戊四氮诱导癫痫大鼠脑N-cadherins和NT-4的变化及灵芝酸的干预作用

目的:观察戊四氮(PTZ)诱导大鼠癫痫后脑神经型钙粘附素(N-cadherins)和神经营养因子-4(NT-4)变化及应用灵芝酸进行干预与治疗,主要研究灵芝酸预防和治疗癫痫的痫性发作的机理。方法:采用健康的雄性Wistar实验大白鼠33只,按随机分配原则分成正常对照组、癫痫模型组和灵芝酸治疗组每组11只。癫痫模型组与灵芝酸治疗组均采用诱导癫痫亚惊厥发作剂量腹腔注射戊四氮制备Wistar大白鼠慢性点燃痫性模型。在实验室冰盒低温条件下迅速提取大鼠脑组织,采用Western-Blotting方法检测大鼠脑N-cadherins和NT-4的变化。结果:实验性诱导癫痫大鼠痫性模型制备成功,灵芝酸治疗组和癫痫模型组实验动物都达到癫痫点燃的实验标准,与癫痫模型组动物相比,灵芝酸组大鼠潜伏期明显延长,但持续时间之间的差别没有显著性。实验检测Western-Blotting结果发现:PTZ诱导癫痫发作后N-cadherins蛋白表达水平明显高于正常对照组,差异有显著性(P<0.01);用灵芝酸干预后,N-cadherins的表达有明显下降,差异有显著性(P<0.01),癫痫模型组大鼠脑NT-4蛋白含量比正常对照组蛋白含量升高,差异具显著性(P<0.05);灵芝酸组大鼠脑NT-4蛋白含量较癫痫模型组蛋白含量升高,差异具显著性(P<0.05)。结论:灵芝酸作用癫痫大鼠后N-cadherins蛋白表达降低,调整机体病变神经元的兴奋性,灵芝酸还能够增强NT-4的表达从而减轻癫痫发作给神经系统造成的损伤,从而起到抗癫痫作用。

Cloning and characterization of an actin gene of Chlamys farreri and the phylogenetic analysis of mollusk actins

An actin gene (CfACT1) was cloned by using RT-PCR, 3’and 5’RACE from hemocytes of the sea scallop Chlamys farreri. The full length of the transcript is 1 535 bp, which contains a long 3’ un-translated region of 436bp and 59bp of a 5’ un-translated sequence. The open reading frame encodes a polypeptide of 376 amino acids. Sequence comparisons indicated that CfACT1 is more closely related to vertebrate cytoplasmic actins than muscle types. Phylogenetic analysis showed that molluscan actins could be generally divided into two categories: muscle and cytoplasmic, although both are similar to vertebrate cytoplasmic actins. It was also inferred that different isotypes existed in muscle or cytoplasma in mollusks. The genomic sequence of CfACT1 was cloned and sequenced. Only one intron was detected: it was located between codons 42 and 43 and different from vertebrate actin genes.

Cortactin/N-cadherin信号轴在病理性心肌肥大中的作用

目的探究皮层肌动蛋白结合蛋白(Cortactin)对异丙肾上腺素(isoprenaline,ISO)诱导的病理性心肌肥大的调控作用及其机制。方法采用ISO刺激新生大鼠心肌细胞(neonatal rat cardiomyocytes,NRCMs)24 h,在细胞水平建立心肌肥大模型;C57BL/6小鼠皮下注射ISO 1周,在动物水平建立心肌肥大模型。采用RT-qPCR检测mRNA的变化;免疫印迹法检测相应蛋白含量的变化;免疫荧光法检测Cortactin的亚细胞定位及表达量的变化;采用腺病毒感染的方法过表达Cortactin,通过转染小干扰RNA敲低Cortactin。结果在细胞和动物水平上,成功建立ISO诱导的心肌肥大模型,均观察到ISO引起Cortactin和N型钙黏连蛋白(N-cadherin)水平降低;过表达Cortactin可逆转ISO导致的N-cadherin蛋白水平的降低及心肌细胞肥大反应;敲低Cortactin则显示相反的效应。结论Cortactin可能联合N-cadherin通过增强心肌细胞之间的连接,发挥抗心肌肥大的作用。

