Protein Disulfide Isomerase and Its Potential Function on Endoplasmic Reticulum Quality Control in Diatom Phaeodactylum tricornutum

PDI is a molecular chaperone and plays an important role in Endoplasmic Reticulum quality control (ERQC).PDI participates in the refolding of the misfolded/unfolded proteins to maintain cellular homeostasis under differentstresses. However, bioinformatic characteristics and potential functions of PDIs in diatom Phaeodactylumtricornutum (Pt) are still unknown so far. Hence, the genome-wide characteristics of PtPDI proteins in P. tricornutumwere first studied via bioinformatic and transcriptomic methods. 42 PtPDI genes were identified from thegenome of P. tricornutum. The motif, protein structure, classification, number of introns, phylogenetic relationship,and the expression level of 42 PtPDI genes under the tunicamycin stress were analyzed. A pair of tandemduplicated genes (PtPDI15 and PtPDI18) was observed in P. tricornutum. The 42 PtPDIs with different genecharacteristics were divided into three independent clades, indicating different evolutional relationships and functionsof these PtPDIs. The 14 up-regulated PtPDI genes under the tunicamycin treatment might have a positiveeffect on the ER quality control of the unfolded/misfolded proteins, while the 7 down-regulated PtPDIs mightnegatively affect the ERQC. The characteristics of all 42 PtPDIs and their proposed working model here providea comprehensive understanding of the PtPDIs gene family. The differential expression of 21 PtPDIs will be usefulfor further functional study in the ERQC.

Cloning and characterization of a novel gene encoding a putative seven-span transmembrane protein localized in endoplasmic reticulum

Objective: To clone and analyze the structure of a novel gene, named EST 1 (endoplasmic reticulum localized seven span transmembrane protein 1) and to analyze the expression pattern and intracellular location of EST 1. Methods: The cDNA library was screened to isolate novel cDNA fragment. The structure of novel gene was analysed by computer software. Expression of EST 1 was analyzed by dot blot and Northern blotting. Intracellular localization was observed after EST 1 enhanced green fluorescence protein (EGFP) fusion gene was transfected into mammalian cells. Results: The full length cDNA of mouse EST 1 was 1 802 bp, with a 1 293 bp open reading frame encoding 431 amino acids. It was predicated that protein encoded by EST 1 contained a signal peptide sequence at the N terminus, seven putative transmembrane domains, and an ER retaining signal at the C terminus. EST 1 EGFP fusion protein showed an ER like intracellular distribution in mammalian cells. Expression pattern analysis showed that EST 1 is expressed in all tissues examined. Conclusion: EST 1 is encoding a putative seven span transmembrane protein localized in endoplasmic reticulum. EST 1 was expressed in all tissues examined, suggesting an essential function of EST 1 in cells.

Yinchenhao decoction attenuates obstructive jaundice-induced liver injury and hepatocyte apoptosis by suppressing protein kinase RNA-like endoplasmic reticulum kinase-induced pathway

BACKGROUND Chronic biliary obstruction results in ischemia and hypoxia of hepatocytes,and leads to apoptosis.Apoptosis is very important in regulating the homeostasis of the hepatobiliary system.Endoplasmic reticulum(ER)stress is one of the signaling pathways that induce apoptosis.Moreover,the protein kinase RNA-like endoplasmic reticulum kinase(PERK)-induced apoptotic pathway is the main way;but its role in liver injury remains unclear.Yinchenhao decoction(YCHD)is a traditional Chinese medicine formula that alleviates liver injury and apoptosis,yet its mechanism is unknown.We undertook this study to investigate the effects of YCHD on the expression of ER stress proteins and hepatocyte apoptosis in rats with obstructive jaundice(OJ).AIM To investigate whether YCHD can attenuate OJ-induced liver injury and hepatocyte apoptosis by inhibiting the PERK-CCAAT/enhancer-binding protein homologous protein(CHOP)-growth arrest and DNA damage-inducible protein 34(GADD34)pathway and B cell lymphoma/leukemia-2 related X protein(Bax)/B cell lymphoma/leukemia-2(Bcl-2)ratio.METHODS For in vivo experiments,30 rats were divided into three groups:control group,OJ model group,and YCHD-treated group.Blood was collected to detect the indicators of liver function,and liver tissues were used for histological analysis.For in vitro experiments,30 rats were divided into three groups:G1,G2,and G3.The rats in group G1 had their bile duct exposed without ligation,the rats in group G2 underwent total bile duct ligation,and the rats in group G3 were given a gavage of YCHD.According to the serum pharmacology,serum was extracted and centrifuged from the rat blood to cultivate the BRL-3A cells.Terminal deoxynucleotidyl transferase mediated dUTP nick end-labelling(TUNEL)assay was used to detect BRL-3A hepatocyte apoptosis.Alanine aminotransferase(ALT)and aspartate transaminase(AST)levels in the medium were detected.Western blot and quantitative real-time polymerase chain reaction(qRT-PCR)analyses were used to detect protein and gene expression levels of PERK,CHOP,GADD34,Bax,and Bcl-2 in the liver tissues and BRL-3A cells.RESULTS Biochemical assays and haematoxylin and eosin staining suggested severe liver function injury and liver tissue structure damage in the OJ model group.The TUNEL assay showed that massive BRL-3A rat hepatocyte apoptosis was induced by OJ.Elevated ALT and AST levels in the medium also demonstrated that hepatocytes could be destroyed by OJ.Western blot or qRT-PCR analyses showed that the protein and mRNA expression levels of PERK,CHOP,and GADD34 were significantly increased both in the rat liver tissue and BRL-3A rat hepatocytes by OJ.The Bax and Bcl-2 levels were increased,and the Bax/Bcl-2 ratio was also increased.When YCHD was used,the PERK,CHOP,GADD34,and Bax levels quickly decreased,while the Bcl-2 levels increased,and the Bax/Bcl-2 ratio decreased.CONCLUSION OJ-induced liver injury and hepatocyte apoptosis are associated with the activation of the PERK-CHOP-GADD34 pathway and increased Bax/Bcl-2 ratio.YCHD can attenuate these changes.

