Neural progenitor cells derived from fibroblasts induced by small molecule compounds under hypoxia for treatment of Parkinson’s disease in rats

Neural progenitor cells(NPCs) capable of self-renewal and differentiation into neural cell lineages offer broad prospects for cell therapy for neurodegenerative diseases. However, cell therapy based on NPC transplantation is limited by the inability to acquire sufficient quantities of NPCs. Previous studies have found that a chemical cocktail of valproic acid, CHIR99021, and Repsox(VCR) promotes mouse fibroblasts to differentiate into NPCs under hypoxic conditions. Therefore, we used VCR(0.5 mM valproic acid, 3 μM CHIR99021, and 1 μM Repsox) to induce the reprogramming of rat embryonic fibroblasts into NPCs under a hypoxic condition(5%). These NPCs exhibited typical neurosphere-like structures that can express NPC markers, such as Nestin, SRY-box transcription factor 2, and paired box 6(Pax6), and could also differentiate into multiple types of functional neurons and astrocytes in vitro. They had similar gene expression profiles to those of rat brain-derived neural stem cells. Subsequently, the chemically-induced NPCs(ciNPCs) were stereotactically transplanted into the substantia nigra of 6-hydroxydopamine-lesioned parkinsonian rats. We found that the ciNPCs exhibited long-term survival, migrated long distances, and differentiated into multiple types of functional neurons and glial cells in vivo. Moreover, the parkinsonian behavioral defects of the parkinsonian model rats grafted with ciNPCs showed remarkable functional recovery. These findings suggest that rat fibroblasts can be directly transformed into NPCs using a chemical cocktail of VCR without introducing exogenous factors, which may be an attractive donor material for transplantation therapy for Parkinson’s disease.

Variaiton in the composition of small molecule compounds in the egg yolks of Asian Short-toed Larks between early and late broods

The egg yolks of birds contain most of the maternally derived materials required for embryo development and are an important factor influencing embryo development and offspring viability.Individual variation in egg-laying date frequently occurs in passerines inhabiting highly seasonal environments.Females laying in early and late stages of the breeding season encounter different environment temperatures and food conditions,which can affect the levels of metabolities in their bodies,thereby altering the transmission of these materials to the eggs.We test a hypothesis that yolk small molecule compounds of Asian Short-toed Lark(Alaudala cheleensis)could vary between early(mid-May)and late(mid-June)broods.Using the UHPLC-MS/MS method,683 compounds belonging to 21 compound groups are detected in the yolks.The contents of 18 compounds are significantly different between early and late broods.Ten differential compounds are significantly higher in the early laid eggs,among whichγ-aminobutyric acid,creatine,prostaglandins,palmitoleic acid,linoleic acid,and trans linoleic acid are related to low environment temperature response.The eggs laid in late stage exhibit significantly higher levels of 5-L-glutamyl-L-alanine andγ-glutamate-leucine,1,3-dimethyluric acid and mannose,which may be attributed to females in the late group consuming more insects.We suggest conducting a comprehensive investigation to reveal the yolk small molecule compounds mediated maternal effects on offspring phenotypes under varying ecological conditions.

A Clinical and Animal Experiment Integrated Platform for Small-Molecule Screening Reveals Potential Targets of Bioactive Compounds from a Herbal Prescription Based on the Therapeutic Efficacy of Yinchenhao Tang for Jaundice Syndrome