异土木香内酯通过miR-5195-3p/KLF5/N-cadherin对肝纤维化的作用机制

目的探讨异土木香内酯(isoalantolactone,IAL)通过miR-5195-3p/KLF5/N-cadherin对肝纤维化的作用机制。方法将SD大鼠随机分为正常组、模型组、IAL低、中、高剂量(IAL组)、IAL+miR-5195-3p mimic组和奥贝胆酸组,每组20只。采用ELISA法检测炎症因子水平,HE染色和天狼星红染色观察病理学改变情况和纤维化分级、评分情况,比色法测定氧化和抗氧化酶水平;qRT-PCR和Western blotting检测肝脏组织各因子mRNA和相关蛋白表达。结果与正常组相比,模型组ALT、AST、IL-6、IL-β、TNF-α、MDA、KLF5 mRNA、N-cadherin mRNA水平及肝纤维化分级、Ishak评分明显升高(P均<0.05)。与模型组相比,IAL低、中、高剂量组和奥贝胆酸组的以上各项指标依次显著降低(P均<0.05)。与正常组相比,模型组的SOD、GSH-Px、miR-5195-3p水平明显降低(P均<0.05),与模型组相比,IAL低、中、高剂量组和奥贝胆酸组的以上指标水平依次显著升高(P均<0.05)。与正常组相比,模型组大鼠肝脏小叶炎症明显升高,并发生严重损伤,与模型组相比,IAL低、中、高剂量组和奥贝胆酸组肝脏病理状态依次减轻。荧光素酶报告发现,KLF5是miR-5195-3p的重要靶点。与IAL组相比,IAL+miR-5195-3p mimic组肝脏损伤明显减轻,纤维化程度明显改善。结论IAL可以明显改善肝功能及炎症因子水平,增强肝脏的抗氧化能力,减轻肝脏脂质过氧化损伤程度,对肝脏纤维化具有一定的防治疗作用,这可能与IAL通过提高miR-5195-3p表达,降低KLF5和N-cadherin水平有关。

PCDH17 restricts dendritic spine morphogenesis by regulating ROCK2-dependent control of the actin cytoskeleton,modulating emotional behavior

Proper regulation of synapse formation and elimination is critical for establishing mature neuronal circuits and maintaining brain function.Synaptic abnormalities,such as defects in the density and morphology of postsynaptic dendritic spines,underlie the pathology of various neuropsychiatric disorders.Protocadherin 17(PCDH17)is associated with major mood disorders,including bipolar disorder and depression.However,the molecular mechanisms by which PCDH17 regulates spine number,morphology,and behavior remain elusive.In this study,we found that PCDH17 functions at postsynaptic sites,restricting the number and size of dendritic spines in excitatory neurons.Selective overexpression of PCDH17 in the ventral hippocampal CA1 results in spine loss and anxiety-and depression-like behaviors in mice.Mechanistically,PCDH17 interacts with actin-relevant proteins and regulates actin filament(F-actin)organization.Specifically,PCDH17 binds to ROCK2,increasing its expression and subsequently enhancing the activity of downstream targets such as LIMK1 and the phosphorylation of cofilin serine-3(Ser3).Inhibition of ROCK2 activity with belumosudil(KD025)ameliorates the defective F-actin organization and spine structure induced by PCDH17 overexpression,suggesting that ROCK2 mediates the effects of PCDH17 on F-actin content and spine development.Hence,these findings reveal a novel mechanism by which PCDH17 regulates synapse development and behavior,providing pathological insights into the neurobiological basis of mood disorders.

Scinderin promotes glioma cell migration and invasion via remodeling actin cytoskeleton

BACKGROUND Glioma is one of the most common intracranial tumors,characterized by invasive growth and poor prognosis.Actin cytoskeletal rearrangement is an essential event of tumor cell migration.The actin dynamics-related protein scinderin(SCIN)has been reported to be closely related to tumor cell migration and invasion in several cancers.AIM To investigate the role and mechanism of SCIN in glioma.METHODS The expression and clinical significance of SCIN in glioma were analyzed based on public databases.SCIN expression was examined using real-time quantitative polymerase chain reaction and Western blotting.Gene silencing was performed using short hairpin RNA transfection.Cell viability,migration,and invasion were assessed using cell counting kit 8 assay,wound healing,and Matrigel invasion assays,respectively.F-actin cytoskeleton organization was assessed using F-actin staining.RESULTS SCIN expression was significantly elevated in glioma,and high levels of SCIN were associated with advanced tumor grade and wild-type isocitrate dehydrogenase.Furthermore,SCIN-deficient cells exhibited decreased proliferation,migration,and invasion in U87 and U251 cells.Moreover,knockdown of SCIN inhibited the RhoA/focal adhesion kinase(FAK)signaling to promote F-actin depolymerization in U87 and U251 cells.CONCLUSION SCIN modulates the actin cytoskeleton via activating RhoA/FAK signaling,thereby promoting the migration and invasion of glioma cells.This study identified the cancer-promoting effect of SCIN and provided a potential therapeutic target for the treatment of glioma.