NDRG2调控IRE1α-XBP1介导内质网应激逆转ER+乳腺癌他莫昔芬耐药

他莫昔芬(tamoxifen,TAM)作为雌激素受体阳性(estrogen receptor,ER+)乳腺癌的一线化疗药物使大多数患者受益,但原发性和继发性耐药问题严重影响临床治疗效果。深入研究ER+乳腺癌TAM耐药机制,改善治疗效果是当前亟待解决的问题。抑癌因子NDRG2(N-myc downstream regulated gene 2,NDRG2)在肿瘤发生发展中发挥重要作用,但是否参与ER+乳腺癌TAM耐药尚不清楚。本研究旨在探明NDRG2在ER+乳腺癌TAM耐药中发挥的作用和机制。通过RT-PCR与免疫印迹分析对比TAM敏感型和耐药型ER+乳腺癌细胞发现,NDRG 2的mRNA转录水平和蛋白质翻译水平在TAM耐药细胞中表达显著下调,且与耐药能力负相关(P<0.001);CCK-8细胞毒性实验和软琼脂克隆形成实验证实,在耐药细胞中过表达NDRG2可显著降低TAM药物半抑制浓度IC 50和软琼脂克隆形成率(P<0.001),逆转耐药表型。分子机制上,X-box结合蛋白1(X-box binding protein 1,XBP1)mRNA剪切实验与内质网相关降解(endoplasmic-reticulum associated degradation,ERAD)报告蛋白的结果显示,过表达NDRG2可增强耐药细胞中剪切型XBP1s mRNA转录与ERAD报告蛋白CD3ε-YFP表达(P<0.001),引发耐药细胞内质网强应激反应;免疫印迹检测结果显示,过表达NDRG2可显著提高耐药细胞中内质网应激感受器肌醇需要激酶1α(inositol requiring enzyme 1,IRE1α)的磷酸化水平及其下游因子,例如内质网EIP辅助因子(endoplasmic reticulum-localized DnaJ 4,ERdj4)、PKR蛋白激酶的细胞抑制剂(cellular Inhibitor of the PKR protein kinase,P58 IPK)、α甘露糖苷酶样应激蛋白(er degradation enhancingαmannosidase likeprotein,EDEM)和蛋白质二硫键异构酶家族A成员5(protein disulfide isomerase family a member 5,PDIA5)的表达水平(P<0.001)。小鼠异种移植瘤研究进一步证实,在耐药细胞中过表达NDRG2可增强TAM治疗效果,显著抑制耐药移植瘤生长(P<0.001)。以上研究结果表明,通过提高耐药细胞中NDRG2表达,增强TAM治疗引发的内质网强烈应激,可逆转ER+乳腺癌细胞耐药性,改善TAM治疗效果。研究结果为解决ER+乳腺癌TAM耐药问题提供了新的思路和有价值的潜在药物靶点。

