RPA1低表达对辐射抵抗人鼻咽癌CNE-2R细胞侵袭迁移及细胞周期的影响*

目的: 探讨复制蛋白A1(RPA1)沉默对人鼻咽癌CNE-2R细胞侵袭、迁移及细胞周期的影响。方法: 采用shRNA技术构建RPA1低表达的CNE-2R细胞模型并通过RT-PCR和Western blot实验验证。选用空白对照组(CNE-2R)、阴性对照组(NC-shRNA)、RPA1低表达组(RPA1-shRNA)3组细胞完成后续实验,通过CCK8和克隆形成实验检测细胞增殖能力、Transwell实验检测侵袭能力、划痕实验检测迁移能力,流式细胞术检测细胞周期;Western blot实验检测Chk2、p-Chk2、Cdc25c和p-cdc25c蛋白的表达。结果: 与CNE-2R和NC-shRNA组比较,RPA1-shRNA组细胞的RPA1mRNA和蛋白质均显著降低(P＜0.01和＜0.05);RPA1-shRNA组组细胞的增殖、侵袭、迁移能力显著下降(P均＜ 0.05),细胞周期被阻滞在G2/M期(P＜0.01);RPA1-shRNA组细胞Chk2、Cdc25c的表达低于CNE-2R和NC-shRNA组细胞(P＜0.05), 而p-Chk2、p-cdc25c的表达高于其它两组(P ＜0.05)。结论: RPA1低表达抑制辐射抵抗人鼻咽癌CNE-2R细胞的增殖、迁移以及使细胞周期阻滞于G2/M期。     

定量测定黑鲷生长激素受体mRNA的液相杂交/RNase保护法

目的　建立液相杂交 核糖核酸酶保护测定 (RibonucleaseProtectionAssay,RPA)技术定量检测黑鲷(Sparusmacrocephalus)生长激素受体mRNA水平。方法　将黑鲷生长激素受体cDNA片断亚克隆至pGEM T载体 ,制备特异性的放射性反义RNA探针及正义RNA ,将反义RNA探针与正义RNA及样品总RNA进行液相杂交 ,用RNaseA和RNaseT1 降解杂交产物的单链RNA ,双链杂交体得到保护 ,然后检测杂交体的分子大小及放射强度。结果　检测出了黑鲷生长激素受体反义RNA探针与正义RNA及样品总RNA的特异性杂交片断 ,由此建立了定量测定黑鲷生长激素受体mRNA的液相杂交 RNase保护法 ,并采用该方法在黑鲷的多种组织中检测出了生长激素受体基因的表达 ,表达水平在肝脏中最高。该结果与我们曾采用生长激素放射受体分析法对黑鲷生长激素受体所研究的结果相吻合。结论　为进一步深入研究鱼类生长激素受体分子内分泌的调控理论提供了有力手段。     

猴B病毒抗体ELISA检测试剂盒的制备及准确性评价

目的:以金标准试剂盒为参照,对新研制的猴B病毒抗体ELISA检测试剂盒进行准确性评价,以期为我国实验灵长类动物产业提供优质廉价的检测试剂。方法:利用3个批次共表达猴B病毒gD和g B蛋白的细胞上清分别制备ELISA检测试剂盒,与金标准试剂盒(VRL的ELISA试剂盒)同时对667只食蟹猴血清进行检测,考察新研制剂盒的准确性;然后,挑选强阳性、弱阳性、阴性样品,用另外2个批次ELISA试剂盒检测,考察不同批次试剂盒检测结果的稳定性。结果:金标准试剂盒对667只食蟹猴血清抗体检测结果为阳性280份、阴性387份,而新研制的ELISA试剂盒结果为阳性282份、阴性385份;与金标准相比,阳性样品的符合率为98.93%(277/280),阴性样品的符合率为98.97%(383/387),总体符合率为98.95%(660/667)。对100份强阳性样品、70份弱阳性样品、108份阴性样品及7份差异样品的重复检测表明,除1份弱阳性(金标准为阴性)检测为阴性外,其余样品与前期一致。结论:与金标准相比,新研制的试剂盒具有较高的准确性,可用于猴B病毒抗体的检测。     

明胶碳料的研制及其在明胶液化试验中的应用

自行研制明胶碳粒用于细菌鉴定中的明胶液化试验。将明胶碳粒加入基质中经３７℃ １６￣２０ｈ培养后,如细菌具有明胶酶,其质即变黑色,反之明胶碳粒在基质中沉淀,基质不变色,经接种标准菌株和临床常见菌２０３株,试验结果表明,该明胶碳粒使用简便,容易判定,特异性强,灵敏度高,质量稳定,可以代替国外同类产品。有助于提高细菌鉴定工作效率和质量,降低检测成本,具有很好的应用价值。     

明胶碳粒的研制及其在明胶液化试验中的应用

自行研制明胶碳粒用于细菌鉴定中的明胶液化试验。将明胶碳粒加入基质中,经３７℃１６～２０ｈ培养后,如细菌具有明胶酶,基质即变黑色,反之明胶碳粒在基质中沉淀,基质不变色。经接种标准菌株和临床常见菌２０３株,试验结果表明,该明胶碳粒使用简便,容易判定,特异性强,灵敏度高,质量稳定,可以代替国外同类产品。有助于提高细菌鉴定工作效率和质量,降低检测成本,具有很好的应用价值。     

