慢病毒载体介导的肝细胞基因转移

慢病毒(lentivirus)具有可以感染低分裂细胞的独特优势,且基因转移效率高,操作相对简便,在多种疾病的基因治疗、转基因动物的制备等方面成为引人瞩目的病毒载体。本文就慢病毒载体(lentiviral vector,LV)的特点、构建、应用,尤其是慢病毒载体对于肝细胞的基因转移进行综述。     

慢病毒载体介导的肝细胞基因转移

慢病毒（lentivirus）具有可以感染低分裂细胞的独特优势,且基因转移效率高,操作相对简便,在多种疾病的基因治疗、转基因动物的制备等方面成为引人瞩目的病毒载体。本文就慢病毒载体（lentiviral vector,LV）的特点、构建、应用,尤其是慢病毒载体对于肝细胞的基因转移进行综述。     

ALEX1基因过表达慢病毒载体的构建和鉴定

目的:探讨构建Arm重复蛋白1(ALEX1)基因过表达慢病毒表达载体,感染乳腺癌细胞系SK-BR3后观察ALEX1的表达,为研究ALEX1在乳腺癌中的作用及机制奠定基础。方法:利用DNA重组技术将ALEX1基因插入到慢病毒穿梭质粒LV5中,获得重组慢病毒质粒V-ALEX1,经酶切鉴定及DNA测序鉴定后,用Lipofectamine 2000将重组质粒和慢病毒包装质粒系统(p Gag/Pol、pRev、p VSV-G)转染293T细胞中包装成重组慢病毒颗粒,经流式细胞术测定重组慢病毒滴度。将成功包装的ALEX1过表达慢病毒载体(LV5-ALEX1)和阴性对照载体(LV5-NC),感染SK-BR3细胞(MOI为10)48~72 h,Realtime PCR和Western blot法分析ALEX1表达情况。结果:酶切鉴定结果显示:产生约1 500 bp的片段,片段大小与ALEX1基因cDNA大小一致。DNA测序比对说明测序结果与预期ALEX1基因序列完全一致,证实ALEX1基因正确插入载体中,成功构建ALEX1基因过表达载体。经293T细胞包装后,成功获得病毒滴度为4.25×10~8TU/ml的重组慢病毒LV5-ALEX1。Realtime PCR和western blot结果显示:与感染LV5-NC的SK-BR3细胞相比,LV5-ALEX1感染可明显增加ALEX1 mRNA及蛋白的表达水平。结论:成功构建ALEX1基因过表达慢病毒载体,ALEX1基因过表达慢病毒能够感染乳腺癌SK-BR3细胞,可使外源基因获得稳定过表达。     

HIV-Ⅰ慢病毒载体的研究进展与应用

基因治疗是治疗肿瘤、遗传病等难治性疾病的最有效手段之一,但目前的基因转移方法存在一定的局限性,成为基因治疗和广泛应用的障碍.以1型人类免疫缺陷病毒(HIV-1)为基础构建的慢病毒载体(LV)具有感染分裂及非分裂细胞、使目的 基因产物表达稳定、表达时间长、载体自身免疫原性小等优点,尤其是LV能有效诱导免疫应答,抑制肿瘤生长,诱导移植免疫耐受等,是很有发展潜力的体内基因治疗新载体.     

慢病毒载体构建及结构优化

慢病毒载体目前是基因治疗中研究较多的载体 ,与通常使用的逆转录病毒载体和腺病毒载体比较 ,它有感染非分裂期细胞及容纳外源性目的基因片段大等优点。对其结构的优化主要集中在提高生物安全性、提高转基因表达、转基因表达的调控及载体靶向性等方面。本文介绍了慢病毒载体构建及结构优化方面的研究进展作一综述     

慢病毒载体介导的转基因整合位点研究方法

慢病毒载体整合到宿主细胞的染色体上可以长期稳定表达,目前已成为基因治疗和转基因动物载体研究的热点.慢病毒载体整合的位置效应是影响外源基因表达的重要因素,慢病毒载体整合位点的研究是探索外源基因整合机制的手段之一.转基因整合位点研究的方法主要有5种:荧光原位杂交、个体基因组文库筛选法、反向PCR、接头PCR、锚定PCR.近来的研究后发现整合位点之间可能存在一些共同特征.     