Dynamics of perinuclear actin ring regulating nuclear morphology

Cells are capable of sensing and responding to the extracellular mechanical microenvironment via the actin skeleton.In vivo,tissues are frequently subject to mechanical forces,such as the rapid and significant shear flow encountered by vascular endothelial cells.However,the investigations about the transient response of intracellular actin networks under these intense external mechanical forces,their intrinsic mechanisms,and potential implications are very limited.Here,we observe that when cells are subject to the shear flow,an actin ring structure could be rapidly assembled at the periphery of the nucleus.To gain insights into the mechanism underlying this perinuclear actin ring assembly,we develop a computational model of actin dynamics.We demonstrate that this perinuclear actin ring assembly is triggered by the depolymerization of cortical actin,Arp2/3-dependent actin filament polymerization,and myosin-mediated actin network contraction.Furthermore,we discover that the compressive stress generated by the perinuclear actin ring could lead to a reduction in the nuclear spreading area,an increase in the nuclear height,and a decrease in the nuclear volume.The present model thus explains the mechanism of the perinuclear actin ring assembly under external mechanical forces and suggests that the spontaneous contraction of this actin structure can significantly impact nuclear morphology.

Analysis and Review of Downregulated Actin Cytoskeletal Proteins in Non-Small Cell Lung Cancer

Actin, a highly conserved protein, plays a dominant role in Non-small cell lung cancer (NSCLC). Late diagnosis and the aggressive nature of NSCLC pose a significant threat. Studying the clinic pathological properties of NSCLC proteins is a potential alternative for developing treatment strategies. Towards this, 35 downregulated actin cytoskeletal proteins on NSCLC prognosis and treatment were studied by examining their protein-protein interactions, gene ontology enrichment terms, and signaling pathways. Using PubMed, various proteins in NSCLC were identified. The protein-protein interactions and functional associations of these proteins were examined using the STRING database. The focal adhesion signaling pathway was selected from all available KEGG and Wiki pathways because of its role in regulating gene expression, facilitating cell movement and reproduction, and significantly impacting NSCLC. The protein-protein interaction network of the 35 downregulated actin cytoskeleton proteins revealed that ACTG1, ACTR2, ACTR3, ANXA2, ARPC4, FLNA, TLN1, CALD1, MYL6, MYH9, MYH10, TPM1, TPM3, TPM4, PFN1, IQGAP1, MSN, and ZXY exhibited the highest number of interactions. Whereas HSPB1, CTNNA1, KRT17, KRT7, FLNB, SEPT2, and TUBA1B displayed medium interactions, while UTRN, TUBA1B, and DUSP23 had relatively fewer interactions. It was discovered that focal adhesions are critical in connecting membrane receptors with the actin cytoskeleton. In addition, protein kinases, phosphatases, and adapter proteins were identified as key signaling molecules in this process, greatly influencing cell shape, motility, and gene expression. Our analysis shows that the focal adhesion pathway plays a crucial role in NSCLC and is essential for developing effective treatment strategies and improving patient outcomes.