过表达丝裂原诱导基因6通过抑制内质网应激减少棕榈酸诱导的肝细胞脂质蓄积

目的:探讨过表达丝裂原诱导基因6(MIG6)通过调节肝细胞内质网应激影响棕榈酸(PA)诱导的肝细胞脂肪变性的作用及其机制。方法:通过使用高脂饲料喂养20只C57BL/6J小鼠26周构建小鼠非酒精性脂肪性肝炎模型,将小鼠随机分为2组:普通饮食对照组和高脂饮食组,每组10只,留取其肝脏进行后续实验。使用PA诱导HepG2细胞和AML12细胞的脂质蓄积,将细胞分为2组:BSA(10%BSA)组和PA(500μmol/L)组。通过给HepG2细胞和AML12细胞分别转染人和小鼠MIG6过表达质粒诱导肝细胞MIG6过表达,根据不同的干预条件分为4组:阴性对照质粒+10%BSA(negative+BSA)组、MIG6过表达质粒+10%BSA(MIG6+BSA)组、阴性对照质粒+PA(negative+PA)组和MIG6过表达质粒+PA(MIG6+PA)组,每组每次干预至少设3个生物学复孔。使用油红O染色评估肝细胞脂质蓄积情况;通过RT-qPCR检测肝组织MIG6的mRNA表达水平,Western blot检测肝细胞MIG6、脂肪酸合酶(FASN)和胆固醇调节元件结合蛋白1(SREBP1)等脂质合成相关分子及内质网应激标志物葡萄糖调节蛋白78(GRP78)和C/EBP同源蛋白(CHOP)的蛋白水平。结果:高脂饮食组小鼠肝组织甘油三酯含量增加,肝组织脂肪变性显著,MIG6的mRNA表达水平显著下调(P<0.01);PA干预促使肝细胞FASN、SREBP1、GRP78和CHOP蛋白水平升高,但是降低MIG6蛋白水平(P<0.01)。转染MIG6过表达质粒显著增加肝细胞MIG6蛋白水平,并抑制GRP78和CHOP的表达(P<0.01),显著减轻PA诱导的肝细胞脂质蓄积并抑制FASN和SREBP1的表达(P<0.01)。以上结果表明过表达MIG6可抑制PA诱导的肝细胞内质网应激并减少其脂质蓄积。结论:过表达MIG6具有抑制内质网应激进而抑制肝细胞脂质蓄积的作用。

转录因子KLF16对非酒精性脂肪肝病脂质代谢的影响

【目的】探究转录因子KLF16在非酒精性脂肪肝病(NAFLD)中的表达改变以及其对脂质代谢的影响。【方法】通过高脂饮食诱导小鼠构建NAFLD的动物模型。将小鼠分为正常饮食组(ND)和高脂饮食组(HFD);油酸诱导小鼠肝脏原代细胞构建NAFLD细胞模型。将细胞分为对照组(Control)和油酸诱导组(OA);实时荧光定量PCR(RT-qPCR)和蛋白免疫印迹技术(Western blot)方法检测NAFLD动物和细胞模型中KLF16的表达变化。通过小鼠尾静脉注射腺相关病毒(AAV)和细胞瞬时转染siRNA构建KLF16敲减的体内外模型。油红染色,苏木素伊红染色(HE)等方法检测KLF16敲减前后细胞NAFLD模型中脂质沉积的变化;RT-qPCR方法检测KLF16敲减前后细胞和动物NAFLD模型中脂质代谢关键基因表达的变化;Western blot方法检测KLF16敲减前后细胞NAFLD模型中内质网应激蛋白的表达。【结果】KLF16在HFD组和OA组表达水平上调,KLF16敲低后OA组脂质沉积加重,肝细胞内TC水平组间无变化(P>0.05),TG水平出现不同程度的上升(P<0.05,P<0.001)。同时引发脂质代谢关键基因的改变,KLF16表达改变也引发OA组中内质网应激蛋白表达改变。【结论】转录因子KLF16可能通过内质网应激缓解非酒精性脂肪肝病的脂质沉积。

The emerging role of nitric oxide in the synaptic dysfunction of vascular dementia

With an increase in global aging,the number of people affected by cerebrovascular diseases is also increasing,and the incidence of vascular dementia-closely related to cerebrovascular risk-is increasing at an epidemic rate.However,few therapeutic options exist that can markedly improve the cognitive impairment and prognosis of vascular dementia patients.Similarly in Alzheimer’s disease and other neurological disorders,synaptic dysfunction is recognized as the main reason for cognitive decline.Nitric oxide is one of the ubiquitous gaseous cellular messengers involved in multiple physiological and pathological processes of the central nervous system.Recently,nitric oxide has been implicated in regulating synaptic plasticity and plays an important role in the pathogenesis of vascular dementia.This review introduces in detail the emerging role of nitric oxide in physiological and pathological states of vascular dementia and summarizes the diverse effects of nitric oxide on different aspects of synaptic dysfunction,neuroinflammation,oxidative stress,and blood-brain barrier dysfunction that underlie the progress of vascular dementia.Additionally,we propose that targeting the nitric oxide-sGC-cGMP pathway using certain specific approaches may provide a novel therapeutic strategy for vascular dementia.

