沉默miR-8063促进结直肠癌细胞获得多药耐药性及机制研究北大核心CSCD

目的:探讨沉默miR-8063促进结直肠癌SW480、HT29细胞多药耐药性及分子机制。方法:分别采用miR-8063-inhibitor-GFP-PURO慢病毒载体和阴性对照病毒(NC-GFP-PURO)转染SW480、HT29细胞,建立实验组(SW480-sh-miR-8063、HT29-sh-miR-8063)和对照组(SW480-sh-NC、HT9-sh-NC),RT-qPCR检测miR-8063表达;CCK-8测定5-氟尿嘧啶(5-FU)和奥沙利铂(OXA)对两组细胞24 h、48 h及72 h的生长抑制率;RT-qPCR和Western blot检测耐药基因P-gp、ABCG2 mRNA和蛋白表达;Western blot检测WNT/β-catenin信号通路关键指标WNT3A、WNT5A及β-catenin蛋白表达。结果:与对照组相比,实验组miR-8063基因表达显著降低(P<0.01),48 h、72 h时5-FU和OXA对细胞的抑制率显著降低(P<0.01),耐药基因P-gp、ABCG2 mRNA及蛋白表达显著提高(P<0.01),WNT3A、WNT5A及β-catenin蛋白表达显著升高(P<0.01)。结论:沉默miR-8063基因可促进CRC细胞耐药基因P-gp、ABCG2表达,使CRC细胞SW480和HT29获得多药耐药性,其分子机制可能与激活WNT/β-catenin信号通路有关。     

腺病毒介导的shRNA下调PTEN表达对体外活化肝星状细胞增殖与凋亡的影响

目的:探讨腺病毒介导的shRNA下调第10号染色体缺失的磷酸酶和张力蛋白同源物基因(PTEN)表达对体外培养的大鼠活化肝星状细胞(HSCs)增殖和凋亡的影响及其信号转导机制。方法:体外培养活化HSCs,以腺病毒为载体将靶向PTEN的shRNA干扰重组体转染至体外活化的大鼠HSCs;四甲基偶氮唑盐(MTT)法检测HSCs增殖;末端转移酶标记技术(TUNEL)及流式细胞术测定HSCs凋亡;Western blotting方法检测PTEN、Bax、Bcl-2、Akt、p-Akt、ERK1/2及p-ERK1/2蛋白表达情况;实时荧光定量PCR方法检测PTEN、Akt及ERK1 mRNA表达情况。结果:(1)靶向PTEN的RNA干扰重组腺病毒成功感染体外活化HSCs,并在一定范围内呈时间依赖性地促进HSCs增殖,腺病毒感染HSCs后72 h,HSCs凋亡率显著下降(P<0.05);(2)Bax表达降低,Bcl-2表达增加(P<0.05);(3)p-Akt和p-ERK1/2蛋白表达显著增加(P<0.05);而Akt蛋白及其mRNA、ERK1蛋白及其mRNA表达均无显著改变(P>0.05)。结论:RNA干扰下调PTEN基因表达可能通过Bcl-2/Bax途径促进体外活化HSCs增殖并抑制其凋亡,此外,RNA干扰下调PTEN基因表达促进p-Akt和p-ERK1/2表达增多,提示PTEN可能通过影响PI3K/Akt和ERK1/2信号通路而在调控HSCs增殖和凋亡中发挥重要作用。     

氧自由基对3T3-L1脂肪细胞表达PAI-1的调节及其可能机制

目的： 研究脂肪细胞中氧自由基（ROS）对纤溶酶原激活物抑制物-1（PAI-1）表达的调节,并探讨其机制。方法：培养3T3-L1细胞,并诱导其分化成为脂肪细胞,以MTT比色法检测细胞的活性。分别以定量PCR、多重免疫分析及夹心ELISA法检测PAI-1 mRNA和蛋白表达的水平,并采用多重磷酸化蛋白分析系统检测细胞内多种信号分子的蛋白磷酸化水平。结果：H2O2可剂量依赖性地增加PAI-1的产生。并且激活了3T3-L1脂肪细胞中多种信号转导通路,包括ERK1/2、JNK、Akt、p70 S6K及JAK/STAT,其中Akt、JAK/STAT及ERK1/2的活化可能参与到H2O2对于PAI-1的调节过程中。结论： H2O2可能通过磷酸化激活Akt、JAK/STAT及ERK1/2,上调脂肪细胞PAI-1的表达。     