TCAB1基因RNAi慢病毒载体的构建及在人子宫内膜间质细胞中沉默TCAB1基因的效应

目的构建TCAB1基因RNAi慢病毒载体,为子宫内膜异位症(EMs)的基因靶向治疗提供理论依据。方法化学合成3条靶向TCAB1基因的siRNA,转染HEK-293T细胞,24h后采用荧光素酶报告系统筛选有效siRNA,其对TCAB1mRNA有明显抑制作用。针对已经筛选确定的TCAB1基因RNAi有效靶序列,合成靶序列的Oligo DNA,退火形成双链DNA,与经XhoI和HpaI酶切后的LV1载体[含U6启动子和绿色荧光蛋白(GFP)]连接产生LV1-shRNA慢病毒载体,PCR筛选阳性克隆,测序鉴定。用LV1-shRNA、pRev、pVsvg、pcgv四质粒共转染包装细胞HEK-293T细胞,包装产生慢病毒,分别于转染后48h、72h收取病毒上清,感染人子宫内膜间质细胞(ESC),进行RT-qPCR检测TCAB1基因相对表达量,检测LV1-shRNA干扰效率。结果 PCR和测序证实,成功构建TCAB1 shRNA的慢病毒载体LV1-shRNA。结论成功构建TCAB1基因的RNAi慢病毒载体,其转染ESC细胞后显著抑制了TCAB1的表达,为子宫内膜异位症的基因靶向治疗提供了实验基础。     

慢病毒载体及其应用进展

慢病毒载体具有可感染分裂细胞及非分裂细胞,转移基因片段容量较大,目的基因表达时间较长,不易引发宿主免疫反应等诸多优点,因此,慢病毒载体已成为基因治疗中载体研究的热点.我们以HIV-1来源的慢病毒载体为代表综述了慢病毒载体的结构特点,发展,及其应用研究进展.     

多肽作为非病毒转基因载体组件的研究进展

在转基因领域,非病毒转基因载体因为具有许多病毒载体无法比拟的优点而越来越受到重视。但是理想的非病毒转基因载体的构建需要一些功能多肽的参与。这些多肽作为转基因载体的组件,在骨架构建、靶向策略的实施、内体膜的破裂和载体的核转运方面发挥重要作用。就多肽在这些方面的进展作一综述。     

玻璃体注射腺相关病毒2和慢病毒转染大鼠视网膜的比较

目的通过玻璃体注射重组腺相关病毒2载体(r AAV2)和慢病毒载体(LV),比较两者转染成年大鼠视网膜的异同。方法实验组大鼠60只,每组12只玻璃体内分别注射重组腺相关病毒2载体-增强绿色荧光蛋白(r AAV2-EGFP)、重组腺相关病毒2载体-神经突起素-增强绿色荧光蛋白(r AAV2-neuritin-EGFP)、慢病毒载体-红色荧光蛋白(LV-RFP)和慢病毒载体-神经突起素-红色荧光蛋白(LV-neuritin-RFP),对照组注射等量的生理盐水,4周后取材,采用免疫荧光和CTB-FITC检测2种病毒载体在视网膜中转染的细胞及转染率,然后分别采用Real-time PCR和Western blotting检测神经突起素(neuritin)在视网膜中的表达变化。结果 r AAV2能够转染约70%视网膜节细胞(RGCs),LV主要转染色素上皮细胞,RGCs转染率仅为30%;r AAV2-neuritin-EGFP组神经突起素mRNA表达约是r AAV2-EGFP组和对照组的16倍,蛋白表达约为3倍;LV-neuritin-RFP组神经突起素mRNA表达约是LVRFP组和对照组的5.5倍,蛋白表达约为1.7倍。结论 r AAV2玻璃体注射后,转染大部分RGCs且神经突起素表达量高于LV,LV主要转染色素上皮细胞,提示基因治疗眼科疾病和损伤时,涉及到转染RGCs时应采用r AAV2载体,转染色素上皮时宜采用LV载体。     

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.