低氧及HIF－1α基因转染对HepG2细胞基因表达谱的影响

 目的：应用基因芯片技术,研究缺氧及缺氧诱导因子1α(HIF-1α)基因转染对HepG2细胞基因表达谱的影响。方法: 以常氧对照腺病毒(Ad-GFP)转染组（CC组）为对照组,常氧HIF-1α腺病毒(Ad-HIF)转染组（CH组）、低氧对照腺病毒(Ad-GFP)转染组（HC组）、低氧(2%O₂, 24 h) HIF-1α腺病毒(Ad-HIF)转染组（HH组）为实验组,提取HepG2细胞的总RNA,反转录成Cy3和Cy5标记的cDNA探针,与定制的包含1 628个能量代谢相关基因的cDNA芯片进行杂交,筛选出实验组与对照组差异表达基因并进行分析。结果: 与常氧对照腺病毒转染组相比较,常氧HIF-1α腺病毒转染组3个基因表达下调;低氧对照腺病毒转染组10个基因表达上调;低氧HIF-1α腺病毒转染组6个基因表达上调、1个基因表达下调。这些差异表达基因编码蛋白的功能涉及能量代谢、信号转导、细胞凋亡、转录和HIF-1调节、生物转化、酸碱平衡调节、细胞黏附分子等方面。结论: 低氧可以上调HepG2细胞能量代谢相关基因表达,HIF-1的转录调节作用可能是其重要环节。     

不同氧体积分数对人胚胎干细胞基因表达谱的影响

背景：低氧培养人胚胎干细胞的相关研究主要集中在干细胞多能性的维持及分化方面,而低氧对人胚胎干细胞基因表达水平的影响研究甚少。 目的：观察不同氧体积分数对人胚胎干细胞基因表达谱的影响。 方法：分别在低氧(体积分数5%O2)和常氧(体积分数21%O2)的条件下持续培养FY-hES-7细胞,收集晚期(第52代)细胞,运用全基因组表达谱芯片技术检测不同氧体积分数组FY-hES-7细胞的基因表达谱,并进行差异基因的功能富集分析及通路分析。 结果与结论：与常氧组相比,低氧组表达上调(>2倍)的基因1 840个,表达下调(>2倍)的基因1 676个。利用基因本体分析发现低氧组表达上调基因与细胞表面受体信号转导、免疫反应、离子转运、生物代谢及细胞活动等功能相关,而表达下调基因则与转录调控,依赖DNA的转录调控、神经分化、细胞形态、胚胎形态、胚胎器官及各系统发育等功能相关。通路分析发现低氧组表达上调的基因与细胞因子受体的相互作用、免疫反应以及造血系统等通路的改变相关,而表达下调的基因则多与胚胎发育及肿瘤通路改变相关。不同氧体积分数下长期培养的人胚胎干细胞基因表达谱存在明显的差异,低氧组出现差异表达基因的功能分析表明低氧有助于人胚胎干细胞的自我更新,防止分化及降低成瘤性。     

GLI-1信号途径参与低氧诱导的宫颈癌Hela细胞侵袭的作用机制研究

目的探讨GLI-1在低氧微环境介导的宫颈癌Hela细胞侵袭中的作用及其机制。方法分别在低氧(1%O2)和常氧(21%O2)环境下体外培养宫颈癌Hela细胞,采用Transwell侵袭试验检测各组细胞侵袭能力的变化;同时采用RT-PCR和Western blot法检测细胞中GLI-1 mRNA和蛋白表达。结果低氧微环境能够增强宫颈癌Hela细胞的侵袭能力,同时在低氧环境下,GLI-1基因能够被激活。阻断GLI-1信号途径后,Hela细胞侵袭能力显著降低,且伴随着GLI-1基因的表达下调。结论在低氧条件下,Hela细胞侵袭能力增强,其机制可能是通过激活GLI-1的表达增强来实现的。     

