FLT-1启动子在血管内皮细胞中特异生物活性的检测及其意义

目的:构建带有组织特异性FLT-1启动子的真核表达载体,检测其在转染的人脐静脉内皮细胞（HUVEC）中对荧光素酶报告基因表达的驱动能力。方法：采用PCR扩增FLT-1启动子,插入到pGL3-Basic-luc载体中,构建携带FLT-1启动子的真核表达载体pGL3-FLT-Basic-luc,经脂质体法转染HUVEC、HepG2、NIH3T3和HEK293细胞,于转染48h后采用双荧光报告系统检测荧光素酶表达活性。 结果:酶切及测序证实构建的pGL3-FLT-Basic-luc载体中含有序列正确的FLT-1基因启动子,双荧光报告系统检测显示,转染的HUVEC细胞其荧光素酶活性明显高于HEK293细胞（P<0.01）,而转染的HepG2和NIH3T3细胞中未检测出荧光素酶表达。结论:克隆的FLT-1启动子具有较高的血管内皮特异性转录活性,可作为血管疾病靶向基因治疗的启动子来源。图     

人热休克因子1对抗增殖蛋白基因启动子转录调控的研究

目的：研究人热休克因子1（hHSF1）对抗增殖蛋白（pmhibitin）动子转录活性的影响。方法：采用PCR方法扩增hHSF1全长编码序列,构建peDNA3．1（＋）-hHSFl真核表达载体,将peDNA3．1（＋）-hHSF1和人prohibitin基因启动子表达载体pGL3-prohibitin共同转染HEK293细胞,采用双荧光素酶报告基因检测系统,检测双荧光索酶活性,分析hHSF1对抗增殖蛋白基因启动子的转录调控作用。结果：成功构建peDNA3．1（＋）-hHSF1真核表达载体,荧光素酶活性测定发现hHSF1明显上调pGL3-prohibitin转录。结论：hHSF1对prohibitin具有转录调控作用。     

小鼠TIE2基因启动子的克隆及转录活性分析

克隆小鼠TIE2基因的启动子并分析其转录活性.设计并合成引物,以小鼠肝脏组织DNA为模板,巢式PCR扩增小鼠TIE2基因启动子区.将扩增获得的系列截短片段克隆入荧光素酶(Luc)报告基因表达载体pGL3-Basic中,构建系列启动子区转录活性报告质粒pGL3-TIE2-Luc.报告质粒与内参质粒共转染SVEC4-10、NIH3T3、HUVEC及NIT-1细胞系,48h后收获细胞检测双荧光素酶的表达情况.构建的pGL3-TIE2-Luc系列报告质粒经过酶切鉴定及DNA测序分析都显示正确;转染4种细胞系后进行双荧光素酶活性检测的结果表明,TIE2基因启动子区域(-2056-+1)具有较强的转录活性.成功构建小鼠TIE2基因启动子报告质粒,证实TIE2基因上游区域(-2056-+1)具有较强的启动子活性.     

Dppa2 基因启动子区Oct4 结合位点突变对其活性的影响

目的：探讨Dppa2 基因5'' 端启动子区Oct4 结合位点突变对Dppa2基因启动子活性的影响。方法：PCR 扩增包括Oct4 结合 位点的Dppa2 基因5'' 端转录起始点上游-2439～+293 bp 的启动子序列,片段长度为2732 bp。将该片段连接到pGL3-Basic 载体, 构建野生型pGL3-2439表达载体。采用定点突变法,将-1959～-1957 位碱基的GCA突变成TAG,构建Oct4 结合位点突变型 pGL3-mo2439 表达载体。用上述两种表达载体、PGL3-basic 载体和Oct4 表达载体分别瞬时转染HEK 293 细胞。细胞培养48 h 后,利用双荧光素酶报告系统测定各组细胞表达的荧光素酶的相对活性。结果：经琼脂糖凝胶电泳及测序鉴定,证实野生型 (pGL3-2439)和突变型(pGL3-mo2439)载体构建成功。荧光素酶活性测定结果显示,转染Dapp2 基因启动子野生型pGL3-2439 表 达载体的细胞组荧光素酶的相对活性为16.307,突变型pGL3-mo2439 表达载体的细胞组荧光素酶的相对活性为10.634。Oct4 结 合位点突变后,Dppa2 基因启动子区转录活性较野生型降低了35 %。结论：Dppa2基因5''端启动子区-1959～-1957 位的Oct4 结 合位点突变可能导致Dppa2 基因启动子活性下降     