A herbal prescription in traditional Chinese medicine(TCM)has great complexity,with multiple components and multiple targets,making it extremely challenging to determine its bioactive compounds.Yinchenhao Tang(YCHT)has been extensively used for the treatment of jaundice disease.Although many studies have examined the efficacy and active ingredients of YCHT,there is still a lack of an in-depth systematic analysis of its effective components,mechanisms,and potential targets—especially one based on clinical patients.This study established an innovative strategy for discovering the potential targets and active compounds of YCHT based on an integrated clinical and animal experiment platform.The serum metabolic profiles and constituents of YCHT in vivo were determined by ultra-performance liquid chromatography–quadrupole time-of-flight mass spectrometry(UPLC-Q-ToF-MS)-based metabolomics combined with a serum pharmacochemistry method.Moreover,a compound–target–pathway network was constructed and analyzed by network pharmacology and ingenuity pathway analysis(IPA).We found that eight active components could modulate five key targets.These key targets were further verified by enzyme-linked immunosorbent assay(ELISA),which indicated that YCHT exerts therapeutic effects by targeting cholesterol 7a-hydroxylase(CYP7A1),multidrug-resistance-associated protein 2(ABCC2),multidrug-resistance-associated protein 3(ABCC3),uridine diphosphate glucuronosyl transferase 1A1(UGT1A1),and farnesoid X receptor(FXR),and by regulating metabolic pathways including primary bile acid biosynthesis,porphyrin and chlorophyll metabolism,and biliary secretion.Eight main effective compounds were discovered and correlated with the key targets and pathways.In this way,we demonstrate that this integrated strategy can be successfully applied for the effective discovery of the active compounds and therapeutic targets of an herbal prescription.

Transient Folate Deprivation in Combination with Small-molecule Compounds Facilitates the Generation of Somatic Cell-derived Pluripotent Stem Cells in Mice

Induced pluripotent stem cells （iPSCs） can be propagated indefinitely, while maintaining the capacity to differentiate into all cell types in the body except for the extra-embryonic tissues. This iPSC technology not only represents a new way to use individual-specific stem cells for regenerative medicine but also constitutes a novel method to obtain large numbers of disease-specific cells for biomedical re- search. However, the low efficiency of reprogramming and genomic integration of oncogenes and viral vectors limit the potential application of iPSCs. Chemical-induced reprogramming offers a novel ap- proach to generating iPSCs. In this study, a new combination of small-molecule compounds （SMs） （so- dium butyrate, A-83-01, CHIR99021, Y-27632） under conditions of transient folate deprivation was used to generate iPSC. It was found that transient folate deprivation combined with SMs was sufficient to permit reprogramming from mouse embryonic fibroblasts （MEFs） in the presence of transcription factors, Oct4 and Klf4, within 25 days, replacing Sox2 and c-Myc, and accelerated the generation of mouse iPSCs The resulting cell lines resembled mouse embryonic stem （ES） cells with respect to proliferation rate, morphology, pluripotency-associatedmarkers and gene expressions. Deprivation of folic acid, combined with treating MEFs with SMs, can improve the inducing efficiency of iPSCs and reduce their carcino- genicity and the use of exogenous reprogramming factors.

Compound 3k治疗骨关节炎:调控氧化应激通路改善软骨细胞糖酵解的作用机制

背景:骨关节炎现已被认为是一种代谢性疾病,既往研究表明糖酵解在骨关节炎的发生发展中起重要作用。Compound 3k作为一种新型糖酵解小分子抑制剂,具有抗炎及抗肿瘤等功效,因此可靶向糖酵解,有望为骨关节炎治疗提供新的思路。目的:基于缺氧诱导因子1α/活性氧的氧化应激通路探究Compound 3k在糖酵解过度活跃所导致的骨关节炎中的作用机制。方法:取对数生长期的ATDC5成软骨细胞,用10 ng/mL白细胞介素1β作用24 h诱导骨关节炎体外细胞模型,以CCK-8法检测不同浓度(0.25,0.5,1,2.5,5,10,15μmol/L)Compound 3k的细胞毒性,选出合适浓度进行后续实验。将软骨细胞随机分为对照组、模型组、治疗组,模型组以10 ng/mL的白细胞介素1β诱导,治疗组以Compound 3k预刺激2 h后与白细胞介素1β共培养,用CCK-8法检测各组细胞增殖情况;用ELISA试剂盒检测各组细胞炎症水平;用试剂盒检测各组细胞活性氧、细胞外乳酸脱氢酶及葡萄糖含量;qRT-PCR及Western blot检测相关炎症因子白细胞介素6、肿瘤坏死因子α及糖酵解相关基因葡萄糖转运蛋白1、甘油醛3-磷酸脱氢酶、单羧酸转运蛋白1和缺氧诱导因子1α的表达水平。结果与结论:①与对照组相比,模型组细胞增殖活性下降、糖酵解水平活跃,表现为细胞外乳酸脱氢酶含量增加(P<0.001),葡萄糖含量减少(P<0.001),相关炎症因子白细胞介素6(P<0.0001)及肿瘤坏死因子α(P<0.001),糖酵解相关基因葡萄糖转运蛋白1(P<0.001)、甘油醛3-磷酸脱氢酶(P<0.001)、单羧酸转运蛋白1(P<0.001)及缺氧诱导因子1α(P<0.001)的表达水平均上调,并伴随氧化应激,活性氧过量产生。②与模型组相比,Compound 3k的治疗有效提高细胞增殖活性,抑制过度活跃的糖酵解水平的同时,抑制了骨关节炎软骨细胞炎症(P<0.001)及糖酵解相关基因的表达(P<0.001),且抑制氧化应激,缺氧诱导因子1α的表达水平下调(P<0.0001),活性氧水平下降。③上述结果证实,Compound 3k抑制了白细胞介素1β诱导的软骨细胞炎症,其机制可能与糖酵解及缺氧诱导因子1α/活性氧介导的氧化应激有关。