溃疡性结肠炎内质网应激相关基因的综合分析

目的基于生物信息学和孟德尔随机化分析研究溃疡性结肠炎(ulcerative colitis,UC)具有诊断和治疗潜力的内质网应激(endoplasmic reticulum stress,ERS)相关基因,并进行相关基因的基因集富集分析(Gene Set Enrichment Analysis,GSEA)、基因本体论(Gene Ontology,GO)和京都基因和基因组百科全书(Kyoto Encyclopedia of Genes and Gnomes,KEGG)分析,构建ceRNA调控网络。方法从高通量基因表达数据库(Gene Expression Omnibus,GEO)获取UC患者和健康对照者的结肠组织基因表达谱及临床信息,采用差异表达分析法筛选与UC相关的差异基因,从GeneCards数据库下载ERS相关基因集,差异基因和ERS相关基因取交集得到差异ERS相关基因,利用GO和KEGG对差异ERS相关基因进行分析。采用Lasso回归、支持向量机(support vector machine,SVM)、随机森林树三种方法筛选并取交集得到UC疾病特征基因,利用表达数量性状位点(expression quantitative trait locus,eQTL)暴露数据和UC结局数据进行孟德尔随机化分析,得到具有诊断和治疗潜力的目标基因。采用GSEA、基因集变异分析(Gene Set Variation Analysis,GSVA)和CIBERSORT免疫细胞浸润分析探索目标基因与免疫细胞组成之间的相关性。预测与目标基因表达相关的miRNA和lncRNA,并构建ceRNA调控网络。结果孟德尔随机化结果显示,基因ANXA5增加UC发病风险。GSEA结果显示,目标基因主要富集通路包括趋化因子信号通路、细胞因子受体相互作用通路、造血细胞调控信号通路、JAK/STAT信号通路、利什曼原虫感染等。GSVA结果显示,补体和凝血级联等通路上调,缬氨酸亮氨酸和异亮氨酸降解等通路下调。免疫细胞浸润分析结果显示,基因ANXA5正调控中性白细胞、活化肥大细胞等,负调控静止树突状细胞、巨噬细胞M2等。鉴定出3个关键miRNA和15个lncRNA,并绘制出ceRNA调控网络。结论本研究预测了ERS相关基因ANXA5增加UC发病风险,鉴定出的miRNA、lncRNA或可作为UC的生物标志物。

From contraction to gene expression:nanojunctions of the sarco/endoplasmic reticulum deliver site-and function-specific calcium signals

Calcium signals determine, for example, smooth muscle contraction and changes in gene expression. How calcium signals select for these processes is enigmatic. We build on the "panjunctional sarcoplasmic reticulum" hypothesis, describing our view that different calcium pumps and release channels, with different kinetics and affinities for calcium, are strategically positioned within nanojunctions of the SR and help demarcate their respective cytoplasmic nanodomains. SERCA2 b and Ry R1 are preferentially targeted to the sarcoplasmic reticulum(SR) proximal to the plasma membrane(PM), i.e., to the superficial buffer barrier formed by PM-SR nanojunctions, and support vasodilation. In marked contrast, SERCA2 a may be entirely restricted to the deep, perinuclear SR and may supply calcium to this sub-compartment in support of vasoconstriction. Ry R3 is also preferentially targeted to the perinuclear SR, where its clusters associate with lysosome-SR nanojunctions. The distribution of Ry R2 is more widespread and extends from this region to the wider cell. Therefore, perinuclear Ry R3 s most likely support the initiation of global calcium waves at L-SR junctions, which subsequently propagate by calcium-induced calcium release via Ry R2 in order to elicit contraction. Data also suggest that unique SERCA and Ry R are preferentially targeted to invaginations of the nuclear membrane. Site- and function-specific calcium signals may thus arise to modulate stimulus-response coupling and transcriptional cascades.

