SOCS1沉默增强树突状细胞抗喉癌免疫效应

目的研究SOCS1沉默的树突状细胞特异性抗肿瘤作用机制,并探讨RNAi技术在喉癌基因治疗中的应用前景,为树突状细胞的临床应用提供新思路和理论依据。方法以细胞因子GM-CSF、IL-4和TNF-α体外诱导扩增外周血单核细胞来源的DC,倒置显微镜下观察DC形态特征;构建RNAi载体转染DC,Western blot检测SOCS1的表达情况,筛选抑制SOCS1表达的有效靶序列;流式细胞术检测DC表面分子CD83、CD86和HLA-DR的表达;ELISA法分析上清中IFN-γ的含量;MTT法评估DC刺激T细胞增殖的能力及诱导细胞毒性T细胞的杀伤活性。结果 DC体外诱导培养成功;设计的RNAi载体经测序验证无误。干扰序列5可显著下调SOCS1表达水平;SOCS1沉默联合喉癌Hep-2抗原致敏的DC可显著上调表面分子标志CD83(85.61±0.96)%、CD86(96.86±1.20)%和HLA-DR(98.02±0.94)%的表达;该组DC能有效刺激T细胞增殖,增加IFN-γ的分泌量,最终增强CTL的特异性杀伤作用,效靶比为50:1时其杀伤活性显著高于对照组(P<0.01)。结论 SOCS1沉默并负载喉癌Hep-2抗原的DC可以产生高效而特异性的抗喉癌免疫应答。     

人树突状细胞SOCS1基因RNA干扰慢病毒载体的构建及鉴定

目的构建人树突状细胞（hDCs）信号传导通路抑制因子1（SOCS1）基因RNA干扰（RNAi）慢病毒载体。方法根据人树突状细胞SOCS1基因（NM-0037）,筛选出一个靶序列,设计并合成包含正反义靶序列的互补单链寡核苷酸,与经BamH和Xho酶切后的慢病毒载体质粒pRNA-Lenti-增强型绿色荧光蛋白（EGFP）（含U6启动子和EGFP）连接产生pRNA-Lenti-SOCS1-EGFP慢病毒重组质粒,与慢病毒包装混合物共转染293T细胞,包装产生慢病毒,收集病毒上清,采取系列稀释法测定慢病毒滴度。然后转染hDCs,通过荧光显微镜观察细胞转染情况,利用荧光实时定量PCR和Westernblot检测干扰组、阴性对照组、空白对照组SOCS1的表达情况。结果将目的序列成功连接到载体上,并经测序分析证实载体构建成功。荧光实时定量PCR及Westernblot检测显示慢病毒重组质粒感染hDCs后,与空白对照组及阴性对照组比较,siRNA组mRNA和SOCS1蛋白的表达量显著降低,差异均有统计学意义（P〈0.05）。结论构建的pRNA-Lenti-SOCS1-EGFP慢病毒载体可有效地抑制hDCs的SOCS1的表达,为进一步研究DCs增强抗肿瘤免疫应答效应奠定基础。     

RNAi沉默WT1基因对人肝癌细胞HepG2的影响

目的:探讨利用RNA干扰沉默WT1基因对人肝癌细胞HepG2细胞生长,凋亡的影响,为肝癌的基因治疗提供理论依据。方法:利用脂质体将siRNA真核表达载体转入HepG2细胞中,并检测转染效率;利用免疫细胞化学染色方法检测HepG2细胞中WT1蛋白水平的表达,测定基因沉默效果;用MTT法测定HepG2细胞生长曲线;电镜下观察细胞凋亡形态,流式细胞仪检测凋亡率。结果:利用脂质体为载体将siRNA真核表达载体转染HepG2细胞,转染率达70%以上,转染后细胞中WT1蛋白表达水平下降;RNA干扰沉默WT1基因可抑制细胞增殖,促进细胞凋亡。结论:靶向WT1的序列特异性siRNA可显著抑制WT1基因的表达;下调HepG2细胞WT1基因表达可抑制细胞增殖,促进细胞凋亡。     