HIV/AIDS患者NK细胞趋化因子受体表达研究

目的:探讨中国HIV/AIDS患者NK细胞表面趋化因子受体CXCR4、CCR5表达情况。方法:采用流式细胞仪分析HIV/AIDS患者外周血NK细胞表面趋化因子受体CCR5和CXCR4的表达。结果:未治疗典型HIV/AIDS患者NK细胞表面趋化因子受体CXCR4和CCR5与正常对照无显著差异(P >0 0 5 ) ,HIV长期不进展者NK细胞CCR5受体低于未治疗的典型HIV/AIDS患者(P <0 0 5 ) ,与正常对照相比无显著差异(P =0 0 5 ) ;HAART治疗组NK细胞趋化因子受体CCR5表达显著低于未治疗典型HIV/AIDS患者(P <0 0 1)。结论:趋化因子受体CCR5在NK细胞上表达的变化与疾病的不同阶段密切相连,对NK趋化因子受体的检测有助于艾滋病疾病进程的研究     

MDA-MB-231乳腺癌细胞两种低氧模型的建立与比较分析

目的:由于实体瘤内的肿瘤细胞常处于低氧环境,本实验拟探究两种常用低氧模型对乳腺癌细胞影响的差异。方法:以不同浓度(0~300μmol/L)CoCl2处理人乳腺癌MDA-MB-231细胞不同时间(24 h、48 h和72 h)建立化学低氧模型,以不同程度低氧(1%、2%和5%O2)处理MDA-MB-231细胞不同时间(4 h、16 h和24 h)建立物理低氧模型。采用CCK-8法检测细胞活力,Western blot检测低氧诱导因子1α(HIF-1α)、Bcl-2及基质金属蛋白酶2(MMP-2)的蛋白表达;流式细胞术检测细胞凋亡;Transwell和划痕实验检测细胞的侵袭及迁移能力。结果:综合不同时间和浓度的HIF-1α蛋白表达结果,最终以100μmol/L CoCl2处理24 h建立化学低氧模型,以2%O 2处理24 h建立物理低氧模型;物理低氧细胞的HIF-1α蛋白表达明显高于化学低氧,且物理低氧细胞处于复氧环境后HIF-1α蛋白快速降解;物理低氧细胞凋亡增加,抗凋亡蛋白Bcl-2表达降低,而化学低氧细胞无明显变化;两种低氧细胞的MMP-2蛋白表达均升高,且化学低氧细胞的侵袭及迁移能力增强。结论:两种低氧细胞模型构建成功,并且两种低氧细胞在HIF-1α蛋白表达与稳定性、细胞活力、凋亡、侵袭及迁移等方面均存在差异。这些差异可为低氧模型的选择和研究低氧环境下的肿瘤细胞特性提供参考。     

低氧诱导因子-1对机体氧分压水平调节的研究进展

低氧诱导因子-1(HIF-1)是研究促红细胞生成素过程中发现的一种DNA结合蛋白,广泛存在于哺乳动物细胞内,是机体适应氧变化的主要转录调节因子。HIF-1被激活时通过调控组织、细胞中的氧耐受相关基因的表达,使这些基因的产物蛋白含量增加,发挥生物学效应,增强机体对氧变化的耐受能力。HIF-1是氧感受家族中的主要转录因子,低氧状态下HIF-1的表达对于细胞的生存起到重要作用。高氧状态下HIF-1的表达也对细胞的生存起作用,高氧与低氧对机体的影响有许多相似之处。机体在不同氧分压变化条件下HIF-1的作用及机制如何?HIF-1是否是一个广泛的氧压调节因子?本文就此进行综述。     

