组织芯片与基因芯片在肿瘤研究中的联合应用

组织芯片是指固定在载玻片上的组织微点阵,可同时进行分析大量同一实验指标(DNA、RNA及蛋白质)的研究;基因芯片是指固相载体上的高密度DNA微点阵,可检测异常组织与正常组织差异表达基因。组织芯片与基因芯片配合使用在寻找肿瘤基因中有很好的互补作用。利用基因芯片检测出差异表达的侯选肿瘤基因,然后,利用组织芯片迅速筛选鉴定肿瘤基因。从而大大简化和加快从基础研究到临床应用的进程。     

微流控芯片技术在免疫分析中的应用

微流控芯片已广泛用于生物医学、高通量药物合成筛选、环境监测和生物战剂侦检等领域,本文就微流控芯片在免疫分析中的应用做一综述。1微流控芯片技术分析概述微流控芯片技术是通过微细加工技术在芯片上构建由储液池、微反应室、微管道等微功能元件构成的微流路系统,加载生物样品和反应液后,在压力泵或者电场作用下形成微流路,于芯片上进行一种或连续多种的反应,达到对样品高通量快速分析的目的。微流控芯片技术由于具有高度集成性,可在一张芯片上完成采样、稀释、加试剂、反应、分离和检测等多种功能,又被称为微型全分析系统(micro total a…     

精子发生障碍患者Y染色体微缺失的研究

目的研究无精子症和少精子症患者Y染色体上无精子症因子(azoospermicfactor,AZF)微缺失情况,建立Y染色体微缺失的分子诊断的临床筛查方法,分析原发性无精子症和少精子症患者与Y染色体微缺失的关系。方法采用多重PCR、凝胶电泳技术对56例无精子症和少精子症患者的10个STS位点或基因进行检测与筛查。结果20例精子密度正常的生育男性未检测出Y染色体微缺失;56例无精子症和少精子症患者中有9例有AZF区域的微缺失,总缺失率16.1%(9/56),AZFc/DAZ区发生微缺失频率较高。结论Y染色体微缺失是导致男性不育患者精子发生障碍的重要原因之一,AZF侯选基因在精子发生过程中可能起重要作用。     

组织芯片联合基因芯片在肿瘤研究中的应用

组织芯片是指固定在载玻片上的组织微点阵 ,可同时进行分析大量同一实验指标 (DNA、RNA及蛋白质 )的研究 ;基因芯片是指固相载体上的高密度DNA微点阵 ,可检测异常组织与正常组织差异表达基因。组织芯片与基因芯片配合使用在寻找肿瘤基因中有很好的互补作用。利用基因芯片检测出差异表达的候选肿瘤基因 ,然后 ,利用组织芯片迅速筛选鉴定肿瘤基因。从而大大简化和加快从基础研究到临床应用的进程。     

无精子症、严重少精子症患者染色体核型与Y染色体AZF微缺失的相关性分析

目的对无精子症、严重少精子症患者染色体核型与Y染色体AZF微缺失的相关性进行分析,以探讨Y染色体AZF微缺失检测在男性不育中的应用价值。方法对无精子症、严重少精子症患者进行细胞遗传学分析,根据分析结果分为染色体核型正常组及异常组。采用PCR方法对各样本Y染色体AZF所在区域的6个序列标签位点（STS）进行扩增,琼脂糖凝胶电泳进行扩增产物的检测。结果在所分析的76例无精子症、严重少精子症患者中,染色体核型异常组27例,其中未检测到有Y染色体AZF缺失的存在;染色体核型正常组49例,其中1例无精症患者发现有Y染色体AZF缺失的存在。结论染色体核型异常与AZF微缺失无相关性。Y染色体AZF微缺失是造成男性不育的原因之一。Y染色体AZF微缺失检测在无精子症、严重少精子症患者中具有重要价值,可明确无精子症、严重少精子症的病因,从而避免不必要的治疗。     

微流体芯片在乙型肝炎病毒基因型检测中的应用

目的：探讨微流体芯片检测乙型肝炎病毒基因型的价值。方法：应用微流体芯片及基因测序技术检测41例慢性乙型肝炎患者感染的乙型肝炎病毒基因型；比较两种方法检测结果的一致性。结果：41例HBV患者中B基因型21例（51．2％），C基因型20例（48．8％），未检测到B＋C混合基因型感染。微流体芯片检测结果与基因测序结果完全一致。结论：微流体芯片技术可以准确快速检测乙型肝炎病毒基因型，对乙型肝炎病毒的分子流行病学研究具有重要意义。     