P27特异性siRNA重组腺病毒载体的构建及功能鉴定

目的 构建针对P27的siRNA腺病毒载体及相应的对照病毒载体,并鉴定重组腺病毒在小鼠胰岛中对内源性p27基因表达的影响.方法 合成针对p27的siRNA的靶DNA序列及相应的阴性对照序列,连接至pShuttle-H1质粒中,然后用Not Ⅰ和Hind Ⅲ限制性内切酶将H1-siRNA片段从pShuttle-H1-siRNA质粒上双酶切下来,克隆至空载pAdTrack穿梭质粒上,与腺病毒骨架质粒pAdeasy-1在BJ5183细菌中进行同源重组,转染QBI-293A细胞,包装得到pAd-P27-siRNA和pAd-NC重组腺病毒.用病毒体外感染小鼠胰岛,Western印迹法检测P27蛋白表达水平.结果 重组腺病毒载体经测序鉴定正确,制备的病毒感染效率高,能有效抑制小鼠胰岛中P27的表达.结论 成功构建了针对P27的siRNA重组腺病毒载体,为进一步研究P27在胰岛β细胞生长中的作用提供了基础.     

siRNA表达载体稳定沉默肺癌A549细胞MEKK3基因的细胞株建立

目的 构建人MEKK3基因的小干扰RNA(siRNA)表达载体并建立稳定沉默MEKK3基因表达的肺癌A549细胞克隆株.方法 设计并合成针对人MEKK3基因4个不同部位siRNA靶点的模板DNA序列,以BamH Ⅰ及Hind Ⅲ酶切位点分别克隆入pSilencer 4.1-CMV hygro载体,测序鉴定后以脂质体分别转染人A549细胞,Western印迹检测它们对MEKK3表达的抑制,并选择抑制效率最高的表达载体转染入A549细胞,潮霉素筛选后获得含该载体的抗性细胞克隆株,荧光实时定量PCR检测该细胞克隆株中MEKK3表达抑制.结果 测序鉴定表明4个MEKK3 siRNA表达载体正确无误;Western印迹结果显示对MEKK3的表达有抑制作用,其中pSilencer4.1-MEKK3 siRNA2的抑制率达84%;荧光实时定量PCR结果表明,pSilencer4.1-MEKK3 siRNA2可稳定沉默MEKK3 mRNA的表达,有效率达83%.结论 成功地构建了针对人MEKK3基因的siRNA表达载体,并成功地建立了能高效且稳定地沉默MEKK3基因表达的肺癌A549细胞克隆株.     

livinβ-siRNA表达载体的构建及其在人星形胶质瘤细胞中的稳定表达

目的构建livinβ-siRNA表达载体,并观察其在人星形胶质瘤细胞(U251)中对livinβ基因表达的抑制。方法根据livinβ序列设计2条livinβ-siRNA特异性序列,PCR扩增方法合成2条siRNA链,与质粒pGenesil-11重组构建livinβ-siRNA表达载体,脂质体法转染人星形胶质瘤细胞(U251),RT-PCR和Western blot检测细胞中livinβ基因及蛋白表达。结果对重组质粒的双酶切鉴定和测序结果证明,livinβ-siRNA表达载体pGenesil-11-livinβ-siRNA构建成功;RT-PCR检测转染组细胞中livinβmRNA表达水平明显低于对照组及空载体组,Western blot检测转染组细胞中livin蛋白表达水平明显低于对照组及空载体组,差异均具有统计学意义(P<0.05)。结论本实验成功构建了livinβ-siRNA表达载体pGenesil-11-livinβ-siRNA,并且该载体能在真核细胞人星形胶质瘤细胞(U251)中稳定表达,并能抑制livinβ基因表达。     

STAT3-siRNA对胃癌细胞株SGC-7901增殖与凋亡的影响

目的： 探讨siRNA沉默STAT3基因表达对胃癌细胞的生长抑制作用。 方法: 用已构建pGCsilencerH1/STAT3 siRNA 重组质粒,转染胃癌细胞SGC-7901,采用Western blotting、RT-PCR技术观察重组质粒对STAT3基因表达的影响;用MTT法观察重组质粒对胃癌细胞的生长抑制作用;用流式细胞术和TUNEL法检测重组质粒诱导的胃癌细胞凋亡。 结果: 用pGCsilencerH1/STAT3-siRNA重组质粒转染人胃癌细胞,Western blotting及RT-PCR 结果证实重组质粒在蛋白及mRNA水平分别显著地抑制STAT3基因表达,STAT3-siRNA抑制率分别为85.43%及80.75%,与对照组相比,有显著差异（P<0.01）。MTT和流式细胞术结果证明上述重组质粒可显著抑制胃癌细胞的生长（抑制率为63%）并诱导胃癌细胞凋亡。 结论: pGCsilencerH1/STAT3-siRNA可抑制STAT3在胃癌细胞中表达,并抑制胃癌细胞生长,促进细胞凋亡。     