SERPINE1在胃癌中的表达及对胃癌细胞免疫逃逸和β-catenin/N-cadherin信号通路的作用机制研究

目的 探讨丝氨酸蛋白酶抑制剂E1(SERPINE1)在胃癌中的表达及对胃癌细胞免疫逃逸及β-连环蛋白/神经钙黏蛋白(β-catenin/N-cadherin)信号通路的作用机制。方法 选择2020年1月至2022年1月在定州市人民医院接受胃癌手术的78例患者作为研究对象,收集其胃癌组织及癌旁组织,采用免疫组织化学法检测组织中SERPINE1表达情况。采用人胃癌细胞系SGC-7901细胞,经不同处理后分为对照组(SGC-7901细胞)、oe-SERPINE1组(pcDNA3.1-SERPINE1载体)、oe-NC组(空pcDNA3.1载体)、SERPINE1 siRNA组(SERPINE1 siRNA)及si-NC组(si-NC)。采用实时荧光定量PCR法检测各组细胞中SERPINE1 mRNA的表达水平,采用Transwell法检测各组细胞的侵袭数目,采用划痕实验检测各组细胞的迁移距离,采用蛋白质印迹法检测各组细胞中程序性死亡受体-1(PD-1)、程序性死亡配体-1(PD-L1)、β-catenin、N-cadherin的蛋白表达水平。结果 胃癌组织和癌旁组织中SERPINE1阳性表达率分别为88.46%和19.23%,差异有统计学意义(P<0.05)。oe-NC组细胞的SERPINE1 mRNA表达水平、侵袭数目、迁移距离,以及PD-1、PD-L1、β-catenin、N-cadherin蛋白表达水平与对照组差异均无统计学意义(P均>0.05)。oe-SERPINE1组细胞的上述指标均较oe-NC组显著升高(P均<0.05)。si-NC组细胞的上述指标均较oe-SERPINE1组显著降低(P均<0.05)。SERPINE1siRNA组细胞的上述指标均较si-NC组显著降低(P均<0.05)。结论 SERPINE1在胃癌组织中呈高表达,下调SERPINE1表达可显著抑制胃癌细胞的侵袭和迁移,抑制PD-1、PD-L1表达从而阻止肿瘤细胞发生免疫逃逸,这与抑制β-catenin/N-cadherin信号通路的活性相关。

益肾通癃汤对前列腺癌模型裸鼠N-cadherin、Vimentin蛋白表达的影响

目的:探究益肾通癃汤对前列腺癌(PCa)皮下种植瘤模型裸鼠N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)表达的影响。方法:用前列腺癌PC-3细胞悬液建立PCa皮下种植瘤模型裸鼠,造模成功后将其随机分为模型组、益肾通癃汤低剂量组、益肾通癃汤中剂量组、益肾通癃汤高剂量组,每组10只。各给药组灌胃给予相应药物,模型组灌胃给予等体积生理盐水,2次/d,连续3周。给药结束后,观察裸鼠一般状态,测量体质量及瘤体大小,称重并计算抑瘤率;运用HE染色观察各组裸鼠种植瘤病理学改变;ELISA法检测各组裸鼠血清中N-cadherin、Vimentin的水平;Western blotting检测各组裸鼠种植瘤组织中N-cadherin、Vimentin蛋白表达情况;PCR检测各组裸鼠种植瘤组织中N-cadherin m RNA、Vimentin m RNA表达情况。结果:与模型组比较,益肾通癃汤低、中、高剂量组裸鼠状态均可,瘤体质量,血清中N-cadherin、Vimentin水平,种植瘤组织中N-cadherin、Vimentin的蛋白及m RNA相对表达量均降低(P<0.05),瘤体细胞少量坏死;与益肾通癃汤低剂量组比较,益肾通癃汤中、高剂量组裸鼠一般状态可,瘤体质量,血清中N-cadherin、Vimentin水平、种植瘤组织中N-cadherin、Vimentin蛋白及mRNA相对表达量均降低(P<0.05),瘤体细胞部分坏死;与益肾通癃汤中剂量组比较,益肾通癃汤高剂量组裸鼠一般状态良好,瘤体质量,血清中N-cadherin水平,种植瘤组织中N-cadherin、Vimentin蛋白相对表达量,种植瘤组织中Vimentin mRNA相对表达量均降低(P<0.05),瘤体细胞坏死多;益肾通癃汤高剂量组裸鼠血清中Vimentin水平、种植瘤组织中N-cadherin mRNA相对表达量与益肾通癃汤中剂量组比较,差异无统计学意义(P>0.05);益肾通癃汤低、中、高剂量组裸鼠抑瘤率分别为26.67%、42.45%、66.26%。结论:益肾通癃汤对人前列腺癌PC-3细胞皮下种植瘤模型裸鼠N-cadherin、Vimentin蛋白的表达具有一定的抑制作用,其机制可能与抑制肿瘤上皮间质转化(EMT)过程相关。

Regulation of actin cytoskeleton via photolithographic micropatterning

Actin cytoskeleton plays crucial roles in various cellular functions.Extracellular matrix(ECM)can modulate cell morphology by remodeling the internal cytoskeleton.To define how geometry of ECM regulates the organization of actin cytoskeleton,we plated individual NIH 3T3 cells on micropatterned substrates with distinct shapes and sizes.It was found that the stress fibers could form along the nonadhesive edges of T-shaped pattern,but were absent from the opening edge of V-shaped pattern,indicating that the organization of actin cytoskeleton was dependent on the mechanical environment.Furthermore,a secondary actin ring was observed on 50μm circular pattern while did not appear on 30μm and 40μm pattern,showing a size-dependent organization of actin cytoskeleton.Finally,osteoblasts,MDCK and A549 cells exhibited distinct organization of actin cytoskeleton on T-shaped pattern,suggesting a cell-type specificity in arrangement of actin cytoskeleton.Together,our findings brought novel insight into the organization of actin cytoskeleton on micropatterned environments.

