphorbia factor L_(1),L_(2),L_(3),L_(7a),L_(7b)and L_(8)could significantly reduce the relative luciferase activity(RLU)of LXRα,and the expression level of LXRαprotein was significantly down-regulated.Conclusion:Euphorbia factor L_(1),L_(2),L_(3),L_(7a),L_(7b)and L_(8)can inhibit the expression of LXR protein level,which may be achieved by inhibiting the transcriptional activity of LXR.

新型PCV2衣壳融合蛋白在HEK293F细胞瞬时表达研究

猪圆环病毒2型(Porcine Circovirus type 2,PCV2)衣壳(Capsid)蛋白是制备抗猪圆环2型病毒亚单位疫苗的有效免疫抗原,能在大肠杆菌及杆状病毒/昆虫细胞表达系统中获得表达,然而在哺乳细胞中的表达研究仍然缺乏。瞬时表达条件考察结果显示,采用宿主HEK293F细胞及PEI-40kDa转染试剂能获得35%病毒PCV2 Capsid基因细胞转染效率。转染试剂PEI(Polyethylenimine)与DNA比例差异会影响复合物形成大小及形态,复合物形成15~80 nm颗粒有利于转染效率的提高。实验以免疫逃逸型猪圆环病毒2b型NDSU41513病毒株,设计了新型PCV2 Capsid (△1-41aa)-Fc(pig) protein(PCFP)分子。在3 L反应器中成功实现了HEK293F细胞瞬时表达PCFP,表达水平达到3.8 mg/L。PCFP蛋白能够自主装形成约为41 nm类病毒颗粒,诱导小鼠机体内产生强烈的体液免疫反应,具有很高的应用潜力。

采用Cell-SELEX技术的核酸适配体在肿瘤靶向治疗的研究进展

阐述细胞-配体指数富集系统进化(Cell-SELEX)技术特点,以及通过该技术筛选得到的核酸适配体在肿瘤靶向治疗中的应用进展和挑战,通过查阅近年的相关文献,综述核酸适配体作为药物及药物载体在肿瘤靶向治疗中的应用研究进展。结果表明:基于Cell-SELEX技术筛选得到的核酸适配体在肿瘤靶向治疗中的疗效显著,可开发成为肿瘤靶向治疗的潜力药物及良好的药物载体。

293T细胞生产慢病毒工艺优化

随着细胞治疗的快速发展,大规模慢病毒的生产成为工艺环节中的瓶颈,因此,优化293T高滴度和高纯度的CAR慢病毒载体生产工艺显得至关重要。本研究旨在优化包装慢病毒的293T贴壁细胞,节省时间,节约成本,提高慢病毒包装的能力。同时对优化慢病毒载体中出现悬浮细胞结团生长的现象进行探索,检测其影响结团的因素。分别采用快速、慢速驯化方式将293T贴壁细胞驯化为悬浮培养,并比较其细胞形态、细胞密度、细胞活率、慢病毒包装能力和冻存复苏后稳定一致性,筛选出最优的悬浮驯化条件。通过调节Ca^(2+)浓度和EDTA添加量来研究比较细胞结团生长状况。结果证明,使用无血清培养基OPM-293 CD05溶剂(medium)可以将293T贴壁细胞快速驯化为293T悬浮细胞,并能制备出慢病毒滴度且优于贴壁细胞的包装滴度(^(*)P<0.05)。Ca^(2+)浓度会影响细胞结团大小,添加EDTA能有效分离分散非必要的细胞抱团生长。研究结果显示,传统293T贴壁细胞可以使用无血清培养基OPM-293 CD05溶剂快速驯化成悬浮细胞;在一定范围内,Ca^(2+)浓度越高细胞所结团块及粒径越大,EDTA添加量越高细胞所结团块及粒径变小。这为优化慢病毒载体包装工艺和悬浮培养条件,同时为体外规模化细胞培养放大和生产奠定了理论基础,具有一定的实用价值。

The combined application of stem cells and three-dimensional bioprinting scaffolds for the repair of spinal cord injury