miR-181b-5p 对于卡波西肉瘤细胞SLK细胞增殖和凋亡的影响

目的：探讨Dppa2 基因5'' 端启动子区Oct4 结合位点突变对Dppa2基因启动子活性的影响。方法：PCR 扩增包括Oct4 结合 位点的Dppa2 基因5'' 端转录起始点上游-2439～+293 bp 的启动子序列,片段长度为2732 bp。将该片段连接到pGL3-Basic 载体, 构建野生型pGL3-2439表达载体。采用定点突变法,将-1959～-1957 位碱基的GCA突变成TAG,构建Oct4 结合位点突变型 pGL3-mo2439 表达载体。用上述两种表达载体、PGL3-basic 载体和Oct4 表达载体分别瞬时转染HEK 293 细胞。细胞培养48 h 后,利用双荧光素酶报告系统测定各组细胞表达的荧光素酶的相对活性。结果：经琼脂糖凝胶电泳及测序鉴定,证实野生型 (pGL3-2439)和突变型(pGL3-mo2439)载体构建成功。荧光素酶活性测定结果显示,转染Dapp2 基因启动子野生型pGL3-2439 表 达载体的细胞组荧光素酶的相对活性为16.307,突变型pGL3-mo2439 表达载体的细胞组荧光素酶的相对活性为10.634。Oct4 结 合位点突变后,Dppa2 基因启动子区转录活性较野生型降低了35 %。结论：Dppa2基因5''端启动子区-1959～-1957 位的Oct4 结 合位点突变可能导致Dppa2 基因启动子活性下降。     

探讨穿心莲内酯对HIV-1感染辅助受体CXCR4启动子作用机制的研究

穿心莲内酯具有明显的抗病毒作用,对HIV-1(Human immunodeficiency virus type 1)具有明显的抑制作用,本研究探讨了穿心莲内酯影响CXCR4启动子活性的作用机制。首先构建双荧光素酶报告基因载体pFireRlucCXCR4(C-X-C chemokine receptor 4),并转染入人HEK293T细胞;利用CCK8法检测穿心莲内酯对人HEK293T细胞细胞毒性作用;双荧光素酶报告基因技术检测穿心莲内酯对CXCR4启动子活性的影响;MTT法检测穿心莲内酯对人T淋巴细胞Jurkat细胞活性影响;实时荧光定量PCR检测穿心莲内酯对人T淋巴细胞Jurkat细胞表面CXCR4 mRNA和蛋白表达的影响。PCR分别扩增了CXCR4启动子（777 bp）和Rluc(1 997 bp)表达单元,通过测序和酶切鉴定双荧光素酶报告基因载体插入正确;穿心莲内酯作用于转染pFireRluc-CXCR4HEK293T细胞,双荧光素酶结果显示：穿心莲内酯能够下调CXCR4启动子活性,差异具有显著性（P<0.05）。穿心莲内酯作用于人T淋巴细胞Jurkat细胞后,qPCR...     

丙型肝炎病毒5’非翻译区不同结构域对其启动子活性的影响

在前期工作已证实HCV基因组5’UTRDNA序列具有启动子活性的基础上,分别构建5’UTR不同结构域的DNA序列驱动虫荧光素酶基因表达的质粒pGL3-5’UTR,转染HepG2细胞以及用全长的5’UTRcDNA构建质粒分别转染HepG2、Hela、HEK293、L02细胞,用双荧光素酶检测系统检测虫荧光素酶的表达水平,逆转录聚合酶链反应检测虫荧光素酶基因mRNA水平,并与相应对照作比较。结果显示当四个结构域都具备时,荧光素酶相对活性为5.91±0.65,为SV40启动子的18.7%;失去结构域I以后,荧光素酶相对活性为9.52±0.32;失去结构域I和II以后,荧光素酶相对活性为2.64±0.25;失去结构域III和IV以后,荧光素酶相对活性为0.32±0.09;失去结构域IV以后,荧光素酶相对活性为2.72±0.45,逆转录聚合酶链反应结果与之相符;全长5’UTRcDNA在L02、hepG2、HEK293、Hela细胞中荧光素酶相对活性分别为0.75,0.49,0.23,0.14。结果提示结构域III是HCV5’UTRDNA序列具备启动子活性的核心结构,结构域I对其5’UTRDNA序列的启动子活性具有抑制效应,而结构域II和IV可增强5’UTRDNA序列的启动子活性;HCV5’UTRcDNA的启动子功能无组织特异性,但在正常的肝细胞(L02)中表达最高。     

解偶联蛋白UCP1启动子荧光素酶报告基因载体的构建

目的:构建解偶联蛋白UCP1启动子荧光素酶报告基因载体,为寻找调控UCP1表达的小分子化合物提供有效工具。方法:从小鼠基因组DNA中PCR扩增小鼠UCP1启动子上游2000 bp序列,并将该序列连接到荧光素酶报告基因载体p GL3-basic中,构建p GL3-UCP1启动子。测序正确后,提取质粒,然后将上述载体与p RL-TK载体共转染至HEK293细胞、小鼠白色脂肪前体细胞和小鼠棕色脂肪前体细胞,48 h后裂解细胞检测荧光素酶的活性。结果:通过PCR成功扩增获得了目的片段,并将其克隆至p GL3-basic中。与细胞内源UCP1表达水平相似,荧光素酶报告系统表明构建的p GL3-UCP1在棕色脂肪细胞中启动子活性最高,在白色脂肪细胞中活性较低,在HEK293细胞中基本没有活性。同时β3肾上腺素受体激动剂CL 316,243同样能够上调p GL3-UCP1的启动子活性。结论:成功构建了小鼠UCP1启动子荧光素酶报告基因载体,并证明在棕色脂肪细胞中,该启动子具有很强的启动子活性,而在白色脂肪和HEK293细胞中,启动子活性很低。该启动子报告系统有望为寻找激活UCP1的小分子化合物提供重要平台。     