低氧对人成熟树突状细胞MMP9/TIMP1及CCR7 表达的影响

目的： 观察低氧对人单核细胞来源成熟树突状细胞（mDCs）趋化因子受体（CCR7）,基质金属蛋白酶-9（MMP-9）及其组织抑制剂（TIMP-1）表达的影响,以期为改进DCs疫苗体内迁移能力低下提供新策略。方法： 分离制备人外周血单核细胞（PBMCs）,采用体外常规培养体系 ［粒-巨噬细胞集落刺激因子（GM-CSF）与白细胞介素-4（IL-4）共同刺激］ 诱导DCs,于诱导后第5 d加入TNF-α促成熟,48 h后将mDCs置低氧环境下（1％ O2、5％ CO2、94％ N2）分别继续培养6 h、12 h,设常氧对照组（21% O2、5% CO2）,收获细胞及培养上清;采用RT-PCR技术检测MMP-9、TIMP-1及CCR7的表达;明胶酶谱法检测培养上清中MMP-9的水平与活性,流式细胞术检测mDCs表面CCR7的表达。结果： RT-PCR及酶谱实验结果显示,低氧6 h、12 h处理组mDCs MMP-9水平显著下降;与对照组比较,各低氧处理组mDCs均未检测到TIMP-1 mRNA表达的变化;转录及细胞表面表达分析结果显示,与常氧对照组比较,低氧6 h处理组趋化因子受体CCR7表达显著降低,而12 h处理组其表达显著回升至近常氧组水平。结论： 低氧下调mDCs MMP-9表达,破坏MMP-9/TIMP-1平衡,可能是DCs疫苗体内迁移能力低下的主要因素之一。趋化因子受体CCR7在mDCs对低氧应答中可能起重要作用。     

HIF-1α/SDF-1信号轴在低氧诱导祖细胞迁移和黏附中的作用

目的: 分析低氧对大鼠肺动脉内皮细胞低氧诱导因子(HIF-1α)及间质细胞衍生因子(SDF-1)表达的影响,探讨二者的相互关系(即是否存在HIF-1α/SDF-1信号轴)及其在低氧诱导祖细胞迁移和黏附中的作用。方法: 免疫磁珠分离纯化SD大鼠外周血CD34/CXCR4阳性祖细胞,免疫荧光、Western blotting及ELISA法检测不同低氧时间HIF-1α和SDF-1在大鼠肺动脉内皮细胞的表达,迁移和黏附实验检测常氧组、低氧组、HIF-1α抑制剂2-甲氧雌二醇(2ME2)组、SDF-1中和抗体组及同时加2ME2和SDF-1中和抗体组祖细胞的迁移指数和黏附率。结果: HIF-1α和SDF-1的表达与低氧时间有关,低氧12 h时二者表达到峰值(均P<0.01),应用2ME2可使SDF-1表达下调(P<0.05),应用2ME2或SDF-1中和抗体后均下调祖细胞的迁移指数和黏附率(P<0.05)。结论: 低氧可诱导肺动脉内皮细胞HIF-1α及受其调控的下游因子SDF-1的表达,提示HIF-1α与SDF-1存在信号调节关系。HIF-1α/SDF-1信号轴在介导祖细胞迁移和黏附至肺动脉内皮细胞的过程中起重要作用。     

miR-199a-5p抑制低氧条件下大鼠气道平滑肌增殖和低氧诱导因子1α表达

目的探讨miR-199a-5p在常氧及低氧环境下的大鼠气道平滑肌细胞(ASMC)中的表达以及miR-199a-5p对ASMC增殖的调控和低氧诱导因子1α(HIF-1α)表达的影响。方法用贴壁法体外培养大鼠ASMC,在常氧及低氧条件下培养24 h,实时荧光定量PCR(qRT-PCR)检测ASMC中miR-199a-5p含量;人工合成大鼠miR-199a-5p的拟似物和抑制物,用脂质体转入低氧培养的ASMC,qRT-PCR检测miR-199a-5p及HIF-1αmRNA的表达,Western blot法检测HIF-1α蛋白的表达,CCK-8法检测ASMC的增殖情况。结果与常氧组相比,低氧处理促进细胞增殖,低氧组HIF-1αmRNA及HIF-1α蛋白的相对水平显著升高;低氧组miR-199a-5p的量较常氧组减少;转染miR-199a-5p拟似物可以显著减缓低氧条件下ASMC的增殖,而其抑制物可促进低氧条件下的细胞增殖。拟似物组HIF-1α蛋白较对照组降低,抑制物组则升高,而低氧条件下各组HIF-1αmRNA水平无明显差异。结论 miR-199a-5p能抑制低氧条件下ASMC的增殖和HIF-1α蛋白的表达。     