Spinal cord injury is considered one of the most difficult injuries to repair and has one of the worst prognoses for injuries to the nervous system.Following surgery,the poor regenerative capacity of nerve cells and the generation of new scars can make it very difficult for the impaired nervous system to restore its neural functionality.Traditional treatments can only alleviate secondary injuries but cannot fundamentally repair the spinal cord.Consequently,there is a critical need to develop new treatments to promote functional repair after spinal cord injury.Over recent years,there have been seve ral developments in the use of stem cell therapy for the treatment of spinal cord injury.Alongside significant developments in the field of tissue engineering,three-dimensional bioprinting technology has become a hot research topic due to its ability to accurately print complex structures.This led to the loading of three-dimensional bioprinting scaffolds which provided precise cell localization.These three-dimensional bioprinting scaffolds co uld repair damaged neural circuits and had the potential to repair the damaged spinal cord.In this review,we discuss the mechanisms underlying simple stem cell therapy,the application of different types of stem cells for the treatment of spinal cord injury,and the different manufa cturing methods for three-dimensional bioprinting scaffolds.In particular,we focus on the development of three-dimensional bioprinting scaffolds for the treatment of spinal cord injury.

Cell reprogramming therapy for Parkinson’s disease

Parkinson’s disease is typically characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta.Many studies have been performed based on the supplementation of lost dopaminergic neurons to treat Parkinson’s disease.The initial strategy for cell replacement therapy used human fetal ventral midbrain and human embryonic stem cells to treat Parkinson’s disease,which could substantially alleviate the symptoms of Parkinson’s disease in clinical practice.However,ethical issues and tumor formation were limitations of its clinical application.Induced pluripotent stem cells can be acquired without sacrificing human embryos,which eliminates the huge ethical barriers of human stem cell therapy.Another widely considered neuronal regeneration strategy is to directly reprogram fibroblasts and astrocytes into neurons,without the need for intermediate proliferation states,thus avoiding issues of immune rejection and tumor formation.Both induced pluripotent stem cells and direct reprogramming of lineage cells have shown promising results in the treatment of Parkinson’s disease.However,there are also ethical concerns and the risk of tumor formation that need to be addressed.This review highlights the current application status of cell reprogramming in the treatment of Parkinson’s disease,focusing on the use of induced pluripotent stem cells in cell replacement therapy,including preclinical animal models and progress in clinical research.The review also discusses the advancements in direct reprogramming of lineage cells in the treatment of Parkinson’s disease,as well as the controversy surrounding in vivo reprogramming.These findings suggest that cell reprogramming may hold great promise as a potential strategy for treating Parkinson’s disease.

Mechanism of inflammatory response and therapeutic effects of stem cells in ischemic stroke:current evidence and future perspectives

Ischemic stroke is a leading cause of death and disability worldwide,with an increasing trend and tendency for onset at a younger age.China,in particular,bears a high burden of stroke cases.In recent years,the inflammatory response after stroke has become a research hotspot:understanding the role of inflammatory response in tissue damage and repair following ischemic stroke is an important direction for its treatment.This review summarizes several major cells involved in the inflammatory response following ischemic stroke,including microglia,neutrophils,monocytes,lymphocytes,and astrocytes.Additionally,we have also highlighted the recent progress in various treatments for ischemic stroke,particularly in the field of stem cell therapy.Overall,understanding the complex interactions between inflammation and ischemic stroke can provide valuable insights for developing treatment strategies and improving patient outcomes.Stem cell therapy may potentially become an important component of ischemic stroke treatment.