Inhibiting phosphatase and actin regulator 1 expression is neuroprotective in the context of traumatic brain injury

Studies have found that the phosphatase actin regulatory factor 1 expression can be related to stroke,but it remains unclear whether changes in phosphatase actin regulatory factor 1 expression also play a role in traumatic brain injury.In this study we found that,in a mouse model of traumatic brain injury induced by controlled cortical impact,phosphatase actin regulatory factor 1 expression is increased in endothelial cells,neurons,astrocytes,and microglia.When we overexpressed phosphatase actin regulatory factor 1 by injection an adeno-associated virus vector into the contused area in the traumatic brain injury mice,the water content of the brain tissue increased.However,when phosphatase actin regulatory factor 1 was knocked down,the water content decreased.We also found that inhibiting phosphatase actin regulatory factor 1 expression regulated the nuclear factor kappa B signaling pathway,decreased blood-brain barrier permeability,reduced aquaporin 4 and intercellular adhesion molecule 1 expression,inhibited neuroinflammation,and neuronal apoptosis,thereby improving neurological function.The findings from this study indicate that phosphatase actin regulatory factor 1 may be a potential therapeutic target for traumatic brain injury.

Bioprocess-inspired Actin Biomineralized Hematite Mesocrystals for Energy Storage

Biomineralization is a biological process of synthesizing inorganic minerals within organisms.It has been found that intracellular proteins are involved in the room temperature synthesis process of anatase Ti O2in living mussels.Here,we used intracellular actin to synthesize hematite by biomineralization.Biomineralized hematite has a nano spindle structure with a particle size of approximately 150 nm.The microstructure indicates that the prepared hematite is a mesocrystals composed of ordered arrangement and assembly of primary nanoparticles.In addition,hematite mesocrystals exhibit good lithium storage performance as electrode materials for lithium batteries.The discharge specific capacity of the battery remained at 560.7 m Ah·g^(-1)after 130 cycles at a current density of 200 m A·g^(-1).This work expands the synthesis methods of hematite by biomineralization,and provides a new strategy for preparing inorganic materials by intracellular proteins.

Holistic Approach in the Treatment of Actinic Keratosis: Benefits and Disadvantages of 5-Fluorouracil, Imiquimod, Diclofenac and Curaderm

Background Actinic keratosis is the most prevalent premalignant skin disorder in the white population. Current guidelines provide no clear recommendations about preferred treatments. Methods The parameters;effectiveness, treatment duration, recurrence, side effects and cost of treatment were investigated for three frequently used topical therapies which were then compared with a most recent developed topical therapy. Published clinical data obtained from the literature was used to compare these parameters for 5-fluorouracil, imiquimod and diclofenac and relate them with the newly developed Curaderm. Results A wide variation in the concentrations of the active anti-keratotic ingredients, application frequency, duration of treatment, recurrence rates and cost of treatment exist between the different topical therapies. The efficacy rates and side effects were less variable. Overall, Curaderm is the most suitable treatment for actinic keratosis. Clinical evidence is presented illustrating the effects of Curaderm on field-directed treatments and solitary treatments of actinic keratoses. Conclusions Current medical guidelines do not provide clear recommendations on which treatment approach for actinic keratosis is preferred. Direct head-to-head comparison between treatments with emphasis on efficacy, safety, treatment duration, compliance, convenience, cosmetic outcome, patient acceptance and cost should be available to the patient, the practising physician, healthcare system and should assist in therapeutic treatment guidelines and policymaking. Given the very favourable profiles of these parameters with Curaderm when compared with other home-based treatments, it should be considered that Curaderm is first-in-line.

