lncRNA SND1-IT1靶向miR-185-5p调控胃癌细胞增殖、迁移和侵袭的机制研究

目的 探讨长链非编码RNA(lncRNA)葡萄球菌核酸酶结构域包含蛋白1内含子转录本1( SND1-IT1)靶向miR-185-5p对胃癌细胞增殖、迁移和侵袭的影响.方法 实时荧光定量PCR(RT-qPCR)检测人胃黏膜上皮正常细胞和胃癌细胞中lnc SND1-IT1和miR-185-5p的表达.将胃癌细胞AGS分为对...     

AQP1基因沉默对前列腺癌PC-3细胞的影响

目的:探讨水通道蛋白1(AQP1)基因沉默对在前列腺癌PC-3细胞的作用.方法:培养PC-3细胞至对数生长期,随机分为质粒转染组和质粒空白组.针对AQP1基因设计合成小片段RNA,并构建高效表达载体.利用转染试剂Lipo-fectamineTM2000用脂质体介导的基因转染技术将质粒转染入PC-3细胞.RT-PCR检测PC-3细胞AQP1mRNA的表达情况及激光共聚焦观察室观察、拍照AQP1表达的情况,转染后72h.将鼠龄为6周的60只BALB/C-nu/nu雄裸鼠随机分成两组,其中正常对照组30只,转染组30只.转染组把转染后的前列腺癌PC-3细胞注射裸鼠腋窝处皮下;对照组把未经转染的前列腺癌PC-3细胞注射到裸鼠腋窝处皮下.每日观察肿瘤生长及体重情况,裸鼠饲养6周处死,完整取出移植瘤瘤体测量体积和重量.结果:与质粒空白组相比,转染组应用RNA干扰技术后AQP-1在前列腺癌PC-3细胞系中mRNA的水平降低,与对照组相比,转染组裸鼠移植瘤体积为(573.39±175.24) mm3明显小于对照组(1482.50±327.86) mm3,(P＜0.05).转染组肿瘤重量为(0.55±0.11)g明显小于对照组(1.31±0.29)g,差别具有统计学意义(P＜0.05).结论:AQP-1广泛的表达在正常前列腺组织和前列腺癌组织中,对内环境稳态起着重要的作用.更多的AQP-1促进了前列腺癌细胞的增长,抑制AQP-1的表达可以使前列腺癌移植瘤生长减慢,体积减小.     

脑内注射白细胞介素-1β对淋巴结细胞应激免疫抑制因子生成的影响

our previous work showed that a suppressive factor( a protein with largemolecular weight in serum was induced by restraint stress in mice and rats,which suppressed Con Ainduced lymphocyte proliferation.It was also found that the generation of serum suppressive factorwas under control of the central nervous system.Our further study showed thatintracerebroventricular(icv )injection of interleukin 1 receptor antagonist(IL-1Ra)antagonised thegeneration of serum suppressive factor induced by restraint stress and icv injection of interleukin-1β(IL-1β)increased the generation of the suppressive factor.Our experiment also showed that the serumsuppressive factor induced by restraint stress was first made in lymph tissue and then released intoblood.The present work was designed to investigate the role of IL-1 in the brain in generation of thesuppressive factor in lymph node in mice.Icv injection of IL-1β( 1 pg/mouse) was shown tosignificantly increase the generation of the suppressive factor in lymph node.Icv injection of IL-1Ra,however ,antagonised generation of the suppressive factor.In mice without restraint stress,both thesuppressive factor in serum and in lymph node were found to be induced in dose-dependent manner byicv injection of IL-1β.Taken together,these results suggest that IL-1β in brain played a veryimportant role in generation of the suppressive factor in lymph node.The positive correlation betweenthe suppressive action of lymph node and of serum added to the evidence that lymph tissue is probablythe source of the serum suppressive factor.     

脑内注射白细胞介素－1β对淋巴结细胞应激免疫抑制因子生成的影响

脑内注射白细胞介素－１β对淋巴结细胞应激免疫抑制因子生成的影响李怡凡，左永昌，邵黎，宋德懋，丁桂凤，范少光（北京医科大学生理学系，北京医科大学免疫学系北京１０００８３）我室以前的工作发现束缚应激后，动物血清内出现一类能抑制淋巴细胞转化的因子，进一步的...     