眼斑双锯鱼(Amphiprion ocellaris)发育中体色花纹时序发生的色素细胞变化和控制基因表达的分析Ⅱ.仔稚幼鱼时期

眼斑双锯鱼(Amphiprion ocellaris)属于鲈形目、雀鲷科、双锯鱼属,是热带珊瑚礁观赏鱼类的首选品种,其不同发育时期各种色素细胞的动态变化及其控制基因表达情况有待深入研究。记录了眼斑双锯鱼仔稚幼鱼体色花纹模式建成的发育过程,对比不同发育时期体色变化的特点,筛选出仔稚幼鱼时期体色花纹变化较为明显的9个发育时期,并利用荧光定量PCR检测了眼斑双锯鱼各发育时期的10个体色控制基因的表达情况。结果显示:眼斑双锯鱼的体色发生存在明显的时序性,仔鱼时期鱼体呈现半透明状,黑色素细胞排列在身体两侧,随着生长发育数量逐渐增多;稚鱼时期,体表开始出现红色素细胞和黄色素细胞,身体慢慢变得不透明,9 dph开始出现第一道条纹,虹彩色素细胞数量逐渐增多,10 dph时期观察到第二道条纹出现;幼鱼时期,三道白色条纹完全形成,体表的橙红色和白色条纹被黑色素细胞分隔开来,界线逐渐清晰,长成完整的花纹。结合荧光定量PCR结果分析发现:在仔稚幼鱼阶段,10个体色控制基因在各发育时期均有表达,不同功能分类的基因在不同发育时期的表达变化趋势差异较大,在仔稚幼鱼前期表达量变化较大的基因主要为TYR、Dct、Ednrb、Sox10等与黑色素细胞迁移、分化、合成相关的基因;随着幼鱼不断的生长发育,白色条纹逐条出现,与虹彩色素细胞相关的Fms、Foxd3等基因也开始出现表达量显著上升的趋势。

Cellular preconditioning and mesenchymal stem cell ferroptosis

In this editorial,we comment on the article published in the recent issue of the World Journal of Stem Cells.They focus on stem cell preconditioning to prevent ferroptosis by modulating the cystathionineγ-lyase/hydrogen sulfide(H_(2)S)pathway as a novel approach to treat vascular disorders,particularly pulmonary hypertension.Preconditioned stem cells are gaining popularity in regenerative medicine due to their unique ability to survive by resisting the harsh,unfavorable microenvironment of the injured tissue.They also secrete various paracrine factors against apoptosis,necrosis,and ferroptosis to enhance cell survival.Ferroptosis,a regulated form of cell death characterized by iron accumulation and oxidative stress,has been implicated in various pathologies encompassing dege-nerative disorders to cancer.The lipid peroxidation cascade initiates and sustains ferroptosis,generating many reactive oxygen species that attack and damage multiple cellular structures.Understanding these intertwined mechanisms provi-des significant insights into developing therapeutic modalities for ferroptosis-related diseases.This editorial primarily discusses stem cell preconditioning in modulating ferroptosis,focusing on the cystathionase gamma/H_(2)S ferroptosis pathway.Ferroptosis presents a significant challenge in mesenchymal stem cell(MSC)-based therapies;hence,the emerging role of H_(2)S/cystathionase gamma/H_(2) S signaling in abrogating ferroptosis provides a novel option for therapeutic intervention.Further research into understanding the precise mechanisms of H_(2)S-mediated cytoprotection against ferroptosis is warranted to enhance the thera-peutic potential of MSCs in clinical settings,particularly vascular disorders.

Effects of mesenchymal stem cell on dopaminergic neurons,motor and memory functions in animal models of Parkinson's disease:a systematic review and meta-analysis