防风提取物对IgE致敏肥大细胞的改善作用及机制研究

[目的]探究防风(Saposhnikovia divaricata,SD)提取物对大鼠嗜碱性白血病细胞RBL-2H3脱颗粒的影响及作用机制。[方法]采用噻唑蓝(methylthialazole tetrazolium,MTT)比色法检测,根据5、25、50、100、200、400μg·mL^(-1)SD提取物对RBL-2H3细胞活性的影响,确定后续实验浓度。以免疫球蛋白E(immunoglobulinE,IgE)诱导建立RBL-2H3细胞脱颗粒模型。设立空白对照组、模型组、低剂量SD提取物组(5μg·mL^(-1))、中剂量SD提取物组(25μg·mL^(-1))、高剂量SD提取物组(50μg·mL^(-1))和地塞米松(dexamethasone,DXMS)组(100μg·mL^(-1)),干预30 min。MTT法检测低、中、高剂量SD提取物对RBL-2H3细胞脱颗粒模型活性的影响。甲苯胺蓝染色观察脱颗粒细胞形态,计算细胞脱颗粒率。免疫荧光染色测定细胞F-肌动蛋白(F-actin)表达。酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测细胞β-氨基己糖苷酶、组胺、白细胞介素-4(interleukin-4,IL-4)、白细胞介素-6(interleukin-6,IL-6)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、干扰素-γ(interferon-γ,IFN-γ)水平。免疫印迹法检测细胞磷脂酰肌醇-3-羟基激酶(phosphoinositide-3 kinase,PI3K)、磷酸化-PI3K(phosphorylation-PI3K,p-PI3K)、蛋白激酶B(protein kinase B,AKT)、磷酸化-AKT(phosphorylation-AKT,p-AKT)、p38丝裂原活化蛋白激酶(p38 mitogen activited protein kinaseelisa,p38MAPK)、磷酸化-p38MAPK(phosphorylation-p38MAPK,p-p38MAPK)、核因子-κB(nuclear factor-κB,NF-κB)、磷酸化-NF-κB(phosphorylation-NF-κB,p-NF-κB)、细胞外调节激酶(extracellular regulated kinases,ERK)、磷酸化-ERK(phosphorylation-ERK,p-ERK)蛋白表达。[结果]低、中、高剂量防风提取物(5、25、50μg·mL^(-1))对RBL-2H3细胞活性无显著影响(P>0.05)。与空白对照组比较,模型组甲苯胺蓝染色细胞数量减少、形态变圆,细胞脱颗粒率显著上升,F-actin表达下降,β-氨基己糖苷酶、组胺、IL-4、IL-6、TNF-α水平升高,IFN-γ水平降低,p-PI3K/PI3K、p-AKT/AKT、p-p38MAPK/p38MAPK、p-NF-κB/NF-κB、p-ERK/ERK表达升高(P<0.01)。与模型组比较,低、中、高剂量SD提取物组和DXMS组细胞F-actin表达增加,β-氨基己糖苷酶、组胺、IL-4、IL-6、TNF-α释放显著下降(P<0.05,P<0.01),IFN-γ释放显著增加(P<0.01),p-PI3K/PI3K、p-AKT/AKT、p-p38MAPK/p38MAPK、p-NF-κB/NF-κB、p-ERK/ERK表达降低(P<0.05,P<0.01);中、高剂量SD提取物组和DXMS组细胞数量增加,形态多呈梭形,细胞脱颗粒率显著下降(P<0.01)。与低剂量SD提取物组比较,高剂量SD提取物组和DXMS组细胞脱颗粒率下降(P<0.01)、F-actin表达增加(P<0.05)、p-p38MAPK/p38MAPK表达降低(P<0.01)。[结论]SD提取物可抑制IgE致敏的RBL-2H3细胞脱颗粒,降低炎性介质水平,其作用机制可能与抑制PI3K/AKT、p38MAPK/NF-κB、ERK蛋白磷酸化有关。

草地贪夜蛾β-Actin基因的原核表达及多克隆抗体制备

草地贪夜蛾是一种重要的农业害虫,其迁飞性强、寄主广、繁殖力高,给我国农业经济带来了严重损失。从分子水平探究草地贪夜蛾生长、发育、繁殖、抗药性形成的内在分子机理,能为虫害的防治、防控提供重要参考。β-Actin作为一种高度保守的看家蛋白常在分子生物学中用作内参去定量目标蛋白的表达水平,因而获得高质量的β-Actin抗体是从事相关分子研究的基本前提。因此,本研究克隆了草地贪夜蛾β-Actin基因部分编码序列,长度为579 bp。利用原核表达系统诱导获得了约37 kD的β-Actin重组蛋白,将纯化后的重组蛋白免疫新西兰大白兔制备得到β-Actin多克隆抗体血清。经ELISA检测,获得的抗血清效价达1∶256000。利用制备的β-Actin抗血清进行Western blot试验,结果显示在42 kD处出现强且单一的蛋白条带,说明制备的β-Actin抗血清能有效识别草地贪夜蛾β-Actin蛋白。综上,本研究制备的β-Actin多克隆抗体效价高、特异性较强,能为草地贪夜蛾后续相关分子研究提供基本条件。