CD31启动子表达载体的构建与功能鉴定

目的:构建CD31启动子的绿色荧光蛋白和萤光素酶真核表达载体,转染至内皮细胞系HUVEC和BEND3细胞中,对其生物学功能和活性进行初步检测,作为鉴定内皮细胞的新方法。方法:从基因组中钓取CD31启动子的核酸序列,将其插入pGL4.0萤光素酶载体和慢病毒表达载体pCDH-copGFP,经限制性内切酶酶切和测序验证后瞬时转染HUVEC和BEND3内皮细胞系及对照MCF7乳腺癌细胞,通过双萤光素酶实验检测其启动子活性,荧光显微镜观察绿色荧光表达。结果:双酶切与测序结果表明表达载体中正确插入了CD31启动子序列;双萤光素酶实验和荧光显微镜检测发现CD31启动子特异性在内皮细胞中表达。结论:CD31启动子的萤光素酶和绿色荧光表达载体构建成功,可作为鉴定内皮细胞的新型技术手段。     

FLT-1启动子荧光素酶报告基因重组体的构建和鉴定

目的：为探索FLT-1启动子靶向调控活性分析,克隆FLT-1启动子基因序列,构建并鉴定肿-1启动子调控的荧光素酶报告基因重组体pGL3-FLT—Basic—luc。方法：应用聚合酶链式反应（PCR）技术扩增FLT-1启动子序列,定向亚克隆至荧光素酶表达载体pGL3-Basic—luc中,构建含有正确目的基因的报告基因重组体pGL3-FIT—Basic—luc,通过限性内切酶酶切、PCR及测序进行鉴定。结果：通过酶切鉴定及基因测定证明,所克隆的基因产物与预期结果-致,序列无碱基突变。结论：成功构建了含有FLT-1启动子基因序列的荧光素酶报告基因真核表达载体,为下-步分析该启动子活性及血管疾病的基因治疗奠定基础。     

Specific Surface Area and Specific Pore Volume Distribution of Tobacco

The specific surface area and the specific pore volume distribution of Japanese tobaccos were measured by means of the low temperature gas adsorption technique utilizing the B.E.T. and the Inkley methods. The specific surface area and the specific pore volume of the micropores less than 300 Å in diameter varied from 6,000 to 17,000 cm²/g and from 0.0012 to 0.0036 cm³/g, respectively, with types of curing in the ascending order of the sun cured, the flue cured and the air cured tobaccos. The both specific values were increased by extracting the tobaccos with water greatly in the case of the flue cured, while slightly in the case of the air cured tobaccos, suggesting that the effect of the curings on the specific values were due to differences in the content of low molecular components. Effectiveness of puffing was also shown. The specific surface area was linearly correlated with the specific volume of the micropores less than 300 Å in diameter, the constant term showing that contribution of the larger pores more than 300 Å in diameter to the specific surface area of tobacco was insignificant.     

Specific prophylaxis of shigellosis

A high level of shigellosis morbidity requires new approaches to the control of bacterial dysentery. According to modern concepts, the outbreaks of Shigella infections are linked both with less intensive epidemic control measures and the objective cyclic character of the epidemic process. In this connection great importance is attached to the development of vaccines for the immunization of high risk groups in territories with unfavorable epidemic conditions and in zones of military conflicts, as well as children of school and pre-school age, who mostly determine seasonal rises of shigellosis morbidity. In this article the data describing new approaches to the creation of new live enteric vaccines on the basis of the knowledge of the genetic control of microbial pathogenicity and the regulation of its expression are presented. Attenuated Shigella mutants, created by different authors and having good prospects to be used for the development of vaccines, are characterized.     

Specific impairments of planning

An information-processing model is outlined that predicts that performance on non-routine tasks can be impaired independently of performance on routine tasks. The model is related to views on frontal lobe functions, particularly those of Luria. Two methods of obtaining more rigorous tests of the model are discussed. One makes use of ideas from artificial intelligence to derive a task heavily loaded on planning abilities. A group of patients with left anterior lesions has a specific deficit on the task. Subsidiary investigations support the inference that this is a planning impairment.     

Specific tetraspanin functions

Relatively little attention has been given to the large family of abundantly expressed transmembrane proteins known as tetraspanins. Now, the importance of tetraspanins is strongly supported by emerging genetic evidence, coupled with new insights into the biochemistry and functions of tetraspanin protein complexes.