Small molecules for mesenchymal stem cell fate determination

Mesenchymal stem cells(MSCs)are adult stem cells harboring self-renewal and multilineage differentiation potential that are capable of differentiating into osteoblasts,adipocytes,or chondrocytes in vitro,and regulating the bone marrow microenvironment and adipose tissue remodeling in vivo.The process of fate determination is initiated by signaling molecules that drive MSCs into a specific lineage.Impairment of MSC fate determination leads to different bone and adipose tissue-related diseases,including aging,osteoporosis,and insulin resistance.Much progress has been made in recent years in discovering small molecules and their underlying mechanisms control the cell fate of MSCs both in vitro and in vivo.In this review,we summarize recent findings in applying small molecules to the trilineage commitment of MSCs,for instance,genistein,medicarpin,and icariin for the osteogenic cell fate commitment;isorhamnetin,risedronate,and arctigenin for pro-adipogenesis;and atractylenolides and dihydroartemisinin for chondrogenic fate determination.We highlight the underlying mechanisms,including direct regulation,epigenetic modification,and post-translational modification of signaling molecules in the AMPK,MAPK,Notch,PI3K/AKT,Hedgehog signaling pathways etc.and discuss the small molecules that are currently being studied in clinical trials.The target-based manipulation of lineage-specific commitment by small molecules offers substantial insights into bone marrow microenvironment regulation,adipose tissue homeostasis,and therapeutic strategies for MSC-related diseases.

Neuroglobin and neuroprotection:the role of natural and synthetic compounds in neuroglobin pharmacological induction

Neuroglobin(Ngb)is a 17 kDa monomeric hexa-coordinated heme protein belonging to the globin family.Ngb is mainly expressed in neurons of the central and peripheral nervous system,although moderate levels of Ngb have been detected in non-nervous tissues.In the past decade,Ngb has been studied for its neuroprotective role in a large number of neurological disorders such as Alzheimer’s disease,Huntington’s disease,brain ischemia and hypoxia.This review discusses and summarizes the natural compounds and the small synthetic molecules capable of modulating Ngb expression that exhibits a protective role against various neurodegenerative diseases.

系统性硬皮病靶向治疗研究进展

系统性硬皮病(SSc)作为一种免疫介导的慢性结缔组织疾病,以皮肤、肺等多器官纤维化为主要临床表现,属于罕见病范畴,但发病率和死亡率在风湿免疫性疾病中均排在前列。目前尚无有效的治疗药物。近年来研究发现,T/B淋巴细胞、成纤维细胞、内皮细胞等相互作用及各种促炎促纤维化信号分子激活在其发生发展中起着关键性作用,在此基础上正在研发系列靶向药物。其中靶向B细胞耗竭的CD20单克隆抗体(利妥昔单抗)、IL-6受体拮抗剂(托珠单抗)、多靶点酪氨酸激酶抑制剂(尼达尼布)等在国际上已被批准用于SSc或系统性硬化病相关间质肺疾病(SSc-ILD)治疗;其他靶向药物如Janus激酶抑制剂、多种白介素抑制剂等在临床试验阶段,部分展现初步应用前景。本文从靶向B淋巴细胞、T淋巴细胞、血管内皮细胞及促炎促纤维化信号分子4个方面综合最新证据,就靶向疗法在SSc中的研究进展进行综述。