脂多糖应激新分子融合蛋白的高表达及纯化

目的在原核表达系统中表达脂多糖应激新分子编码的蛋白质。方法PCR扩增HlrgcDNA编码区,克隆入表达载体pcTAT中,构建成融合6His的表达载体pcTAT-lrg,转化E.coli BL21(DE3),以IPTG诱导。表达产物用SDS-PAGE及凝胶光密度扫描分析,用Ni-NTA亲和层析柱纯化。结果经过IPTG诱导4 h,表达出相对分子质量(Mr)约25 000的蛋白,占菌体总蛋白的51%。表达产物为可溶性的,用Ni-NTA亲和层析柱纯化的表达蛋白达到电泳纯。加入培养液中的纯化蛋白,可在30 min内进入HEK293细胞。结论在E.coli中成功地高表达人Lrg融合蛋白,并对其进行初步纯化,为人Lrg功能的研究打下基础。     

SLCO1B1基因多态性的辛伐他汀个体化用药临床研究分析

辛伐他汀(Simvastatin)是一种临床常用的降血脂药物,其常见的药物不良反应是横纹肌溶解、肌病等肌肉相关症状。SLCO1B1基因多态性是导致辛伐他汀肌肉不良反应个体化差异的主要原因之一。SLCO1B1编码的产物参与辛伐他汀的肝摄取,其功能丧失性变异可引起辛伐他汀的血药水平升高,导致更多的药物暴露。对于服用辛伐他汀的患者,检测SLCO1B1基因型可指导患者的精准用药,但目前尚无研究讨论常规开展SLCO1B1基因检测的临床意义。对于携带功能降低的等位基因患者,推荐服用小剂量的辛伐他汀或者换用其他他汀类(阿托伐他汀、瑞舒伐他汀等)药物,以降低可能的肌肉不良反应风险。本研究综述了SLCO1B1基因与辛伐他汀个体化用药的相关研究,为辛伐他汀的合理应用提供参考。     

重组人甲状旁腺激素1-34的纯化研究

重组人甲状旁腺激素(rhPTH(1-34))在工程菌pET(32a+)-PTH(1-34)/BL21(DE3)中以胞内可溶性融合蛋白形式高效表达。经过高压匀浆破菌处理,取上清经过亲和层析、酶切、阴离子交换层析、反相层析和阳离子交换层析进行分离纯化,得到rhPTH(1-34)原液样品纯度在98%以上,比活性达到1.05×104U/mg。     

野生型DJ-1基因在体外培养SH-SY5Y细胞中高表达对氧化损伤的抑制作用*

目的:研究帕金森病相关基因野生型DJ-1高表达对鱼藤酮及过氧化氢致多巴胺神经元SH-SY5Y细胞氧化损伤的保护作用及机制。方法:将pcDNA3-FLAG-DJ-1质粒转染进入SH-SY5Y细胞,采用Western blot分析观察DJ-1在细胞内的表达,以2,7-二氯荧光黄双乙酸盐(DCFH-DA)作为探针,用流式细胞仪检测鱼藤酮诱导的活性氧水平,以MTT法检测过氧化氢损伤的细胞存活率变化。结果:野生型DJ-1被转染进入SH-SY5Y细胞中,并能高表达。野生型DJ-1的高表达可减少活性氧的生成,0.5μmo.lL-1浓度的鱼藤酮处理细胞48 h后,细胞内存在大量活性氧,M2期细胞明显减少(转染野生型DJ-1的SH-SY5Y细胞71.60%与未转染DJ-1的SH-SY5Y细胞89.19%相比)。并且可以抑制过氧化氢导致的细胞存活率下降,过氧化氢处理1 h后,野生型DJ-1转染的细胞存活率明显高于未转染者,过氧化氢浓度为0.1,0.2和0.5 mmol.L-1,细胞存活率分别由77.71%,66.28%和39.08%上升至93.01%,83.96%和60.15%。结论:野生型DJ-1基因在体外培养SH-SY5Y细胞中高表达对氧化损伤具有抑制作用,该作用可能与降低细胞内活性氧水平有关。     