铁死亡诱导剂RAS合成致死分子3抑制病理性瘢痕成纤维细胞的纤维化

背景:病理性瘢痕主要表现为异常的细胞外基质积累和过度的成纤维细胞增殖,成纤维细胞过度增殖就会产生大量以胶原纤维为主的细胞外基质。因此深入探讨成纤维细胞纤维化在病理性瘢痕形成中的作用,将为揭示病理性瘢痕的机制和生物学治疗提供新思路。目的:探讨铁死亡诱导剂RAS合成致死分子3(RAS-selective lethal small molecule 3,RSL3)对人病理性瘢痕成纤维细胞纤维化的影响。方法:收集10例宁夏医科大学总医院烧伤整形美容科提供的病理性瘢痕组织和同一个体正常皮肤组织,提取人病理性瘢痕成纤维细胞和人正常皮肤成纤维细胞用于后续实验;苏木精-伊红染色观察病理性瘢痕组织和正常皮肤组织的形态;倒置显微镜观察病理性瘢痕成纤维细胞和正常皮肤成纤维细胞的外观形态;免疫荧光实验验证所提取的细胞是否为成纤维细胞;用不同浓度的RSL3(1,3,5,7,9,11,13μmol/L)干预细胞,CCK-8法检测RSL3作用于成纤维细胞的半数抑制浓度(IC_(50));设置对照组(不做处理)和RSL3干预组(用7μmol/L的RSL3干预细胞24 h),qRT-PCR和Western blot检测谷胱甘肽过氧化物酶4、Ⅰ型胶原蛋白、Ⅲ型胶原蛋白和α-平滑肌肌动蛋白的mRNA和蛋白的表达;检测细胞丙二醛浓度;划痕试验检测细胞划痕后24 h剩余划痕面积,并计算剩余划痕面积百分比。结果与结论:①与正常皮肤组相比,病理性瘢痕组的谷胱甘肽过氧化物酶4高表达(mRNA:t=3.252,P<0.01;蛋白:t=5.075,P<0.01);②与正常皮肤成纤维细胞组相比,病理性瘢痕成纤维细胞组的谷胱甘肽过氧化物酶4高表达(mRNA:t=10.32,P<0.01;蛋白:t=26.22,P<0.01);③与对照组相比,RSL3干预组谷胱甘肽过氧化物酶4表达减少(mRNA:t=2.798,P<0.05;蛋白:t=4.643,P<0.01),丙二醛浓度上升(t=2.917,P<0.05),Ⅰ型胶原蛋白(mRNA:t=15.84,P<0.01;蛋白:t=4.610,P<0.01)、Ⅲ型胶原蛋白(mRNA:t=28.86,P<0.01;蛋白:t=7.713,P<0.01)和α-平滑肌肌动蛋白(mRNA:t=2.671,P<0.05;蛋白:t=7.417,P<0.01)的表达减少,迁移能力减弱(t=14.06,P<0.01);④提示RSL3通过抑制谷胱甘肽过氧化物酶4的表达,进而抑制病理性瘢痕成纤维细胞的纤维化和迁移能力。