Novel Mutations in Endoplasmic Reticulum Lipid Raft-associated Protein 2 Gene Cause Pure Hereditary Spastic Paraplegia Type 18

Hereditary spastic paraplegia type 18 （HSP18） is a complicated form ofautosomal recessive HSP characterized by progressive weakness and spasticity of the lower extremities,dysarthria,and cognitive decline. In the year 2011,HSP18,also known as Spastic Paraplegia 18 （SPG18）,was firstly identified due to a candidate gene endoplasmic reticulum lipid raft-associated protein 2 （ERLIN2） on chromosome 8pl 1.2 in one Saudis family.During the past 5 years,another two families with SPG18 due to ERLIN2 mutations have been reported presenting with complicated phenotype. Here,we reported a patient born in a nonconsanguineous family who possessed an autosomal recessive pure form of HSP owing to novel mutations in ERLIN2.Patient was characterized by late-onset spasticity of lower extremities without significant speech involvement or cognitive disability.

急性力竭运动恢复期内大鼠骨骼肌内质网应激水平变化研究

目的:研究急性力竭运动恢复期内不同时间点内质网应激关键基因表达水平,探究急性运动干预对大鼠骨骼肌内质网应激信号通路及细胞凋亡的影响。方法:将48只雄性大鼠随机分为对照组(C)、力竭运动后即刻组(E)、力竭运动后恢复3h组(E3)、力竭运动后恢复8h组(E8)、力竭运动后恢复15h组(E15)及力竭运动后恢复24h组(E24),每组8只。在不同时间点取大鼠血液及腓肠肌组织,血液指标检测乳酸脱氢酶(LDH)、肌酸激酶(CK)及C反应蛋白(CRP)含量,提取骨骼肌总RNA,采用荧光定量PCR法检测大鼠骨骼肌内质网应激基因mRNA水平,包括免疫球蛋白结合蛋白(Bip/GRP78),内质网膜上跨膜蛋白激活转录因子6(ATF-6)、PER样内质网激酶(PERK)、真核生物起始因子2(eIF2α)以及CCAAT/增强子结合蛋白同源蛋白(CHOP)等,采用ELISA检测骨骼肌Bip含量,Western Blot法检测Bip、CRT、CHOP蛋白表达水平。结果:与对照组相比:①E组血液CK、CRP显著升高(P<0.05),E3组血液LDH显著升高(P<0.05);②E组Bip、ATF6、PERK、eIF2α等内质网应激基因转录水平均显著上调,其中内质网应激关键基因Bip mRNA水平及蛋白表达水平在恢复3h时达到峰值;③E组CRT与E24组CHOP蛋白表达水平显著升高(P<0.05)。结论:①急性力竭运动后恢复期骨骼肌内质网应激关键蛋白Bip表达水平及下游相关基因mRNA水平均呈一过性上调趋势,在运动后即刻至3h间出现峰值,随着恢复期时间的延长,Bip蛋白表达水平及各基因转录水平逐渐恢复正常;②急性力竭运动后24h引发细胞凋亡,其在内质网应激效应后发生,具有一定的延迟性。

Mechanisms and treatment strategies of demyelinating and dysmyelinating Charcot-Marie-Tooth disease

Schwann cells,the myelinating glia of the peripheral nervous system,wrap axons multiple times to build their myelin sheath.Myelin is of paramount importance for axonal integrity and fast axon potential propagation.However,myelin is lacking or dysfunctional in several neuropathies including demyelinating and dysmyelinating Charcot-M arie-To oth disease.Charcot-Marie-To oth disease represents the most prevalent inherited neuropathy in humans and is classified either as axonal,demyelinating or dysmyelinating,or as intermediate.The demyelinating or dysmyelinating forms of Charcot-Marie-Tooth disease constitute the majority of the disease cases and are most frequently due to mutations in the three following myelin genes:peripheral myelin protein 22,myelin protein ze ro and gap junction beta 1(coding for Connexin 32) causing Charcot-M arie-Tooth disease type 1A,Charcot-Marie-Tooth disease type 1B,and X-linked Charcot-M arie-Tooth disease type 1,respectively.The resulting perturbation of myelin structure and function leads to axonal demyelination or dysmyelination and causes severe disabilities in affected patients.No treatment to cure or slow down the disease progression is currently available on the market,howeve r,scientific discoveries led to a better understanding of the pathomechanisms of the disease and to potential treatment strategies.In this review,we describe the features and molecular mechanisms of the three main demyelinating or dysmyelinating forms of Charcot-Marie-Tooth disease,the rodent models used in research,and the emerging therapeutic approaches to cure or counteract the progression of the disease.