人剪切修复基因XPD对人肝癌SMMC-7721细胞中Ets-1和Cdk6基因的调控作用

目的:观察野生型人剪切修复基因XPD转染入人肝癌细胞SMMC-7721后,细胞内Ets-1和Cdk6基因的表达变化及对SMMC-7721肝癌细胞增殖的影响。方法:将人工合成的pEGFP-N2-XPD重组质粒通过Lipofectamine 2000TM转染SMMC-7721细胞。设重组质粒转染细胞SMMC-7721-pEGFP-N2-XPD(XPD)组、空载质粒转染细胞SMMC-7721-pEGFP-N2(N2)组、脂质体组、无转染空白对照组。分别用逆转录-聚合酶链反应(RT-PCR)和蛋白印迹法(Western blot)检测细胞中XPD、Ets-1、Cdk6基因mRNA和蛋白质的表达量,并用流式细胞仪检测细胞周期变化,四甲基偶氮唑盐微量酶反应比色法(MTT)检测各组细胞的增殖活力。结果:XPD组中的XPD的mRNA和蛋白质表达较其他3组明显增高(P<0.001),而Ets-1、Cdk6 mRNA和蛋白质表达较其他3组明显减少(P<0.001)。转染pEGFP-N2-XPD重组质粒后细胞停滞在G1期,难于进入S期。转染了野生型XPD的SMMC-7721细胞增殖能力减弱。结论:XPD基因可能通过抑制Ets-1、Cdk6基因的表达影响肝癌细胞的生长。     

骨髓白血病细胞形态与AML1/ETO融合基因阳性的关系

t(8;21)(q22;q22)使21号染色体上的AML1基因与8号染色体上的ETO基因发生融合,形成AML1/ETO融合基因,产生一种嵌合蛋白,作为主要的抑制物改变了正常AML1-CBF(核结合因子)B这种与造血干细胞分化有关的转录复合物的生理功能,导致白血病的发生。急性髓细胞白血病(AML)部分成熟型的M2b亚型是我国学者于50年代末提出的,具有髓外浸润率高、治疗反应好、完全缓解率高、缓解期长等特点,其特异性遗传标志为8号与21号染色体的长臂在二区二带断裂并相互移位[t(8;21)(q22;q22)],AML1基因重排可作为诊断本病的分子标志。以往文献报导M2b出现AML1/ETO融合基因阳性率占95%以上,AML其他各亚型亦可出现,但极为少数。王平等报导AML1/ETO融合基因在M2b中占100%,AML1/ETO融合基因阳性AML在骨髓涂片中能检测到异形中幼粒细胞。肖志坚等总结30例AML1/ETO阳性病例M2b占26例。巩文玉等报道46例AML1/ETO融合基因阳性AMI患儿M2b仅占19.6%。笔者收集35例AML1/ETO融合基因阳性骨髓涂片标本,观察其白血病细胞形态,探讨细胞形态与AML1/ETO融合基因阳性的关系。     

Ginsenoside Rb1 Modulates Level of Monoamine Neurotransmitters in Mice Frontal Cortex and Cerebellum in Response to Immobilization Stress

Cerebral monoamines play important roles as neurotransmitters that are associated with various stressful stimuli. Some components such as ginsenosides (triterpenoidal glycosides derived from the Ginseng Radix) may interact with monoamine systems. The aim of this study was to determine whether ginsenoside Rb1 can modulate levels of the monoamines such as dihydroxyphenylalanine (DOPA), dopamine (DA), norepinephrine (NE), epinephrine (EP), 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydorxytryptamine (5-HT), 5-hydroxindole-3-acetic acid (5-HIAA), and 5-hydroxytryptophan (5-HTP) in mice frontal cortex and cerebellum in response to immobilization stress. Mice were treated with ginsenoside Rb1 (10 mg/kg, oral) before a single 30 min immobilization stress. Acute immobilization stress resulted in elevation of monoamine levels in frontal cortex and cerebellum. Pretreatment with ginsenoside Rb1 attenuated the stress-induced changes in the levels of monoamines in each region. The present findings showed the anti-stress potential of ginsenoside Rb1 in relation to regulation effects on the cerebral monoaminergic systems. Therefore, the ginsenoside Rb1 may be a useful candidate for treating several brain symptoms related with stress.     

人胰岛素样生长因子-l的原核表达和纯化

目的构建高表达、易纯化的人胰岛素样生长因子-1(hIGF-1)工程菌。方法采用pET32a(+)质粒构建带羟胺裂解部位的表达载体,转入大肠杆菌DH5α进行诱导表达。表达产物经镍离子-亲和柱色谱分离后进行羟胺裂解。裂解产物纯化后用MALDI-TOF-MS法测定相对分子质量(Mr)。结果重组质粒序列完全正确。工程菌可表达预计Mr的融合蛋白,经Western blot证实有hIGF-1抗原活性。经镍离子-亲和柱色谱分离、羟胺裂解后得到的肽的Mr为7 640,和理论值相符。结论成功构建了表达hIGF-1的工程菌,为开发hIGF-1奠定了基础。     