天然产物调控氧化应激治疗骨质疏松症

背景:越来越多的研究表明氧化应激在治疗骨质疏松症方面发挥着重要作用,氧化应激会导致氧化活性物质的累积,进而损伤骨相关细胞,最终引起骨吸收和骨生成的失衡,导致骨量减少以及骨微结构的破坏。近年来研究发现,一些天然产物可以调节氧化应激治疗骨质疏松症,其来源广泛、不良反应小等特点在治疗骨质疏松症方面具有明显的优势,疗效可观。目的:讨论天然产物调控氧化应激治疗骨质疏松症的机制,并根据最新的相关研究进展展开综述,以期为今后更多天然产物治疗骨质疏松症的研究提供参考和思路,并为临床上应用天然化合物治疗骨质疏松症提供数据支持。方法:以“oxidative stress,free radical,antioxidant,phytotherapy,plant extracts,medicinal plants,herbal medicine,osteoporosis,bone density,bone loss”“氧化应激,自由基,抗氧化,天然产物,植物提取物,植物药,中药,骨质疏松症,骨密度,骨丢失”为关键词在PubMed、Web of Science、Embase、Cochrane、维普、CBM、万方和中国知网数据库检索2010年1月至2023年2月的相关文献。制定纳入和排除标准,通过阅读文献标题、摘要及全文内容进行筛选,最终纳入64篇相关文献。结果与结论:①一些天然产物具有抗氧化作用,并可以通过改善氧化应激调控成骨分化、调控成骨细胞骨基质矿化、调控破骨细胞介导的骨吸收、调控骨相关细胞的增殖、分化、活性和凋亡,从而影响骨代谢过程;②这些具有抗氧化作用的天然产物通过改善骨重塑平衡起到治疗骨质疏松症的作用;③多种天然产物联合使用改善骨质疏松症的研究还有待探索;④利用天然产物调控氧化应激可能成为今后临床上治疗骨质疏松症的有力武器。

新型4,6-二取代吡啶并嘧啶类化合物的合成及抗肿瘤活性研究

目的设计、合成4,6-二取代吡啶并嘧啶类化合物并进行抗肿瘤活性研究。方法参考MNKs激酶小分子抑制剂的构效关系,结合计算机虚拟对接评价结果,设计了4,6-二取代吡啶并[3,2-d]嘧啶类化合物。以6-氯-2-氰基-3-硝基吡啶为原料,经还原、水解、环合、氯代反应得到关键中间体4,6-二氯吡啶并[3,2-d]嘧啶,然后经亲核取代反应在4位引入取代苯胺,再经Suzuki偶联、酰化以及脱保护等反应得到目标化合物。考察了化合物对MNK1和MNK2激酶的抑制活性及体外肿瘤细胞增殖抑制活性。结果22个4,6-二取代吡啶并[3,2-d]嘧啶类化合物均未见文献报道,其中化合物12r和12u对MNK2表现出显著的抑制活性,其IC_(50)值分别为0.5μmol·L^(-1)和0.1μmol·L^(-1)。化合物12u对人结肠癌HCT-116的细胞增殖具有抑制作用,其GI_(50)值为0.71μmol·L^(-1)。结论4,6-二取代吡啶并[3,2-d]嘧啶类化合物具有一定的MNK2抑制活性,其中化合物12u与先导化合物B21活性相当,对人结肠癌细胞HCT-116的增殖抑制活性优于B21。

