FLT3配基在人骨髓基质细胞系中的基因转移与表达

目的：研究逆转录病毒介导的FL在骨髓基质细胞系HFCL中的表达。方法：采用脂质体法将重组质粒pLF-SN/HFCL和空载体pLXSN/HFCL转染包装细胞PA317,G418筛选抗性克隆,用抗性克隆上清液感染HFCL。RT－PCR和基因组DNA－PCR检测外源基因mRNA水平的表达及染色体的整合,小鼠CFU－GM集落法检测FL生物学活性。结果：在mRNA水平上有FL的表达,染色体基因组中整合有标记neo基因和FL基因。活性测试结果显示转染的骨髓基质细胞分泌FL。结论：提示骨髓基质细胞可作为基因治疗的靶细胞。     

逆转录病毒介导的人血小板生成素(TPO)在骨髓基质细胞中的表达

为探讨逆转录病毒介导的TPO基因在人骨髓基质细胞系HFCL中的表达,利用脂质体法将含TPO基因的逆转录病毒载体导入HF-CL细胞中,RT-PCR和基因组DNAPCR分析证实mRNA水平有表达,基因组中整合有Neo基因和TPO基因。TPO依赖细胞株TD-3检测生物学活性表明转染的骨髓基质细胞分泌TPO。上述结果为进一步研究转基因骨髓基质细胞对造血细胞的调控作用提供了必要的基础资料。     

pcDNA3．1表达载体转染对细胞生长的影响

为了探讨真核表达载体转染对细胞生长的影响,通过脂质体介导将pcDNA3.1( )表达载体DNA转染鼻咽癌细胞系HNE1,G418筛选后,Southern杂交鉴定稳定表达细胞株,以HNE1细胞为对照,观察pcDNA3.1( ）／HNE1克隆细胞的生物学特性;结果显示,在pcDNA3.1( )/HNE1阳性克隆中,一株细胞克隆培养过程中发生自溶性死亡,一株细胞生长明显受到抑制,另一株细胞生长无明显影响,揭示在宿主细胞中pcDNA3.1( )DNA与宿主基因组DNA发生了随机整合,从而表现不同的细胞生物学改变。     

人胃癌细胞系中FHIT基因mRNA的表达及pcDNA3．1-FHIT的构建与转染

目的:检测人胃癌细胞系中FHIT基因mRNA的表达状况及构建pcDNA3.1-FI-IIT真核表达载体.方法:RT-PCR法检测三种不同类型人胃癌细胞系中FHIT基因mRNA的表达,构建真核表达质粒pcDNA3.1-FHIT,通过酶切法、PCR扩增法和DNA序列分析鉴定重组质粒后,用脂质体转染至FHIT基因mR.NA阴性表达人胃癌细胞系MKN-45,经G418筛选后RT-PCR鉴定.结果:FHIT基因在人胃癌细胞系BGC-823中呈阳性表达,在MGC-803、MKN-45细胞系中呈阴性表达.FHIT基因cDNA正确克隆到真核细胞表达栽体pcDNA3.1中,并成功转染FHIT基因mRNA阴性表达人胃癌细胞系MKN-45.结论:FHIT基因在不同类型人胃癌细胞系中表达各异.成功构建pcDNA3.1-FHIT,并转染到FHIT基因mRNA阴性表达人胃癌细胞系MKN-45,使其唧T基因阳性表达.     

伪狂犬病病毒gC基因在IBRS-2细胞中的表达及其对病毒繁殖的影响

 对含伪狂犬病病毒 Ea株 g C全基因的质粒 p UC1 .75进行亚克隆 ,将其完整编码区置于真核表达载体 pc DNA3.1 +的 HCMV启动子 /增强子下游 ,构建了 g C基因真核表达质粒 pc DNA-g C.脂质体转染 IBRS- 2细胞 ,在 G41 8抗性选择下 ,获得多个阳性克隆细胞系 .经 ELISA筛选反应最强的阳性克隆细胞系 ,进一步用间接免疫荧光检测证实 g C基因在 IBRS- 2细胞中得到了正确表达并分布在细胞膜 .以 1 0 0个蚀斑形成单位的伪狂犬病病毒分别接种表达 g C的细胞系和空白载体转染细胞系 .通过测定蚀斑数发现 ,表达 g C的细胞系对病毒的感染具有抑制作用 ,平均抑制率达 59.4%± 1 .3% .     