低氧促进神经干细胞增殖分化过程中低氧诱导因子-1α表达的变化

目的:观察小鼠胎脑皮质神经干细胞(NSCs)在体外低氧条件下细胞增殖与分化以及低氧诱导因子-1α(HIF-1α)的表达情况,分析HIF-1α对NSCs增殖与分化的作用。方法:体外低氧条件下(3%O2)培养扩增胚鼠皮质来源的细胞,实验分低氧1、2、3、4d组,常氧组为对照。免疫细胞化学染色法鉴定神经干细胞及其子代细胞,RT-PCR检测实验组细胞HIF-1α mRNA的表达。结果:低氧4d组细胞呈HIF-1α阳性,其余HIF-1α为阴性。实验组神经元特异性烯醇酶(NSE)平均灰度值进行性增加。各实验组HIF-1α mRNA与对照组相比具有显著性差异。结论:在低氧能促进NSCs的增殖与分化过程中,HIF-1α表达变化可能在其中发挥了重要作用。     

Schizophrenia in a North Swedish geographical isolate, 1900–1977. Epidemiology, genetics and biochemistry

Over 200 schizophrenic patients belonging to three major and interrelated pedigree complexes have been investigated over the past 30 years in a North Swedish geographically isolated population, presently numbering about 6,000. An intensive investigation of a number of biochemical correlates and genetic markers in a few selected families belonging to one of the major pedigrees has indicated new strategies for the current research program.
Schizophrenia, as defined operationally, is significantly associated with decreased activities of two enzymes (1) blood platelet monoamine oxidase, (2) plasma dopamine-β-hydroxylase, and (3) with the genetic marker Gc² (group specific antigen). Both enzymes are subject to genetic variation. A positive score for linkage between schizophrenia and low plasma DBH activity has been calculated, but, so far, available data are insufficient for discrimination between linkage and partial contribution of genetically controlled low plasma DBH to the pathogenesis of the disease. Alternatively, both mechanisms could be involved.
As a model for continued research, schizophrenia is explained as based on a double dominant-recessive genotype (Aabb), representing a vulnerability which in about 50 % of cases develops into clinical schizophrenia. It is suggested that the dominant mutation (A) operates on or affects MAO activity, and that the recessive genotype (bb) is instrumental in low variates of DBH activity and very likely such variates within the normal range of physiological variation. Moreover, it is suggested that the combined effects of MAO- and DBH-reduced efficiency on the metabolism of e.g. dopamine could be an essential pathogenic mechanism for the schizophrenic illness which is segregating in this population.     

Renal dysplasia and asplenia in two sibs

A family is reported in which two sibs, one male and the other female, both died within 24 hours of birth with enlarged polycystic kidneys. Postmortem histology in the second child showed gross renal dysplasia. In both children the pancreas was enlarged, nodular and cystic but the liver appeared macroscopically normal. In the second child, histological examination confirmed pancreatic fibrosis with cystic dilation of ducts, but showed portal fibrosis with bile duct proliferation in the liver.
This combination of findings is very reminiscent of those in a girl and her brother reported by Ivemark et al. (1959). The children reported here also showed absence or hypoplasia of the spleen, cardiac anomalies and other features of the Ivemark syndrome (Ivemark 1955), a quite different, usually sporadic, congenital disorder. It is suggested that the children described here have a distinct lethal congenital disorder, probably inherited in an autosomal recessive manner.     