基于质谱熵的小分子化合物相似性计算方法研究

在二级质谱(MS2)数据检索中,通常利用质谱数据之间的相似性进行检索。针对质谱数据相似性计算中数据不整齐导致的检索效率和准确性不高以及商用软件相似性计算方法单一的问题,研究提出了“拼接填充”与“匹配填充”两种MS2数据对齐方法,并基于信息熵采用质谱熵相似性计算方法进行相似性检索。首先对归一化后的原始质谱数据进行特征提取,保留能突出质谱数据特征的数据,再分别采用两种数据对齐方法对质谱数据进行预处理;然后基于信息熵方法,分别计算未知质谱与已知质谱混合后的虚拟质谱与两者质谱的熵差,获得未知质谱与已知质谱的相关系数即相似性;最后选择小分子化合物的质谱数据集进行实例验证。结果表明:两种质谱数据预处理方法能够解决相似性计算中质谱长度不等的问题,基于质谱熵的相似性计算方法稳定且结果可靠,适用于小分子化合物的相似性检索,同时也为商用软件的谱图相似性计算提供了新的方案。

tPA溶栓引起的出血性转化机制及小分子化合物干预作用研究进展

组织型纤溶酶原激活剂(tissue type plasmin activator,tPA)是美国食品药品监督管理局唯一批准的用于急性缺血性卒中治疗的药物,但由于治疗时间窗狭窄以及会导致严重的出血性转化(hemorrhagic transformation,HT),其临床应用受到限制。本文拟从血脑屏障破坏、神经炎症、氧化应激以及亚硝酸应激等方面对HT发展的机制及近7年来发表在国内外期刊上的小分子化合物对HT保护的研究进展予以综述,为缺血性中风的新药开发和药物联用提供一定参考。

植物根际促生菌:作用机制与未来

能够促进植物生长的植物根际细菌统称为植物根际促生菌(PGPR)。PGPR的应用可以减少农药化肥的使用,有助于农业可持续性发展。过去二十多年中,围绕PGPR功能与作用机理相关的理论与应用研究取得了显著进展。本文旨在总结PGPR的促生与生防机制,重点探讨PGPR在参与植物营养元素的改善与摄取、产生植物激素、分泌信号小分子、合成挥发性有机化合物促进植物生长以及合成抗生素类物质拮抗植物病原菌五个方面的生物学功能,并探讨了PGPR在基础与应用两方面的后续研究方向,以期为未来PGPR相关研究提供理论参考。

小分子药物筛选技术研究现状及其应用进展

小分子药物筛选技术伴随着药物的发现正在不断更新和拓展,药物筛选技术的创新可以提高研发效率和成功率、缩短研发周期、降低成本。从基于已知活性化合物和高通量筛选等传统筛选技术,到基于结构的药物发现、基于片段的药物发现、DNA编码化合物库、蛋白降解靶向联合体等新技术,小分子药物筛选技术在不断拓宽小分子药物的市场潜力。该文将介绍目前小分子药物筛选技术整体现状,系统综述各技术及其优劣势,为小分子药物筛选新技术的发展提供重要参考。

菊科橐吾属植物中艾里莫芬烷型倍半萜化合物细胞毒性研究进展

艾里莫芬烷型倍半萜是由15个碳原子组成的双环倍半萜,主要从菊科植物橐吾属植物中提取,具有抗肿瘤、抗菌、抗炎等生物活性。艾里莫芬烷型倍半萜的基本结构类型主要包括内酯环、二聚体、呋喃环、降碳型和C19等。本文综述了艾里莫芬烷型倍半萜的相关结构及其潜在的细胞毒性,阐述其特定化学结构特点对抗肿瘤活性的影响,以期为研究其抗肿瘤的临床治疗效果提供参考。