CRM1 Is a Direct Cellular Target of the Natural Anti-cancer Agent Plumbagin

Plumbagin, a naphthoquinone derived from the medicinal plant Plumbago zeylanica, has been shown to exert anti-cancer and anti-proliferative activities in vitro as well as in animal tumor models. However, the mechanism underlying its anti-tumor action still remains unclear. CRM1 is a nuclear export receptor involved in the active transport of tumor suppressors whose function is altered in cancer due to increased expression and overactive transport. We showed that CRM1 is a direct cellular target of plumbagin. The nuclei of cells incubated with plumbagin accumulated tumor-suppressor proteins and inhibited the interactions between CRM1 and these proteins. Particularly, we demonstrated that plumbagin could specifically react with the conserved Cys⁵²⁸ of CRM1 but not with a Cys⁵²⁸ mutant peptide through Mass spectrometric analysis. More importantly, cancer cells that are transfected with mutant CRM1 (C528S) are resistant to the inhibitory effects of plumbagin, demonstrating that the inhibition is through direct interaction with Cys⁵²⁸ of CRM1. The inhibition of nuclear traffic by plumbagin may account for its therapeutic properties in cancer and inflammatory diseases. Our findings could contribute to the development of a new class of CRM1 inhibitors.     

酮咯酸氨丁三醇超前镇痛对儿童下腹腔镜手术应激反应的影响

目的:研究酮咯酸氨丁三醇超前镇痛对儿童下腹腔镜手术应激反应及血浆血栓素A2(TXA2)和血小板颗粒膜蛋白(GMP140)水平的影响。方法:将60例2~12岁ASA Ⅰ~ Ⅱ级择期行下腹手术的患儿以抽签法随机分为观察组和对照组各30例,观察组给予酮咯酸氨丁三醇进行超前镇痛,对照组不给予药物进行超前镇痛,比较两组患儿的应激反应、氧化损伤、血小板活化、不良反应情况。结果:(1)观察组术后30 min、6 h皮质激素、肾上腺素、去甲肾上腺素水平均低于对照组(P均<0.05),而术后12 h上述指标与对照组比较差异无统计学意义(P>0.05);(2)观察组术后30 min GMP140水平低于对照组(P<0.05);(3)观察组不良反应发生率6.67%(2/30),低于对照组的13.33%(4/30)。结论:对于儿童下腹腔镜手术,采用酮咯酸氨丁三醇超前镇痛能有效抑制应激反应和血小板激活,同时降低不良反应发生率。     

A Comparative Analysis of Perceptions of Pharmacy Students’ Stress and Stressors across Two Multicampus Universities

Objective. To compare perceived levels of stress, stressors, and academic self-efficacy among students at two multicampus colleges of pharmacy.Methods. A survey instrument using previously validated items was developed and administered to first-year, second-year, and third-year pharmacy students at two universities with multiple campuses in spring 2013.Results. Eight hundred twenty students out of 1115 responded (73.5% response rate). Institutional differences were found in perceived student stress levels, self-efficacy, and stress-related causes. An interaction effect was demonstrated between institution and campus type (main or branch) for perceived stress and self-efficacy although campus type alone did not demonstrate a direct effect. Institutional and campus differences existed in awareness of campus counseling services, as did a few differences in coping methods.Conclusion. Stress measures were similar for pharmacy students at main or branch campuses. Institutional differences in student stress might be explained by instructional methods, campus support services, institutional climate, and nonuniversity factors.     

GTPase Rac Regulates Conidiation,AFB1 Production and Stress Response in Pathogenic Fungus Aspergillus flavus

As a member of the Rho family, Rac plays important roles in many species, including proliferation, differentiation, apoptosis, DNA damage responses, metabolism, angiogenesis, and immunosuppression. In this study, by constructing Rac-deleted mutants in Aspergillus flavus, it was found that the deletion of Rac gene led to the decline of growth and development, conidia production, AFB1 toxin synthesis, and seed infection ability of A. flavus. The deletion of Rac gene also caused the disappearance of A. flavus sclerotium, indicating that Rac is required for sclerotium formation in A. flavus. The sensitivity of Rac-deficient strains responding to cell wall stress and osmotic pressure stress increased when compared to A. flavus WT. The Western blot result showed that mitogen-activated serine/threonine-protein kinase Slt2 and mitogen-activated protein kinase Hog1 proteins were no longer phosphorylated in Rac-deficient strains of A. flavus, showing that Rac may be used as a molecular switch to control the Slt2-MAPK cascade pathway and regulate the osmotic Hog-MAPK cascade pathway in A. flavus in response to external stress. Altogether, these results indicated that Rac was involved in regulating the growth and development, conidia formation and AFB1 synthesis, and response to cell wall stress and osmotic pressure stress in A. flavus.