稳定表达可诱导型CKS2 shRNA的卵巢癌OVCAR3细胞系的建立与鉴定

目的建立稳定表达可诱导型CKS2 shRNA的卵巢癌OVCAR3细胞系并研究CKS2敲除对卵巢癌OVCAR3细胞增殖的影响。方法根据siRNA的原理设计2对靶向CKS2的shRNA序列,构建慢病毒表达质粒(p LVTHM/CKS2 shRNA重组质粒);先用p LV-t TR/KRAB-Red空载体制备慢病毒并感染OVCAR3细胞;在此基础上再用p LVTHM/CKS2 shRNA重组质粒制备慢病毒并感染上述OVCAR3细胞;对上述两次慢病毒感染的OVCAR3细胞,用强力霉素(DOX)处理72 h后,用real time PCR及Western blot检测CKS2 mRNA及蛋白的表达;用MTT法检测细胞增殖。结果构建靶向CKS2的shRNA慢病毒表达质粒,包装出慢病毒并感染OVCAR3细胞;建立稳定表达可诱导型CKS2 shRNA的OVCAR3细胞系,通过DOX诱导,可明显抑制该细胞系中CKS2在mRNA及蛋白水平的表达(P0.05,P0.01);CKS2敲除可明显抑制OVCAR3细胞的增殖。结论建立稳定表达可诱导型CKS2 shRNA的OVCAR3细胞系,DOX诱导的CKS2敲除可明显抑制OVCAR3细胞增殖。     

IQGAP1在人骨髓瘤细胞中过表达且与ERK相互作用

 目的：探讨含IQ模序的GTP酶活化蛋白1(IQ motif-containing GTPase-activating protein 1,IQGAP1)在骨髓瘤细胞中的过表达及其机制。方法：RT-PCR和Western blotting 检测RPMI8226和U266细胞IQGAP1表达;使用不同的寡核苷酸序列构建沉默人IQGAP1的shRNA质粒（clone 1、clone 2、clone 3和clone 4）、转染RPMI8226细胞,RT-PCR和Western blotting检测其IQGAP1表达;Western blotting检测RPMI8226-shIQGAP1组 (clone 1)、RPMI8226-shRNA阴性对照组和RPMI8226未转染组细胞p-ERK1/2、ERK1/2、Akt、p-AKT、STAT3和p-STAT3水平,免疫共沉淀确定IQGAP1和ERK的关系。结果：IQGAP1在RPMI8226和U266骨髓瘤细胞株中过表达,使用shRNA表达质粒沉默RPMI8226细胞IQGAP1 后,IQGAP1表达减少,在有或无血管内皮生长因子（VEGF）/白细胞介素6（IL-6）作用下检测RPMI8226-shIQGAP1组 (clone 1) 细胞增殖明显下降。进一步检测3组细胞p-ERK1/2、ERK1/2、AKT、p-AKT、STAT3和p-STAT3蛋白水平,与RPMI8226-shRNA阴性对照组和RPMI8226未转染组相比,RPMI8226-shIQGAP1组ERK1/2磷酸化水平下降70.2%,免疫共沉淀法明确在RPMI8226细胞中IQGAP1和ERK存在相互作用。结论：IQGAP1在骨髓瘤细胞中的过表达可能与MAPK途径的ERK相互作用有关。     

幽门螺杆菌毒素相关蛋白CagA上调胃泌素基因表达

目的 对幽门螺杆菌分泌的cagA蛋白能否调控胃泌素基因表达及作用机制进行研究.方法 用含有cagA基因的真核表达载体——pcDNA3.1ZEO(-)/cagA7转染AGS和SGC-7901细胞;同时培养幽门螺杆菌NCTC11637后感染AGS和SGC-7901细胞;加入JAK2信号通路抑制剂AG490和ERK信号通路抑制剂U0126,实时荧光定量PCR检测转染和感染细胞中胃泌素mRNA的表达.结果 用PeDNA3.1ZEO(-)/eagA7转染和NCTC11637感染胃癌细胞AGS和SGC-7901后胃泌素mRNA表达最显著增加(P＜0.05),但加入AG490和U0126后胃泌素mRNA的表达量显著降低(P＜O.05).结论 cagA上调胃泌素基因的表达,ERK/MAPK和JAK/STAT信号通路参与了CagA对胃泌素表达的调控.     