基于内质网应激应答的胶质瘤风险模型构建

目的探索内质网应激应答(ERS)相关调控基因表达对胶质瘤(不包括胶质瘤母细胞瘤)病理分级、预后和恶性进展表型的预测价值。方法排除重复和无生存期的样本后,分别选取美国癌症基因组图谱和中国脑胶质瘤基因组图谱中胶质瘤临床样本测序数据作为训练集和验证集。通过差异基因富集和蛋白互作分析,构建与预后相关的ERS风险回归模型。ROC曲线分析、实时荧光定量PCR和免疫组化验证该模型对胶质瘤预后及恶性进展表型的预测价值。结果研究结果显示,7个ERS相关风险因子表达随胶质瘤级别上升而上调,并精准预测患者不良预后(在1、3和5年预测准确度均高于0.7),差异均有统计学意义。进一步验证显示,ERS风险基因与胶质瘤恶性表型标志物CD44呈正相关,与临床有利预后标志物GPR158呈负相关(P<0.05)。最后,临床样本的基因表达和免疫组化分析证实,ERS相关风险因子在较高级别胶质瘤中高表达,与CD44表达量呈正相关(P<0.05)。结论基于ERS应答的风险回归模型具备病理分级和预后的能力,并与胶质瘤的恶性进展表型呈正相关,可为胶质瘤精准靶向诊疗提供参考。

Charcot-Marie-Tooth-1A and sciatic nerve crush rat models:insights from proteomics

The sensorimotor and histological aspects of peripheral neuropathies were already studied by our team in two rat models:the sciatic nerve crush and the Charcot-Marie-Tooth-1A disease.In this study,we sought to highlight and compare the protein signature of these two pathological situations.Indeed,the identification of protein profiles in diseases can play an important role in the development of pharmacological targets.In fact,Charcot-Marie-Tooth-1A rats develop motor impairments that are more severe in the hind limbs.Therefore,for the first time,protein expression in sciatic nerve of Charcot-Marie-Tooth-1A rats was examined.First,distal sciatic nerves were collected from Charcot-Marie-Tooth-1A and uninjured wild-type rats aged 3 months.After protein extraction,sequential window acquisition of all theoretical fragment ion spectra liquid chromatography and mass spectrometry was employed.445 proteins mapped to Swiss-Prot or trEMBL Uniprot databases were identified and quantified.Of these,153 proteins showed statistically significant differences between Charcot-Marie-Tooth-1A and wild-type groups.The majority of these proteins were overexpressed in Charcot-Marie-Tooth-1A.Hierarchical clustering and functional enrichment using Gene Ontology were used to group these proteins based on their biological effects concerning Charcot-Marie-Tooth-1A pathophysiology.Second,proteomic characterization of wild-type rats subjected to sciatic nerve crush was performed sequential window acquisition of all theoretical fragment ion spectra liquid chromatography and mass spectrometry.One month after injury,distal sciatic nerves were collected and analyzed as described above.Out of 459 identified proteins,92 showed significant differences between sciatic nerve crush and the uninjured wild-type rats used in the first study.The results suggest that young adult Charcot-Marie-Tooth-1A rats(3 months old)develop compensatory mechanisms at the level of redox balance,protein folding,myelination,and axonogenesis.These mechanisms seem insufficient to hurdle the progress of the disease.Notably,response to oxidative stress appears to be a significant feature of Charcot-Marie-Tooth-1A,potentially playing a role in the pathological process.In contrast to the first experiment,the majority of the proteins that differed from wild-type were downregulated in the sciatic nerve crush group.Functional enrichment suggested that neurogenesis,response to axon injury,and oxidative stress were important biological processes.Protein analysis revealed an imperfect repair at this time point after injury and identified several distinguishable proteins.In conclusion,we suggest that peripheral neuropathies,whether of a genetic or traumatic cause,share some common pathological pathways.This study may provide directions for better characterization of these models and/or identifying new specific therapeutic targets.