Parkinson’s disease is chara cterized by the loss of dopaminergic neurons in the substantia nigra pars com pacta,and although restoring striatal dopamine levels may improve symptoms,no treatment can cure or reve rse the disease itself.Stem cell therapy has a regenerative effect and is being actively studied as a candidate for the treatment of Parkinson’s disease.Mesenchymal stem cells are considered a promising option due to fewer ethical concerns,a lower risk of immune rejection,and a lower risk of teratogenicity.We performed a meta-analysis to evaluate the therapeutic effects of mesenchymal stem cells and their derivatives on motor function,memory,and preservation of dopamine rgic neurons in a Parkinson’s disease animal model.We searched bibliographic databases(PubMed/MEDLINE,Embase,CENTRAL,Scopus,and Web of Science)to identify articles and included only pee r-reviewed in vivo interve ntional animal studies published in any language through J une 28,2023.The study utilized the random-effect model to estimate the 95%confidence intervals(CI)of the standard mean differences(SMD)between the treatment and control groups.We use the systematic review center for laboratory animal expe rimentation’s risk of bias tool and the collaborative approach to meta-analysis and review of animal studies checklist for study quality assessment.A total of 33studies with data from 840 Parkinson’s disease model animals were included in the meta-analysis.Treatment with mesenchymal stem cells significantly improved motor function as assessed by the amphetamine-induced rotational test.Among the stem cell types,the bone marrow MSCs with neurotrophic factor group showed la rgest effect size(SMD[95%CI]=-6.21[-9.50 to-2.93],P=0.0001,I^(2)=0.0%).The stem cell treatment group had significantly more tyrosine hydroxylase positive dopamine rgic neurons in the striatum([95%CI]=1.04[0.59 to 1.49],P=0.0001,I^(2)=65.1%)and substantia nigra(SMD[95%CI]=1.38[0.89 to 1.87],P=0.0001,I^(2)=75.3%),indicating a protective effect on dopaminergic neurons.Subgroup analysis of the amphetamine-induced rotation test showed a significant reduction only in the intracranial-striatum route(SMD[95%CI]=-2.59[-3.25 to-1.94],P=0.0001,I^(2)=74.4%).The memory test showed significant improvement only in the intravenous route(SMD[95%CI]=4.80[1.84 to 7.76],P=0.027,I^(2)=79.6%).Mesenchymal stem cells have been shown to positively impact motor function and memory function and protect dopaminergic neurons in preclinical models of Parkinson’s disease.Further research is required to determine the optimal stem cell types,modifications,transplanted cell numbe rs,and delivery methods for these protocols.

Emerging strategies for nerve repair and regeneration in ischemic stroke:neural stem cell therapy

Ischemic stroke is a major cause of mortality and disability worldwide,with limited treatment options available in clinical practice.The emergence of stem cell therapy has provided new hope to the field of stroke treatment via the restoration of brain neuron function.Exogenous neural stem cells are beneficial not only in cell replacement but also through the bystander effect.Neural stem cells regulate multiple physiological responses,including nerve repair,endogenous regeneration,immune function,and blood-brain barrier permeability,through the secretion of bioactive substances,including extracellular vesicles/exosomes.However,due to the complex microenvironment of ischemic cerebrovascular events and the low survival rate of neural stem cells following transplantation,limitations in the treatment effect remain unresolved.In this paper,we provide a detailed summary of the potential mechanisms of neural stem cell therapy for the treatment of ischemic stroke,review current neural stem cell therapeutic strategies and clinical trial results,and summarize the latest advancements in neural stem cell engineering to improve the survival rate of neural stem cells.We hope that this review could help provide insight into the therapeutic potential of neural stem cells and guide future scientific endeavors on neural stem cells.

TP53BP2基因真核表达载体的构建及在人胚肾Expi293F细胞中蛋白表达、纯化及活性鉴定

目的构建人肿瘤抑制因子p53结合蛋白2(tumor suppressor p53-binding protein 2,TP53BP2)的重组真核表达载体,转染人胚肾Expi293F细胞,获得高纯度的重组人全长TP53BP2蛋白并对其进行生物学活性鉴定。方法利用UniProt网站查询TP53BP2基因序列,并进行Expi293F表达系统序列优化,通过同源重组连接至pcDNA3.1(+)-P2Ae GFP载体并进行双酶切和测序鉴定,通过转染试剂聚乙烯亚胺(polyethylenimine,PEI)将pcDNA3.1(+)-P2A-eGFPTP53BP2质粒瞬时转染至Expi293F细胞,荧光显微镜观察转染效率,收集实验组及对照组细胞,利用免疫印记试验(Western blot,WB)检测TP53BP2重组蛋白表达水平。通过His标签纯化试剂盒及Superdex 20010/300GL层析柱进行蛋白纯化,十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDSPAGE)对纯化后重组蛋白进行鉴定。利用免疫共沉淀(Co-immunoprecipitation,Co-IP)检测重组人全长TP53BP2蛋白与p65蛋白结合情况。利用免疫荧光(immunofluorescence,IF)检测重组人全长TP53BP2蛋白与p65蛋白共定位。利用表面等离子体共振(surface-plasmon resonance,SPR)技术,检测纯化后的重组人全长TP53BP2蛋白与TP53BP2抗体的相互作用。结果经测序和双酶切鉴定,重组质粒pcDNA3.1(+)-P2A-eGFP-TP53BP2构建成功。经荧光显微镜观察结果显示转染效率约为60%,WB结果表明TP53BP2蛋白在Expi293F细胞中过表达,证明转染成功。SDS-PAGE结果表明纯化后重组蛋白纯度在90%以上,证明纯化成功。Co-IP结果表明,TP53BP2重组蛋白可与p65蛋白相互作用。IF结果表明,His标签蛋白、TP53BP2蛋白及p65蛋白存在共定位,表明三者之间存在相互作用。SPR结果表明,纯化的重组人TP53BP蛋白与TP53BP2抗体具有较好的结合活性。以上结果均证明重组人全长TP53BP2蛋白具有生物学活性。结论成功构建了TP53BP2基因真核表达载体并在人胚肾Expi293F细胞中成功表达出具有生物学活性的重组人全长TP53BP2蛋白,为进一步研究TP53BP2的结构和功能奠定了基础。