人p65真核表达载体的构建及鉴定

目的:构建带Myc标签的人p65真核表达载体,对其在人宫颈癌He La细胞中的定位进行检测,并研究p65对核因子κB(NF-κB)转录活性的影响。方法:构建带Myc标签的p65真核表达载体pc DNA3.1-Myc-p65,质粒测序验证正确后脂质体法瞬时转染人胚肾HEK293T细胞,Western印迹鉴定p65的表达;细胞免疫荧光实验检测p65的亚细胞定位;重组质粒与NF-κB-Luc报告基因质粒共转染HEK293T细胞,加TNFα刺激后检测萤光素酶报告基因的活性。结果:测序结果证实pc DNA3.1-Myc-p65真核表达载体构建成功;脂质体法转染HEK293T细胞后检测到Myc-p65蛋白的表达;细胞免疫荧光实验显示Myc-p65蛋白定位于He La细胞的细胞质中,当加入TNFα刺激后大部分Myc-p65蛋白进入细胞核;萤光素酶活性检测结果显示,提高细胞内p65水平或加入TNFα刺激均可明显激活NF-κB信号通路的活性。结论:构建了带Myc标签的p65真核表达载体,并使其在HEK293T细胞中表达;正常情况下,Myc-p65蛋白定位于宫颈癌He La细胞的细胞质中,TNFα促进Myc-p65入核;Myc-p65能够以TNFα不依赖的方式激活NF-κB信号通路。pc DNA3.1-Myc-p65真核表达载体的构建,为进一步筛选NF-κB的相互作用蛋白及功能研究奠定了基础。     

乳腺癌细胞株MCF-7-PC-1-46的建立

以人前列腺癌C4-2细胞基因组DNA为模板,扩增出PC-1基因N端编码46个氨基酸残基及其上游非编码区共599bp的DNA序列,将其正向克隆到真核表达载体pIRES2中,并在脂质体介导下,转染人乳腺癌细胞MCF-7,经G418筛选获得阳性单克隆,细胞扩大培养后,进行PCR和RT-PCR分析,检测外源PC-1基因在靶细胞中的整合与转录,PCR和RT-PCR结果表明,稳定转梁细胞株MCF-7-PC-1-46具有外源目的基因的整合和相应mRNA的高表达,说明成功建立了稳定表达外源PC-1基因N端46个氨基酸的人乳腺癌细胞株,为进一步研究PC-1基因的生物学功能提供了实验材料。     

人溶酶体酸性β-葡萄糖脑苷脂酶基因的克隆及其在COS7细胞中的表达

从人胎盘组织提取总RNA,采用RT-PCR扩增人溶酶体酸性β-葡萄糖脑苷脂酶(Lysosomal acid β-glucosidase,GlcCerase)基因编码区的全部序列,并克隆到pMD-19T载体上,构建克隆载体pMD-GlcCerase.经测序验证后.将GlcCerase亚克隆至表达载体pEGFP-C1上,构建了人GlcCerase绿色荧光蛋白真核表达载体pEGFP-GlcCerase.采用脂质体法将其瞬时转染至COS7细胞系后,在细胞中检测到了GlcCerase基因,并在细胞裂解产物中检测到了GlcCerase生物活性的表达.GlcCerase基因的克隆及其表达,为进一步了解GlcCerase基因的功能以及利用转基因动物乳腺生物反应器高效生产GlcCerase奠定了基础.     

重组人apoE基因表达活性和转基因小鼠研究

人apoE基因组DNA,去除其自身启动子,代之以小鼠金属硫蛋白启动子,重组质粒经脂质体介导转入小鼠NIH／3T3细胞后,以人apoE基因组DNA／EcoRⅠ片段为探针检测mRNA表达,可见apoEmRNA杂交信号很强,经重金属诱导后杂交信号更强,表明MT启动子功能良好,pME表达正常．将人apoE基因组DNA用显微注射法导入小鼠受精卵雄性原核,再将胚胎移植入假孕母鼠输卵管内,仔鼠分娩四周后,自鼠尾提取DNA,鉴定人apoEDNA在小鼠染色体上的整合,最终得到有人apoE基因整合的转基因首建鼠．     

维甲酸核受体RARα真核表达载体的构建、突变纠正及表达

目的构建维甲酸核受体RARα真核表达载体,并检测其在人肺腺癌细胞A549中表达。方法从小鼠巨噬细胞RAW264.7中提取总RNA,以RT-PCR法扩增RARαcDNA,克隆至真核表达载体pDsRed1-C1中,测序结果显示RARα第1040位A→G,导致其编码蛋白的氨基酸发生改变。通过二次PCR将其纠正,重组载体RedC1-RARα转化大肠埃希菌Top10,筛选阳性克隆做酶切及测序鉴定。脂质体瞬时转染A549细胞,在荧光显微镜下观察RARα的表达。RT-PCR法检测RARα的mRNA水平表达。结果通过RT-PCR及二次PCR得到RARαcDNA,构建其真核表达载体,脂质体瞬时转染A549细胞得到了成功表达,RARα基因产物定位于细胞核内。结论成功构建维甲酸核受体RARα真核表达载体,且证实RARα编码蛋白定位于细胞核内,本研究结果为进一步探讨结核分枝杆菌固有免疫机制奠定了基础。     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.