年轻化间充质干细胞条件培养基促进小鼠创面愈合的研究

背景间充质干细胞(mesenchymal stem cell,MSC)条件培养基无细胞疗法是创伤修复领域的一种新型治疗方案,但其治疗效果受间充质干细胞状态的影响。目的探究年轻化MSC培养基对小鼠创面愈合的作用。方法建立间充质干细胞复制性衰老(传代至15~20代)模型,检测其衰老指征;使用小分子化合物[丙戊酸(valproic acid,VPA)和RepSox]逆转衰老间充质干细胞并验证逆衰老效果。分别收集衰老MSC培养基与年轻化MSC培养基。用收集到的两种条件培养基培养人成纤维细胞,使用CCK-8实验、Ki-67免疫荧光、划痕实验和Transwell实验检测这两种条件培养基对成纤维细胞增殖、迁移和侵袭能力的影响。分别用衰老MSC培养基、年轻化MSC培养基和0.9%氯化钠注射液治疗C57BL/6小鼠背部皮肤全层缺损模型,评估3种治疗方法对创面愈合的影响。结果与衰老的间充质干细胞相比,经小分子处理后衰老间充质干细胞SA-β-gal阳性率降低(P<0.01),且免疫荧光检测显示其衰老基因P21、P16表达降低(P<0.01)。经年轻化MSC培养基处理的成纤维细胞增殖、迁移和侵袭能力增强(P<0.01)。创伤模型显示,与对照组相比,年轻化MSC培养基治疗组小鼠创面愈合速度加快(P<0.01);年轻化MSC培养基治疗组小鼠创面HE染色显示表皮细胞再上皮化加快(P<0.01)、Masson染色显示创面胶原沉积量增加(P<0.01)、CD31免疫荧光染色显示创面血管量增加(P<0.01)。结论年轻化MSC培养基对小鼠创面修复或有促进作用。

分子印迹聚合物微球制备及其对5α胆甾烷吸附性能研究

采用沉淀聚合法,以胆固醇、去氧胆酸、β-谷固醇为虚拟模板,丙烯酸(AA)为功能单体,偶氮二异丁腈(AIBN)为引发剂,乙二醇二甲基丙烯酸酯(EGDMA)为交联剂,制备甾烷类分子印迹聚合物(MIPs)和空白分子印迹聚合物(NIP)。采用扫描电子显微镜(SEM)、X射线光谱(XRD)、傅里叶变换红外光谱(FT-IR)和比表面积(BET)表征聚合物形貌和结构,并考察其对甾烷类物质的吸附性能。分析结果表明,甾烷类分子印迹聚合物尺寸均一、分散性好,是表面密布孔穴的球形纳米颗粒。吸附性能研究结果表明,MIPs对5α-胆甾烷的吸附能力明显强于NIP,且三种MIPs相比,去氧胆酸、β-谷固醇分子印迹聚合物对5α-胆甾烷的吸附强于胆固醇分子印迹聚合物。通过吸附动力学研究发现,MIPs对5α-胆甾烷的吸附过程符合准二级动力学模型,主要受化学吸附控制;MIPs和NIP的等温吸附符合Langmuir等温吸附模型和Scatchard模型,表明MIPs对5α-胆甾烷具有特异选择性吸附能力,且吸附过程属于单分子层吸附,最大吸附量为0.735 mg/g。表明胆固醇、去氧胆酸、β-谷固醇三种虚拟分子印迹聚合物均对5α-胆甾烷具有较高的分子识别能力及选择性。

小分子组合诱导大鼠胚胎成纤维细胞重编程为功能性神经元

目的:建立通过小分子化合物将大鼠胚胎成纤维细胞(REF)重编程为化学诱导神经元(ciNC)的方法体系,为细胞移植治疗神经退行性疾病提供安全有效的供体细胞。方法:以裴钢研究团队建立的人成纤维细胞直接重编程为神经元的方法为基础,通过更换包被液、缓解氧化应激损伤、添加神经源性保护因子、调整毛喉素浓度和小分子去除实验对诱导培养基和成熟培养基进行优化,并通过免疫荧光法和蛋白质印迹法验证不同诱导阶段细胞的相关蛋白质。结果:以原方案进行诱导时,细胞存活率仅(34.24±2.77)%。包被液更换为基质胶后,细胞存活率上升到(45.41±4.27)%;添加褪黑素后细胞存活率提高至(67.95±5.61)%,(23.43±1.42)%的细胞转变为神经样细胞;添加小分子P7C3-A20后细胞存活率进一步提升至(76.27±1.41)%,(39.72±4.75)%的细胞转变为神经样细胞;毛喉素浓度提升至30μmol/L时,神经样细胞比例达到(55.79±1.90)%;去除SP600125后,(86.96±2.15)%细胞存活,神经样细胞生成率提高至(63.43±1.60)%。以优化后的方案诱导,可经过神经前体细胞将REF诱导为ciNC。其中,神经前体细胞能够高表达神经前体细胞标志物SRY-box转录因子2、配对盒6以及神经元特异性标志物Ⅲ类β-微管蛋白,而成纤维相关蛋白波形蛋白表达量减少。神经前体细胞在成熟培养基中诱导两周后,可见大部分细胞呈神经样细胞形态,诱导后的ciNC高表达Ⅲ类β-微管蛋白和微管相关蛋白2,而成纤维相关蛋白波形蛋白表达减少。结论:优化后的小分子组合在常氧条件下能将REF重编程为ciNC。