Neural stem cells promote neuroplasticity: a promising therapeutic strategy for the treatment of Alzheimer’s disease

Recent studies have demonstrated that neuroplasticity,such as synaptic plasticity and neurogenesis,exists throughout the normal lifespan but declines with age and is significantly impaired in individuals with Alzheimer’s disease.Hence,promoting neuroplasticity may represent an effective strategy with which Alzheimer’s disease can be alleviated.Due to their significant ability to self-renew,differentiate,and migrate,neural stem cells play an essential role in reversing synaptic and neuronal damage,reducing the pathology of Alzheimer’s disease,including amyloid-β,tau protein,and neuroinflammation,and secreting neurotrophic factors and growth factors that are related to plasticity.These events can promote synaptic plasticity and neurogenesis to repair the microenvironment of the mammalian brain.Consequently,neural stem cells are considered to represent a potential regenerative therapy with which to improve Alzheimer’s disease and other neurodegenerative diseases.In this review,we discuss how neural stem cells regulate neuroplasticity and optimize their effects to enhance their potential for treating Alzheimer’s disease in the clinic.

Cell replacement with stem cell-derived retinal ganglion cells from different protocols

Glaucoma,characterized by a degenerative loss of retinal ganglion cells,is the second leading cause of blindness worldwide.There is currently no cure for vision loss in glaucoma because retinal ganglion cells do not regenerate and are not replaced after injury.Human stem cell-derived retinal ganglion cell transplant is a potential therapeutic strategy for retinal ganglion cell degenerative diseases.In this review,we first discuss a 2D protocol for retinal ganglion cell differentiation from human stem cell culture,including a rapid protocol that can generate retinal ganglion cells in less than two weeks and focus on their transplantation outcomes.Next,we discuss using 3D retinal organoids for retinal ganglion cell transplantation,comparing cell suspensions and clusters.This review provides insight into current knowledge on human stem cell-derived retinal ganglion cell differentiation and transplantation,with an impact on the field of regenerative medicine and especially retinal ganglion cell degenerative diseases such as glaucoma and other optic neuropathies.

手动膜片钳检测盐酸罗哌卡因及其右旋异构体对HEK293细胞hERG电流的影响

目的研究比较盐酸罗哌卡因和盐酸罗哌卡因右旋异构体对高表达hERG钾通道的HEK293细胞hERG电流的影响。方法用手动膜片钳检测转染后hERG钾通道稳定表达的HEK293细胞电流,多菲莱德做阳性药,将盐酸罗哌卡因和盐酸罗哌卡因右旋异构体依次稀释成30.00、10.00、3.33、1.11、0.37μmol·L^(-1),依次作用于细胞,记录电流变化,计算抑制率。结果盐酸罗哌卡因0.37、1.11、3.33、10、30μmol·L^(-1)对电流Iherg-tail的抑制率分别为(6.12±0.30)%、(13.04±1.20)%、(19.21±0.33)%、(35.56±0.66)%、(65.37±4.17)%,IC_(50)为19.482μmol·L^(-1)(n=15)。盐酸罗哌卡因右旋异构体0.37、1.11、3.33、10.00、30.00μmol·L^(-1)对电流Iherg-tail的抑制率分别为(4.13±3.43)%、(7.34±5.60)%、(9.49±2.75)%、(16.60±0.87)%、(31.36±1.45)%,IC_(50)>30μmol·L^(-1)(n=15)。阳性对照药品多菲莱德0.00185、0.00556、0.01667、0.05000、0.15000μmol·L^(-1)对电流Iherg-tail的抑制率分别为(7.81±2.77)%、(19.67±1.88)%、(57.16±4.39)%、(89.71±3.55)%、(99.66±0.89)%、IC_(50)为0.015μmol·L^(-1)(n=15)。结论和阳性对照药品多菲莱德比较,盐酸罗哌卡因对hERG通道为弱抑制作用,盐酸罗哌卡因右旋异构体对hERG通道为无明显抑制作用。