The dominant diseases of modernized societies as omega-3 essential fatty acid deficiency syndrome: Substrate beriberi

About 1900, modern food selection and processing caused widespread $\text{epidemics}$ of the B vitamin deficiency diseases of beriberi and pellagra which, for genetic reasons, often expressed as different diseases ranging from bowel and heart disease to dermatoses and psychoses. But the B vitamins merely help convert essential fatty acids (EFA) into the prostaglandin (PG) tissue regulators and it now turns out that, through hydrogenation, milling and selection of w3-poor southern foods, we have also been systematically depleting, by as much as 90%, a newly discovered trace Nordic EFA (w3) of special importance to primates and sole precursor of the PG3(4) series, even as a concurrent fiber deficiency increases body demand for EFA. Since substrate EFA is processed by many B vitamin catalysts, an EFA deficiency will mimic a $\text{panh}$ypovitaminosis B, i.e., a mixture of $\text{substrate}$ beriberi and $\text{substrate}$ pellagra resembling vitamin beriberi and pellagra but exhibiting as even more diverse $\text{endemic}$ disease. This would consitute a second stage of the Modern Malnutrition and explain why some workers now hold the dominant diseases of modermized societies to be new, nutritionally based, pellagraform yet lipid-related and to range, once again, from heart disease to psychosis. It is an assumption that our dominant diseases are unrelated to each other or are merely revealed by our diagnostic acumen and therapeutic success; and that hydrogenating millions of tons of food oils annually, to destroy the rancidity producing w3-EFA, is safe for $\text{primates}$. Extensive beriberiform disease is reported here in 32 typical cases taken from medical practice which responds strikingly to linseed oil supplements (60% w3-EFA) in confirmation of identical results in Capuchins.     

'Very sore nights and days': the child's experience of illness in early modern England, c.1580-1720

Sick children were ubiquitous in early modern England, and yet they have received very little attention from historians. Taking the elusive perspective of the child, this article explores the physical, emotional, and spiritual experience of illness in England between approximately 1580 and 1720. What was it like being ill and suffering pain? How did the young respond emotionally to the anticipation of death? It is argued that children’s experiences were characterised by profound ambivalence: illness could be terrifying and distressing, but also a source of emotional and spiritual fulfilment and joy. This interpretation challenges the common assumption amongst medical historians that the experiences of early modern patients were utterly miserable. It also sheds light on children’s emotional feelings for their parents, a subject often overlooked in the historiography of childhood. The primary sources used in this article include diaries, autobiographies, letters, the biographies of pious children, printed possession cases, doctors’ casebooks, and theological treatises concerning the afterlife.     

Guidelines for the Validation and Use of Immunoassays for Determination of Introduced Proteins in Biotechnology Enhanced Crops and Derived Food Ingredients

Recent advancements in agricultural biotechnology have created a need for analytical techniques to determine introduced proteins in crops enhanced through modern biotechnology techniques. These proteins are expressed in plant tissues and may be present in food ingredients. Immunoassays are ideally suited for protein detection and may be used as both quantitative and threshold methods. Microplate ELISA and lateral flow devices are two of the most commonly used immunoassay formats for agricultural biotechnology applications. This paper provides general background information and a discussion of criteria for the validation and application of immunochemical methods to the analysis of proteins introduced into plants and food ingredients using biotechnology methods. It is the result of a collaborative effort of members of the Analytical Environmental Immunochemical Consortium. This collaborative effort represents the combined expertise of several organizations to reach consensus on establishing guidelines for the validation and use of immunoassays. Further, the paper offers developers and users a consistent approach to adopting the technology as well as aid in producing accurate and meaningful results.     

HLA in North Colombia Chimila Amerindians

HLA-A,-B,-C,-DRB1 and -DQB1 alleles have been studied in Chimila Amerindians from Sabana de San Angel (North Colombian Coast) by using high resolution molecular typing. A frequent extended haplotype was found:HLA-A*24:02-B*51:10-C*15:02-BRB1*04:07-DQB1*03:02 (28.7%) which has also been described in Amerinndian Mayos Mexican population (Mexico, California Gulf, Pacific Ocean). Other haplotypes had already been found in Amerindians from Mexico (Pacific and Atlantic Coast), Peru (highlands and Amazon Basin), Bolivia and North USA. A geographic pattern according to HLA allele or haplotype frequencies is lacking in Amerindians, as already known. Also, five new extended haplotypes were found in Chimila Amerindians. Their HLA-A*24:02 high frequencies characteristic is shared with aboriginal populations of Taiwan; also, HLA-C*01:02 high frequencies are found in New Zealand Maoris, New Caledonians and Kimberly Aborigines from Australia. Finally, this study may show a model of evolutionary factors acting and rising one HLA allele frequency (-A*24:02), but not in others that belong to the same or different HLA loci.     