小分子化合物组合改善多能干细胞向脑类器官定向分化的效率

目的优化多能干细胞(PSCs)定向分化为脑类器官的条件,提高人脑类器官的分化效率。方法利用人胚胎干细胞(hESCs)系H9向人脑类器官诱导分化体系,在早期脑类器官分化到神经祖细胞的发育阶段添加小分子化合物组合,通过形态学观察不同分化阶段脑类器官中的神经祖细胞形成的效率、细胞凋亡的情况以及分化成神经元的效率,通过RT-qPCR检测标志性基因的表达量,综合评估添加的小分子化合物组合对脑类器官形成的影响。结果在脑类器官神经祖细胞发育的关键阶段(1 d~14 d),培养基中依次添加多索啡(dorsomorphine)、A83-01、GSK-3β抑制剂CHIR99021和SMAD抑制剂SB-431542,可使神经祖细胞阶段的相关标志性基因的表达量显著提高,促进特异性神经管样结构形成,脑类器官中心区域的细胞凋亡减少。结论通过使用上述4种小分子化合物组合,可明显提高早期脑类器官的形成效率,减少脑类器官中的细胞凋亡、促进神经元形成,减少培养过程中的组织结构异质性。

一种吡唑并吡啶酮类衍生物对新型冠状病毒SARS-CoV-2的抑制作用研究

目的探究3-(4-氯苯基)-1,4-二苯基-1,4,5,7-四氢-6H-吡唑并[3,4-b]吡啶-6-酮(以下简称为J1)体外抑制新型冠状病毒(SARS-CoV-2)的作用及潜在作用机制。方法采用MTT法评价化合物J1对Vero-E6、ACE2/293T和HRT18细胞的毒性。利用感染性SARS-CoV-2和人冠状病毒OC43(HCoV-OC43)检测化合物J1的抗冠状病毒活性。采用SARS-CoV-2假病毒体外细胞感染模型和时间点加药实验检测J1抑制SARS-CoV-2病毒感染靶细胞的作用阶段。通过S蛋白介导的细胞融合抑制实验检测J1是否通过阻止病毒膜融合而抑制SARS-CoV-2的进入。采用SPR实验和分子对接技术阐明J1与SARS-CoV-2 S蛋白的作用。结果J1对Vero-E6、ACE2/293T和HRT18细胞的毒性较小,半数细胞毒性浓度CC50均大于100μmol·L^(-1)。J1能显著抑制SARS-CoV-2原始株和Delta变异株的活性,半数有效浓度EC50分别为607μmol·L^(-1)和221μmol·L^(-1)。此外,J1可抑制HCoV-OC43病毒的感染,显著减少NP蛋白和mRNA的表达。分子对接结果显示,J1通过氢键作用和疏水作用力与SARS-CoV-2 S蛋白活性位点稳定结合。机制研究结果表明,J1可抑制SARS-CoV-2病毒进入靶细胞,半数抑制浓度IC50为157μmol·L^(-1)。J1浓度依赖性抑制SARS-CoV-2 S蛋白介导的细胞-细胞膜融合,SPR实验证实J1与S蛋白具有较强结合能力。结论吡唑并吡啶酮类衍生物J1通过靶向S蛋白抑制SARS-CoV-2进入靶细胞。