ACTN1在头颈部鳞癌中的表达及其与预后和免疫浸润的关系

目的通过生物信息学方法探讨肌动蛋白a1(ACTN1)在头颈部鳞癌(HNSCC)中的表达及其与预后和免疫细胞浸润的关系。方法从TCGA数据库中下载头颈部鳞癌的数据集及分析ACTN1 mRNA在HNSCC组织中的表达情况,利用GEPIA数据库分析ACTN1 mRNA表达水平与预后的关系;采用HPA数据库分析HNSCC组织及正常组织中ACTN1蛋白表达水平及定位情况,TIMER数据库分析HNSCC组织中ACTN1表达水平与免疫细胞浸润程度的相关性,UALCAN数据库筛选HNSCC组织中与ACTN1的共表达基因,采用DAVID数据库对共表达基因进行GO和KEGG富集分析。结果与正常组织相比,HNSCC组织中ACTN1 mRNA表达水平明显升高,差异有统计学意义(P<0.05);与TP53基因未突变的HNSCC组织相比,TP53基因突变HNSCC组织中ACTN1 mRNA表达水平明显升高,差异有统计学意义(P<0.05)。ACTN1高表达和低表达组患者的中位生存时间分别为32.72个月和57.31个月,K-M曲线显示,ACTN1 mRNA高表达组总生存率相对更差(HR=1.500,P=0.002)。免疫组化结果显示,HPA数据库中4例HSNCC组织中ACTN1均为高表达,而1例正常口腔黏膜组织中ACTN1为低表达,ACTN1在HNSCC组织中主要表达于细胞质中,呈现棕黄色颗粒。在HNSCC中,ACTN1 mRNA表达水平与B细胞(r=-0.168,P<0.001)和CD8^(+)T细胞(r=-0.198,P<0.001)浸润程度呈明显负相关,与CD4^(+)T细胞(r=0.217,P<0.001)、巨噬细胞(r=0.099,P=0.030)、中性粒细胞(r=0.218,P<0.001)和树突状细胞(r=0.165,P<0.001)浸润程度呈显著正相关。在HNSCC中,与ACTN1具有显著正相关性和负相关性基因分别有1501个和258个。GO和KEGG富集分析结果显示,HNSCC组织中与ACTN1共表达的50个基因主要富集于上皮细胞迁移的调控及蛋白质转运、足细胞黏附、细胞-细胞连接等、钙粘着蛋白及肌动蛋白结合、肌动蛋白骨架的调控等。结论ACTN1在HNSCC组织中显著高表达,可作为HNSCC预后不良的标志物,ACTN1可能通过调节免疫细胞浸润参与HNSCC的发生发展。

血清长链非编码RNA肌动蛋白纤维相关蛋白1-反义RNA1水平与钙化性主动脉瓣狭窄病人左心室功能的相关性研究

目的 分析血清长链非编码RNA(lncRNA)肌动蛋白纤维相关蛋白1-反义RNA1(AFAP1-AS1)表达水平与钙化性主动脉瓣狭窄(CAS)病人左心室收缩及舒张功能的相关性。方法 于2020年1月至2021年12月,选取中国中医科学院广安门医院就诊的CAS病人129例作为CAS组[左心室射血分数(LVEF)≥50%],同期该院健康志愿者130例作为对照组。收集病人人口学资料、超声及实验室生化指标,检测血清lncRNA AFAP1-AS1表达。受试者操作特征曲线(ROC曲线)分析血清lncRNA AFAP1-AS1诊断CAS效能。结果 对照组血清lncRNA AFAP1-AS1表达水平(1.15±0.18)低于CAS组(1.58±0.30)(P<0.001)。轻度狭窄者血清lncRNA AFAP1-AS1表达水平(1.37±0.26)低于中、重度狭窄者,而中度狭窄者lncRNA AFAP1-AS1表达水平(1.59±0.30)低于重度狭窄者(1.79±0.34)(P<0.001)。ROC结果显示,血清lncRNA AFAP1-AS1诊断CAS、重度狭窄的曲线下面积分别为0.86[95%CI:(0.82,0.91)]、0.88[95%CI:(0.82,0.94)]。CAS组AVA水平低于对照组(P<0.001),左室舒张末期内径(LVEDD)、左室舒张末期容积(LVEDV)、室间隔厚度(IVST)、左室后壁厚度(LVPWT)、左房前后径(LAD)、主动脉瓣平均压差(PGmean)、主动脉瓣峰值流速(Vmax)水平高于对照组(均P<0.001)。相关性分析显示,血清lncRNA AFAP1-AS1与LVEDD、Vmax、二尖瓣口舒张早期血流速度峰值(E峰)、二尖瓣口舒张晚期血流速度峰值(A峰)、LVEDV、PGmean、LVESD呈正相关(r=0.60、0.66、0.72、0.68、0.56、0.57、0.50,均P<0.001),与LVEF、AVA呈负相关(r=-0.78、-0.62,均P<0.001)。结论 CAS病人血清lncRNA AFAP1-AS1表达水平升高,与CAS病情严重程度以及左心室舒张、收缩功能有关,并可作为无创血清标志物辅助临床诊断CAS。