纳豆抗氧化肽对H2O2诱导HEK293细胞氧化应激损伤的保护作用

目的评价纳豆肽的抗氧化能力以及对对H_(2)O_(2)诱导HEK293细胞氧化应激损伤的保护作用。方法首先以1,1-二苯基-2-三硝基苯(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基(DPPH·)、2,2’-联氮-二(3-乙基-苯并噻唑啉-6-磺酸)二铵盐[2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)ammonium salt,ABTS]阳离子自由基(ABTS^(+)·)、羟自由基(·OH)和超氧阴离子自由基(·O^(2-))清除能力以及总还原能力为指标,测定酶解得到纳豆肽粗品的抗氧化能力。利用超滤技术对纳豆肽进行分离,测定分离后各组分在不同浓度下对DPPH·及ABTS^(+)·清除活性。将活性最强的两个组分作为纳豆抗氧化肽,测定其对H_(2)O_(2)诱导氧化损伤HEK293细胞抗氧化酶含量的影响,评价其对氧化损伤HEK293细胞的保护效果。结果纳豆肽粗品在8.00 mg/mL时具有良好的DPPH·、ABTS^(+)·、·OH和·O^(2-)清除能力以及总还原能力。超滤后得到了相对分子质量分别大于30、10~30、3~10、小于3 kDa的4个纳豆肽组分,其对DPPH·及ABTS^(+)·清除活性均随着质量浓度的增加呈现上升趋势,特别是在8 mg/mL时,相对分子质量为3~10 kDa和小于3 kDa组分表现出最强的抗氧化活性,后续的细胞试验表明,这两个组分能够显著提高氧化应激下HEK293细胞的存活率。在300μg/mL的剂量范围内,小于3 kDa组分的纳豆抗氧化肽可使细胞存活率恢复至未损伤时的水平,而3~10 kDa组分也使损伤细胞的存活率提高了40.8%。同时,纳豆抗氧化肽均能提高氧化损伤HEK293细胞中的超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽过氧化酶(glutathione peroxidase,GPX)和过氧化氢酶(catalase,CAT)等抗氧化酶的含量,<3 kDa组分和3~10 kDa组分的纳豆抗氧化肽分别使SOD含量提高了49.52%和50.80%,GPX含量提高了49.52%和50.81%,CAT含量提高了93.64%和91.97%。结论纳豆肽具有抗氧化潜力,纳豆抗氧化肽可以有效地缓解H_(2)O_(2)诱导的HEK293细胞的氧化应激损伤,为纳豆抗氧化肽在功能食品中的应用提供理论依据。