在肺腺癌组织中低表达的STING可通过抑制内质网应激促进肺腺癌进展

目的:分析干扰素基因刺激因子(STING)在肺腺癌中的表达及其与肺腺癌患者临床特征间的关系,探讨STING与内质网应激的相关性及其在调控肺腺癌进展中的作用机制。方法:利用TIMER数据库分析STING基因在泛癌水平的表达情况,利用UALCAN和HPA数据库分析STING在肺腺癌组织中的表达及其与肺腺癌患者临床特征间的关系,利用Kaplan-Meier生存函数分析STING表达与肺腺癌患者OS率间的关系。利用LinkedOmics数据库对肺腺癌表达谱数据进行STING基因共表达分析,对STING相关差异表达基因(DEG)进行GO功能与KEGG通路富集分析,通过GSEA筛选STING调控肺腺癌的潜在通路。使用STING激动剂diABZI及内质网应激抑制剂TUDCA对肺腺癌A549与H460细胞进行处理,通过qPCR、WB法检测STING及内质网应激相关分子的表达,通过CCK-8法检测细胞增殖活力。结果:肺腺癌组织和细胞中STING的表达水平均显著低于正常肺组织(均P<0.01),STING高表达肺腺癌患者5年OS率显著高于低表达患者(P<0.01),STING的表达与肺腺癌患者的年龄、性别等临床特征密切相关(均P<0.01)。STING高表达在肺腺癌外源性抗原处理及提呈等通路上存在富集(均P<0.01)。使用STING激动剂可显著诱导肺腺癌细胞发生内质网应激(P<0.05),STING诱导活化后肺腺癌细胞增殖活力显著下降(均P<0.01),内质网应激抑制剂能部分恢复STING活化诱导后下降的细胞活力(P<0.05)。结论:STING基因在肺腺癌中低表达,其表达下调与肺腺癌患者预后不良相关,其机制可能是STING通过诱导内质网应激而抑制肺腺癌细胞活力。

COL10A1基因新突变导致Schmid型干骺端软骨发育不全

目的对2021年1月陆军军医大学第二附属医院接诊的1例Schmid型干骺端软骨发育不全(Schmid type metaphyseal chondrodysplasia,SMCD)家系进行疾病表型与基因型分析,结合文献复习探讨其防治手段。方法对先证者进行家系调查及系谱分析,收集先证者及家系成员的临床资料,采用全外显子组测序明确突变基因,并通过Sanger测序验证和家系共分离分析,同时结合ACMG指南进行生物信息学分析来评价变异的致病性。结果在家系患者中发现COL10A1基因存在新的杂合错义突变c.1843T>G(p.Tyr615Asp),该位点所在区域在不同物种之间高度保守,此突变可能通过影响X型胶原(α1)蛋白的三聚化及其与细胞外基质分子结合导致疾病发生。结论发现了1种SMCD的新突变,丰富了SMCD患者的突变谱,为SMCD的预防、诊断及治疗提供理论依据。

mda-7／IL-24通过内质网应激通路诱导肝癌细胞生长抑制和凋亡

目的:观察mda-7/IL-24对不同类型的肝肿瘤细胞及正常肝脏细胞增殖及凋亡的影响并探讨其可能的作用机制。方法:构建携带mda-7基因的重组腺病毒Ad-mda-7,转染肝癌细胞系HepG2、Hep3B、PLC/PRF/5和正常肝细胞L02,MTT法和流式细胞术检测细胞增殖及凋亡情况,Western印迹检测细胞相关蛋白的表达。应用钙蛋白酶抑制剂Ⅰ(ALLN,25μmol/L)预处理上述细胞30min,观察阻断内质网应激通路后上述指标的变化。结果:MTT法和流式细胞术检测结果表明,与感染Ad-GFP比较,Ad-mda-7选择性抑制肝癌细胞生长(P<0.01),诱导肝癌细胞凋亡(P<0.01,其中对HepG2细胞影响最明显),而对正常肝细胞生长无明显影响;ALLN预处理能部分抑制Ad-mda-7的上述作用。Western印迹结果表明Ad-mda-7能诱导HepG2细胞BiP/GRP78、Bax蛋白高表达(P<0.01),caspase-12、caspase-3活化及p38 MAPK磷酸化;ALLN预处理能抑制Ad-mda-7转染引起的Bax蛋白高表达及caspase-12、caspase-3活化;而对BiP/GRP78的高表达及p38 MAPK磷酸化无影响。结论:mda-7/IL-24可能通过内质网应激通路诱导肝癌细胞生长抑制和凋亡。