敲低肝癌细胞STAT3抑制IFN1诱导的HepG2肝癌细胞增殖和迁移

目的筛选并制备信号转导子与转录激活子3短发夹RNA(STAT3 shRNA)慢病毒,感染Hep G2肝癌细胞抑制其STAT3表达,观察对1型干扰素(IFN1)诱导的肝癌细胞增殖和迁移的影响及机制。方法设计并合成4段人STAT3基因的干扰序列(STAT3 shRNA 1~STAT3 shRNA 4)以及对照序列(Ctrl shRNA),与p LKO.1-sp6-pgk-GFP连接构建慢病毒表达载体,随后与骨架质粒ps PAX2、p MD2.G共转染HEK293T细胞制备慢病毒并感染Hep G2细胞。Western blot法检测STAT3的蛋白水平,筛选有效序列。敲低STAT3水平后,CCK-8法检测Hep G2细胞的增殖,TranswellTM迁移和划痕实验检测Hep G2细胞的迁移。同时观察2000 U/m L重组人IFNα2b诱导的肝癌细胞增殖、迁移能力的变化,Western blot法检测IFN相关STAT1信号的磷酸化水平。结果筛选对STAT3敲低效果最显著的STAT3 shRNA1和STAT3 shRNA2,敲低STAT3水平后,肝癌细胞增殖和迁移降低。2000 U/m L IFNα2b对细胞的增殖抑制作用更显著,迁移细胞的数量显著下降。敲低STAT3水平后,肝癌细胞STAT1的磷酸化水平升高,在IFNα2b处理后STAT1磷酸化水平进一步升高。结论敲低肝癌细胞STAT3通过上调STAT1信号通路活化,抑制细胞的迁移、增殖,恢复IFN在肝癌细胞中的应答。     

慢病毒载体抑制FOXM1对卵巢癌SKOV3细胞增殖的影响与分子机制

目的:研究靶向转录因子FOXM1 shRNA慢病毒载体抑制FOXM1表达对人卵巢癌SKOV3细胞增殖的影响及其分子机制。方法:应用感染复数(MOI)为20的靶向FOXM1shRNA慢病毒颗粒感染人卵巢癌SKOV3细胞,MTT比色法检测感染后SKOV3细胞的生长曲线;流式细胞术分析感染72 h后细胞周期变化;实时荧光定量PCR及Western blot法分别检测感染72 h后FOXM1、Cyclin D1、PLK1等基因mRNA及蛋白表达变化。结果:MOI为20的靶向FOXM1 shRNA慢病毒颗粒能显著抑制SKOV3细胞生长,并将细胞阻滞在G0/G1期而S期细胞减少(P<0.01);显著下调FOXM1、CyclinD1、PLK1等基因mRNA及蛋白表达。结论:抑制FOXM1表达对人卵巢癌SKOV3细胞具有显著的增殖抑制作用。其作用与下调Cyclin D1、PLK1等蛋白的表达将细胞阻滞在G0/G1期有关。     

脂肪间充质干细胞条件培养基诱导结肠癌HT29细胞凋亡

目的:观察脂肪间充质干细胞条件培养基(ASC-CM)对结肠癌HT29细胞的增殖抑制作用。方法:将体外培养生长良好的HT29细胞分为两组,一组加入ASC-CM再培养(实验组),另一组加入DMEM/F12培养基再培养(对照组),然后进行如下实验:(1)分别于再培养24h、48h和72h,采用CCK-8检测两组HT29细胞增殖水平,并计算实验组HT29增殖率;(2)于再培养48h采用流式细胞术检测两组HT29细胞凋亡率;(3)再培养48h后,采用实时荧光定量PCR检测两组HT29细胞凋亡因子Caspase-3、Caspase-9、Survivin、XIAP的mRNA表达水平。结果:(1)与对照组比较,实验组HT29细胞24h增殖水平(吸光度OD值)无明显差异(P0.05),培养48h、72h的OD值显著降低(P0.01),且其增殖率下降趋势与培养时间呈明显负相关(r=-0.974,P0.01)。(2)实验组HT29细胞ASC-CM培养48h凋亡率较对照组明显增加(P0.01)。(3)实验组HT29细胞ASC-CM培养48h凋亡因子Caspase-3、Caspase-9mRNA表达较对照组显著升高(P0.01),Survivin、XIAP mRNA表达较对照组显著降低(P0.01)。结论:ASC-CM能有效抑制结肠癌HT29细胞增殖和促进凋亡。     

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.     

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.     

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.     

The case for allele‐specific recognition by the TCR

There is a sharp difference in how one views TCR structure–function–behaviour dependent on whether its recognition of major histocompatibility complex‐encoded restriction elements (R) is germline selected or somatically generated. The generally accepted or Standard model is built on the assumption that recognition of R is by the V regions of the αβ TCR, which is not driven by allele specificity, whereas the competing model posits that recognition of R is allele‐specific. The establishing of allele‐specific recognition of R by the TCR would rule out the Standard model and clear the road to a consideration of a competing construct, the Tritope model. Here, the case for allele‐specific recognition (germline selected) is detailed making it obvious that the Standard model is untenable.     

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.