花生红衣中反式白藜芦醇对HEK293T细胞抗氧化的影响

目的:分析花生红衣中的白藜芦醇成分,探究其主要成分反式白藜芦醇(RES)对人胚胎肾细胞293(HEK293T)抗氧化的影响及其潜在的分子机制。方法:采用超声波辅助酶法提取花生红衣中的白藜芦醇,采用高效液相色谱-质谱联用(HPLC-MS)技术分析花生红衣中白藜芦醇粗提液的组成成分。通过测定细胞活力,检测RES对HEK293T细胞的最高毒性。通过荧光素酶报告基因试验及免疫印迹试验检测其对Keap1-Nrf2-ARE抗氧化信号通路的影响,以DPPH自由基清除率评价其体外抗氧化能力。结果:HPLC-MS结果表明,花生红衣中含有白藜芦醇苷、白皮杉醇和RES。由于白藜芦醇在自然界中主要以反式白藜芦醇形式存在,因此选择RES进行后续试验。细胞存活力检测结果表明,RES对HEK293T细胞的最高无毒浓度为50μmol/L,体外抗氧化作用呈浓度依赖性。荧光素酶报告基因分析表明,RES显著诱导了ARE介导的转录激活。免疫印迹结果显示,RES能诱导Nrf2介导的3个抗氧化蛋白表达【血红素氧合酶1(HO-1)、醌氧化还原酶1(NQO1)和谷氨酸半胱氨酸连接酶(GCLM)】表达。此外,DPPH自由基清除率测定结果表明,RES能有效清除DPPH自由基,具有体外抗氧化能力。结论:花生红衣中白藜芦醇的主要成分RES对HEK293T细胞有抗氧化作用,可通过Keap1-Nrf2-ARE信号通路激活潜在的抗氧化活性。

Metastatic clear cell sarcoma of the pancreas:A rare case report

BACKGROUND Clear cell sarcoma(CCS)is a rare soft-tissue sarcoma.The most common metastatic sites for CCS are the lungs,bones and brain.CCS is highly invasive and mainly metastasizes to the lung,followed by the bone and brain;however,pancreatic metastasis is relatively rare.CASE SUMMARY We report on a rare case of CCS with pancreatic metastasis in a 47-year-old man.The patient had a relevant medical history 3 years ago,with abdominal pain as the main clinical manifestation.No abnormalities were observed on physical examination and the tumor was found on abdominal computed tomography.Based on the medical history and postoperative pathology,the patient was diagnosed with CCS with pancreatic metastasis.The patient was successfully treated with surgical interventions,including distal pancreatectomy and sple-nectomy.CONCLUSION This report summarizes the available treatment modalities for CCS and the importance of regular postoperative follow-up for patients with CCS.

Priming mesenchymal stem cells to develop “super stem cells”

The stem cell pre-treatment approaches at cellular and sub-cellular levels encompass physical manipulation of stem cells to growth factor treatment,genetic manipulation,and chemical and pharmacological treatment,each strategy having advantages and limitations.Most of these pre-treatment protocols are non-combinative.This editorial is a continuum of Li et al’s published article and Wan et al’s editorial focusing on the significance of pre-treatment strategies to enhance their stemness,immunoregulatory,and immunosuppressive properties.They have elaborated on the intricacies of the combinative pre-treatment protocol using pro-inflammatory cytokines and hypoxia.Applying a well-defined multi-pronged combinatorial strategy of mesenchymal stem cells(MSCs),pre-treatment based on the mechanistic understanding is expected to develop“Super MSCs”,which will create a transformative shift in MSC-based therapies in clinical settings,potentially revolutionizing the field.Once optimized,the standardized protocols may be used with slight modifications to pre-treat different stem cells to develop“super stem cells”with augmented stemness,functionality,and reparability for diverse clinical applications with better outcomes.

One-step cell biomanufacturing platform:porous gelatin microcarrier beads promote human embryonic stem cell-derived midbrain dopaminergic progenitor cell differentiation in vitro and survival after transplantation in vivo

Numerous studies have shown that cell replacement therapy can replenish lost cells and rebuild neural circuitry in animal models of Parkinson’s disease.Transplantation of midbrain dopaminergic progenitor cells is a promising treatment for Parkinson’s disease.However,transplanted cells can be injured by mechanical damage during handling and by changes in the transplantation niche.Here,we developed a one-step biomanufacturing platform that uses small-aperture gelatin microcarriers to produce beads carrying midbrain dopaminergic progenitor cells.These beads allow midbrain dopaminergic progenitor cell differentiation and cryopreservation without digestion,effectively maintaining axonal integrity in vitro.Importantly,midbrain dopaminergic progenitor cell bead grafts showed increased survival and only mild immunoreactivity in vivo compared with suspended midbrain dopaminergic progenitor cell grafts.Overall,our findings show that these midbrain dopaminergic progenitor cell beads enhance the effectiveness of neuronal cell transplantation.