Ultracryotomy: development and application (with examples from the yeast cytology) (author's transl)

The preparation steps usually necessary for obtaining ultrathin frozen sections of biological material (chemical prefixation, enclosing, cryoprotective treatment, freezing, sectioning, and post-staining the sections for transmission electron microscopy) are submitted to a critical analysis. The application of cryo-ultramicrotomy, in particularly for cytochemical purposes, is reviewed. Fundamental considerations of chemical prefixation and poststaining are supported by examples from yeast cytology. Furthermore, the efficiency of the cryo-ultramicrotomy (electron optical resolution of ultrastructural details) is demonstrated on yeast cells and protoplasts.     

The universal muscular chamber. A basic unit of the genitourinary, cardiovascular, gastro-intestinal, skeletal, cutaneous, respiratory and other systems, endocameral hypertension; and spasm, the key to their idiopathic diseases and arthropathies

Starting with the integument, we see many organs are contractile sacs or multiples thereof, which tubes or bags constitute the major part of the entire body. Recognition of this basic unit and its characteristics sheds new light, individually and collectively, on many disorders previously considered unrelated. Muscular tears and perforations develop in the walls of these chambers, being no way peculiar to those organs, wherein, hydrochloric acid occurs. So, it is not necessary to explain the absence of excessive acid from patients who exhibit holes in the gastric, uterine, aortic, duodenal, rectal, pulmonary, retina, and other walls. Muscle, not acid is the great common factor relating idiopathic disorders in the gastrointestinal tract to each other and to similar diseases in other systems. When the units are linked together, the lesions tend to appear as arthropathies, i.e. at the joints. Rephrasing common-place observations, frees us from conventional, conceptual cul-de-sacs. An observation is only as good as its interpretation, so all possibilities must be considered, otherwise, we will remain blinded by our misconceptions.     

Der Einfluß von Nahrungsmitteln und Rauchen auf die klinisch-chemische Diagnostik von Phäochromozytom,Neuroblastom und Karzinoid-Syndrom

Zusammenfassung Der Einfluß von verschiedenen Nahrungsmitteln auf Methoden zur Bestimmung von Adrenalin (AD), Noradrenalin (NA), Vanillinmandelsäure (VMS), Metanephrinen (MN), Homovanillinsäure (HVS) und 5-Hydroxyindolessigsäure (5-HIE) im 24 h-Harn zur Diagnose des Phäochromozytoms bzw. Karzinoid-Syndroms wurde untersucht. Die in die Untersuchung einbezogenen Nahrungsmittel waren: Tee, Kaffee, Mandeln, Ananas, Käse, Walnüsse, Vanillepudding, Bananen, Tomaten und Milchschokolade. Außerdem wurde der Einfluß des Zigarettenrauchens auf die Bestimmung von AD, NA, VMS und MN untersucht.Walnüsse führten zu einer starken Erhöhung der 5-HIE-Ausscheidung. Bananen erhöhten die Ausscheidung von AD, NA, VMS, MN und 5-HIE. Kaffee und Ananas bewirkten eine geringe Zunahme der MN-Werte. Rauchen von 20–30 Zigaretten/Tag beeinflußte keine der vier Variablen.Wenn die beschriebenen Methoden benutzt werden, sollte lediglich auf den Verzehr von Bananen und Walnüssen vor und während der Harnsammelperioden verzichtet werden, da die oberen Normgrenzen im Harn überschritten werden könnten. Ein Verzicht auf Kaffee und Ananas in normalen Mengen ist nicht erforderlich. Es besteht kein Anlaß, weiterhin die bisherigen umfangreichen Restriktionen der übrigen Nahrungsmittel beizubehalten.