siRNA沉默Smad3对人KFB细胞增殖、凋亡及合成MMP3的影响

目的 研究siRNA特异性沉默Smad3基因对人瘢痕疙瘩成纤维细胞(keloid fibroblast,KFB)增殖、凋亡及合成基质金属蛋白酶3(matrix metalloproteinase3,MMP3)的影响.方法 设计合成3对Smad3特异性siRNA片段(Smad3-siRNA-Y1,Y2,Y3)和1对无关干扰片段,转染人KFB细胞,RT-PCR和Western blot方法检测Smad3-siRNA片段对Smad3基因的特异性沉默效果,MTT法检测其对细胞增殖的影响,流式细胞仪检测细胞周期及细胞凋亡的变化,免疫细胞化学法检测MMP-3表达量的变化.结果 75 nmol/L Smad3-siRNA-Y1处理KFB细胞48小时后可特异并有效地抑制Smad3基因的表达;抑制细胞增殖,使S期细胞减少,G0/G1幅期细胞增多;凋亡指数增加;细胞表达MMP-3增加.结论 Smad3-siRNA可特异性抑制KFB细胞中Smad3基因表达,明显抑制细胞增殖,诱导细胞凋亡,并加快其合成MMP3.     

沉默癌胚抗原相关细胞黏附分子1基因抑制SHG44人胶质瘤细胞增殖并促进其凋亡

目的采用RNA干扰技术(RNAi)沉默癌胚抗原相关细胞黏附分子1(CEACAM1)基因的表达,观察沉默效果及沉默CEACAM1表达后对SHG44人胶质瘤细胞增殖和凋亡的影响。方法设计并化学合成针对CEACAM1的3对小干扰RNA(siRNA),脂质体法瞬时转染SHG44细胞。流式细胞术(FCM)检测转染效率,采用实时定量PCR(qRT-PCR)检测siRNA转染前后SHG44细胞中CEACAM1 mRNA的表达,Western blot法检测CEACAM1蛋白的表达。CCK-8法检测SHG44细胞细胞增殖活性,annexin V-FITC/PI染色结合FCM检测SHG44细胞凋亡,Western blot法检测cleaved caspase-3和裂解型多聚腺苷酸二磷酸核糖聚合酶(cleaved PARP)的变化。结果 CEACAM1 siRNA转染效率达85%。与空白对照组和阴性对照组比较,qRT-PCR及Western blot结果显示,3组特异性siRNA转染48 h后,在mRNA和蛋白水平上CEACAM1的表达均降低,以CEACAM1-siRNA3效果最明显。siRNA组SHG44细胞的增殖能力较正常对照组明显下降,凋亡细胞比例增加。沉默CEACAM1表达可以上调cleaved caspase-3和cleaved PARP的表达。结论沉默CEACAM1基因表达可有效抑制SHG44胶质瘤细胞的增殖,促进细胞凋亡。     

MEKK3基因siRNA重组腺病毒表达载体的构建及其对胃腺癌AGS细胞MEKK3的表达抑制

目的 构建人MEKK3基因的小干扰RNA(siRNA)重组腺病毒载体表达载体,观察其在胃腺癌AGS细胞中对MEKK3的表达抑制.方法 设计并合成针对人MEKK3基因3个不同部位siRNA靶点的模板DNA序列,以MluⅠ及XhoⅠ克隆入pRNAT-H1.1/Adeno穿梭载体中,得到的质粒用PmeⅠ线性化后在大肠埃项菌BJ5183中与腺病毒骨架质粒pAdEasy-1进行同源重组.卡那霉素筛选后,用PacⅠ酶切鉴定.鉴定正确的质粒经PacⅠ酶切、乙醇沉淀后转染293A细胞,包装得到具有感染能力的pAd-MEKK3-siRNA重组腺病毒.病毒体外转导人胃腺癌AGS细胞,Western印迹法检测其对MEKK3蛋白表达的抑制.结果酶切和测序鉴定表明3个MEKK3 siRNA重组腺病毒表达载体正确无误.Western印迹检测结果显示3个pAd-MEKK3-siRNA重组腺病毒表达载体中有两个可有效地抑制胃腺癌AGS细胞中MEKK3基因的表达,其中以pAd-MEKK3-siRNA3的抑制作用最显著,有效率达89 %.结论 成功构建了针对MEKK3基因的siRNA重组腺病毒载体,为进一步研究MEKK3基因在人胃腺癌AGS细胞中的作用和功能奠定了基础.     

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.