补肾益肺消癥方干预肺纤维化大鼠JNK凋亡信号通路关键分子的表达调控内质网应激的作用机制

目的探讨补肾益肺消癥方干预肺纤维化大鼠内质网应激和JNK细胞凋亡信号通路的作用机制。方法将SD大鼠随机分为空白组、模型组、阳性药物组、中药预防组、中药治疗组、中药防治组,除空白组外,其他组均以博莱霉素致大鼠肺纤维化模型。中药预防组及中药防治组造模1 d后开始给予补肾益肺消癥方12.68 g/(kg·d)灌胃,空白组和模型组予等量生理盐水灌胃。造模28 d后确定造模是否成功,并处死中药预防组大鼠,观察预防性给药对IPF的影响;造模成功后,阳性药物组给予吡非尼酮53.57 mg/(kg·d)灌胃,中药治疗组及中药防治组给予补肾益肺消癥方12.68 g/(kg·d)灌胃,空白组和模型组仍给予等量生理盐水灌胃,持续28 d。HE染色观察各组大鼠肺组织病理变化,real-time PCR及Westernblot检测肺组织中JNK信号通路相关因子GRP78、IRE1α、p-JNK、TRAF2基因表达及蛋白含量的差异。结果肺组织切片HE染色显示,阳性药物组、中药预防组及中药治疗组肺泡轻度破坏,成纤维细胞有一定程度增生;中药防治组肺泡结构完整,有少量成纤维细胞增生,较模型组纤维化程度明显改善;模型组肺组织中GRP78、IRE1α、p-JNK、TRAF2基因表达拷贝数比值及蛋白含量均明显高于空白组(P均<0.05);中药治疗组和中药防治组GRP78基因表达拷贝数比值及蛋白含量和TRAF2基因表达拷贝数比值均明显低于模型组(P均<0.05),阳性药物组和中药防治组IRE1α基因表达拷贝数比值均明显低于模型组(P均<0.05),阳性药物组、中药预防组、中药治疗组和中药防治组IRE1α蛋白含量均明显低于模型组(P均<0.05),中药防治组p-JNK蛋白含量明显低于模型组(P<0.05)。结论通过下调GRP78、IRE1α、p-JNK、TRAF2的水平,调控JNK信号通路,减轻内质网应激,减少细胞凋亡是补肾益肺消癥方延缓肺纤维化的作用机制之一。

内质网氨肽酶2基因rs2549782多态性与妊娠期高血压发病的相关性

目的:探讨内质网氨肽酶2(ERAP2)基因rs2549782多态性与淮安地区汉族女性妊娠期高血压发病的关系。方法:应用病例对照研究,收集2013年6月—2016年4月江苏省淮安市妇幼保健院确诊的汉族妊娠期高血压疾病患者100例为研究对象(病例组),同期住院分娩正常孕妇310例为对照(对照组),应用MassARRAY-IPLEX技术和基质辅助激光解吸离子飞行时间质谱(MALDI-TOF-MS)对ERAP2基因rs2549782位点进行基因分型。结果:2组中rs2549782位点等位基因分布差异无统计学意义(P=0.089),而基因型分布差异有统计学意义(P=0.045)。经校正混杂因素后,Logistic回归分析提示G等位基因(OR=1.64,95%CI:1.09~2.47)以及GT基因型(OR=2.23,95%CI:1.01~4.07)和GG基因型(OR=2.96,95%CI:1.20~7.31)为妊娠期高血压发生的危险因素。结论:ERAP2基因rs2549782位点单核苷酸多态性与淮安汉族妊娠期高血压疾病的发生具有相关性,G等位基因、GT和GG基因型均为妊娠期高血压的危险因素。

顺铂诱导肾小管上皮细胞程序性死亡的机理

目的:探讨顺铂导致细胞凋亡的发生部位和诱导细胞凋亡的信号传导途径,为更好地预防铂类药物对肾脏的毒副作用奠定基础。方法:将含目的基因的Bcl-2(Bcl-acta、Bcl-cb5及Bcl-nt)的pcDNA3.1(+)/Hygromycin质粒及pEGFP-C3质粒,通过共转染方式导入肾小管上皮细胞(RPTC)。并将细胞分为对照组、Bcl-acta组、Bcl-cb5组及Bcl-nt组。细胞经顺铂处理后,应用显微共聚焦和免疫荧光及免疫印记技术,分析各种突变的Bcl-2基因对细胞Bax激活和线粒体细胞色素C释放的保护作用,并进行Hoechst33258染色观察细胞凋亡,综合分析顺铂诱导细胞凋亡的信号传导途径。结果:转染Bcl-cb5组细胞色素C释放百分率为35.74%,明显高于Bcl-nt组(18.7%)和Bcl-acta组(24.6%),Bcl-cb5组细胞色素C释放的细胞数与Bcl-nt组及Bcl-acta组比较明显增多(P<0.05)。未经转染Bcl-2的细胞和经过转染Bcl-cb5的细胞均可见明显的Bax激活和细胞凋亡。而经Bcl-acta和Bcl-nt转染后再进行顺铂处理,24h后比Bcl-cb5基因转染组和对照组的细胞凋亡率明显降低(P<0.05)。结论:转染Bcl-acta和Bcl-nt基因组RPTC抵抗凋亡的能力明显高于转染Bcl-cb5基因组,转染Bcl-cb5基因能够使所表达的蛋白在内质网上定位,但不能发挥抗凋亡的作用。说明RPTC抵抗顺铂诱导的细胞凋亡的主要作用是通过线粒体途径而不是通过内质网起作用的。