实时荧光重组酶聚合酶扩增快速检测临床常见曲霉菌方法的建立

目的:针对曲霉菌属转录间隔区ITS1设计引物、探针,利用实时荧光重组酶聚合酶扩增(Real-time RPA)技术建立一种快速、准确、经济的临床常见曲霉菌检测鉴定方法。方法:利用建立的实时荧光重组酶聚合酶扩增体系对标准菌株及临床标本提取的DNA进行扩增,验证该方法的性能。结果:本研究针对曲霉菌属转录间隔区ITS1设计引物、探针利用RPA试剂盒(荧光型)建立了Real-time RPA扩增体系,在15分钟内即可检测出临床常见的四种曲霉菌;特异性试验结果显示反应体系只对烟曲霉、黄曲霉、土曲霉、黑曲霉四种曲霉呈现出明显的扩增曲线,而其它细菌和真菌均无扩增曲线。灵敏性试验显示最低检出限为10-3 ng/μL。临床验证试验的12份曲霉菌均有较高的扩增效应。结论:本研究建立的Real-time RPA方法能快速、特异、灵敏地检出烟曲霉、黄曲霉、土曲霉、黑曲霉等临床常见曲霉菌,为曲霉菌的快速、现场检测提供了一种新的思路。     

壳寡糖缓解甲萘醌诱导巨噬细胞损伤机制初探

目的:研究壳寡糖对甲萘醌诱导的巨噬细胞氧化损伤的保护作用.方法:通过MTT实验检测相应处理的细胞活力,并通过相应试剂盒检测细胞氧化还原体系中某些相关酶的活力及相应产物含量.结果:壳寡糖能够缓解甲萘醌诱导的细胞损伤,并且发现壳寡糖可以缓解甲萘醌导致的胞内超氧化物歧化酶(SOD),谷胱甘肽过氧化物酶(GSH-PX)活力的降...     

AUT凝胶电泳在牛胰核糖核酸酶折叠研究中的应用

目的:牛胰核糖核酸酶是一种用于蛋白折叠研究的经典模式蛋白,在折叠研究过程中主要使用高效液相色谱用于分离检测不同阶段的蛋白折叠中间体。高效液相色谱具有自动化、分离效果好、样品可回收等优点,同时也存在检测通量较低、仪器设备较为昂贵等不足。AUT凝胶电泳简便、快捷、检测通量较高,本文尝试将其应用于牛胰核糖核酸酶的折叠研究。方法:使用AUT凝胶电泳、酶活性检测、质谱对牛胰核糖核酸酶还原变性过程及产生的折叠中间体进行检测;通过高效液相色谱和质谱对折叠中间单体进行分离检测,并分别进行AUT凝胶电泳检测以解析各折叠中间单体在电泳中的条带位置;通过AUT凝胶电泳和酶切后质谱鉴定各折叠中间单体的二硫键配对方式。结果:AUT凝胶电泳可以有效区分不同条件下的牛胰核糖核酸酶还原变性过程,检测结果与酶活性、质谱结果相符,并可以很好地区分牛胰核糖核酸酶还原变性过程折叠中间体。高效液相色谱将牛胰核糖核酸酶还原变性过程折叠中间体分离为13个色谱峰,并与AUT凝胶电泳中的11个条带位置进行匹配。确认牛胰核糖核酸酶还原变性过程折叠中间单体的二硫键配对方式,并与AUT凝胶电泳条带进行匹配,Cys58-Cys110和Cys26-Cys84构象熵减作用强于Cys40-Cys95和Cys65-Cys72。结论:AUT凝胶电泳适用于检测牛胰核糖核酸酶折叠中间体,可以与高效液相色谱、质谱等检测技术相互补充,共同应用于牛胰核糖核酸酶的折叠研究。     

食品中克罗诺杆菌属双重PCR检测试剂盒的评价

【背景】在食品安全领域,克罗诺杆菌属于需要重点监测的致病菌,当前随着分子检测相关技术的不断发展,研制有关食品中克罗诺杆菌简便、高效的检测产品至关重要。【目的】研制克罗诺杆菌检测的双重PCR检测试剂盒并评价其用于食品中克罗诺杆菌检测的实效性。【方法】优化双重PCR反应体系,反应试剂采用冻干工艺,确立了试剂盒组成,并评价其特异性、灵敏度、重复性、保质期等性能指标。【结果】克罗诺杆菌标准菌株和分离菌株均在目标位置出现两条明显条带,非克罗诺杆菌标准菌株和分离菌株均检测为阴性,纯基因组DNA检测灵敏度为2.3×10-1 ng/μL,纯培养菌检验限为3.2×104CFU/mL;对65份食品样品进行克罗诺杆菌检测,该试剂盒检测结果与标准方法检测结果一致性较高;批内、批间检测重复率均为100%,可在42°C环境放置120 h且其检测效力不受影响,4°C保质期可长达12个月。【结论】该试剂盒性能好,检测结果稳定、可靠,适用于食品中克罗诺杆菌的快速检测。     

介绍一种快速印迹杂交法

本文介绍一种快速的细胞mRNA及DNA印迹法。本方法以细胞数为单位,将细胞破碎后在高浓度NaI存在的条件下直接点样于硝酸纤维滤膜上用于核酸的杂交。用本方法可以检测细胞中特异mRNA和DNA的含量,主要用于细胞中特异mRNA的半定量分析和不同细胞之间特异mRNA表达的比较,也适用于分析比较细胞接受各种刺激前后特异mRNA含量的变化。     

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.