2336例不育男性患者AZF微缺失及其临床分析

目的探讨不育男性患者Y染色体上无精因子基因(AZF)微缺失情况及其临床表现。方法对2005年10月~2014年7月在广西妇幼保健院就诊2 336不育男性患者采用多重聚合酶链反应结合琼脂糖凝胶电泳检测AZF15个序列标签位点。结果 2 336例患者中检测出AZF微缺失138例(5.91%),以c区微缺失率最高为4.92%,检出11种微缺失类型。其中单独AZFc区微缺失表现为少或弱精子症、严重少精子症和或无精子症,单独a区或b区微缺失表现为无精子症或严重少精子症,多个区联合微缺失均表现为无精子症。结论本地区男性不育患者AZF微缺失具有多样性,以c区微缺失为主,单独c区微缺失临床表现异质性较大,a区和b区微缺失临床表现较重,多个区联合微缺失时临床表现最严重。     

细胞周期类分子在人无精子症睾丸中的表达

目的:评价细胞周期类基因在人无精子症及正常睾丸组织中的表达及意义.方法:应用包含有人CDC10等细胞周期类基因在内的cDNA微矩阵芯片对人正常睾丸及无精子症睾丸组织中差异表达基因进行了研究:通过PCR方法获得两种组织mRNA, 再分别用Cy5-dUTP及Cy3-dUTP标记制备cDNA探针.两种探针混合后与人cDNA微矩阵芯片杂交, 经扫描、计算机处理分析比较杂交结果;利用原位杂交技术对芯片杂交结果进行了验证研究.结果:部分细胞周期类基因可能与无精子症相关, 其中CDC7L1 与CDC10基因表达上调, CDK9、 CDC20 以及CLK3基因表达下调.原位杂交证实CDC10在正常睾丸组织生精细胞中表达强于无精子症睾丸组织.结论:细胞周期类分子CDC10、 CDC7L1 、 CDK9、 CDC20及CLK3可能在无精子症的发生与进展过程中起一定的作用.     

Y染色体AZFa区sY86缺失对男性生育能力无影响的一个家系分析

目的 分析一个在Y染色体AZFa区存在sY86缺失,有正常生育能力的家系。方法 经知情同意,对患者进行精液常规分析,精子形态分析,外周血染色体核型分析,对患者及其父亲进行Y染色体微缺失检测,并重新设计引物,用电泳法对扩展位点sY82、sY83、sY1064、sY86、sY84、sY1065、sY1182和sY88进行检测分析,采用AffymtrixCytoScan 750K SNP-Array芯片,来验证患者及其父亲Y染色体微缺失的缺失模式和缺失断点。结果 患者精液常规分析及精子形态正常,Y染色体AZFa区sY86缺失,拓展分析显示sY83、sY1064、sY86缺失,SNP-Array芯片检测结果显示在Yq11.21(14416038-14622345)区段存在206.3kb的缺失,该片段内含sY83、sY1064和sY86位点,但是不含有OMIM基因,患者父亲的缺失位点与缺失片段与患者一致。结论 Y染色体AZFa区sY83、sY1064、sY86位点的缺失,对应区段Yq11.21(14416038-14622345)的缺失是良性的,当发现AZF区域存在部分缺失时,需要进行额外的扩展...     

登革病毒悬液芯片分型检测方法的建立

目的 建立一种基于悬液芯片的登革病毒(dengue virus,DV)检测方法,可对四种血清型登革病毒进行快速检测和鉴定.方法 依据GenBank上4种病毒的基因序列信息,设计并合成相关引物及探针序列.抽提病毒RNA,经反转录后对目的基因进行PCR扩增,产物与核酸探针微球组杂交后于Bio-PlexTM 200系统检测荧光信号值.结果 DV1的悬液芯片检测敏感性约9 DNA拷贝,DV2、DV3、DV4的悬液芯片检测敏感性约90 DNA拷贝.进而将本方法用于检测15份临床标本,其检测结果与分型荧光RT-PCR一致.结论 建立了可同时检测四种血清型登革病毒的悬液芯片检测方法,为快速筛查和鉴定登革病毒提供了新的手段.     

Multicomponent properties of barley stripe mosaic virus ribonucleic acid

RNA extracted from purified barley stripe mosaic virus (BSMV) was analyzed by density gradient centrifugation and polyacrylamide gel electrophoresis. All strains tested had a prominent 21.3 S RNA, which was separated into two components by gel electrophoresis. Most preparations of North Dakota 18 and Argentina Mild strains had an additional 19.5 S component, which was resolved into one or two RNA species by gel electrophoresis. Some preparations also contained variable amounts of two or three minor RNA components migrating slowly in gels; these components sedimented at about 30 S in sucrose gradients. The two 19.5 S components were more variable than other components, and occasionally one or both disappeared during virus subculture. Double-stranded RNA isolated from infected plants migrated more slowly in polyacrylamide gels than did single-stranded RNA isolated from purified virions, but both types of RNA from the same strain had the same number of major RNA components. The RNAs of BSMV migrated more slowly in gels than expected from their sedimentation rates if brome mosaic virus RNAs and barley ribosomal RNAs were used as standards, which suggests that BSMV RNAs and the standard RNAs differ in secondary configuration. Mixtures of the separated RNA components were more infectious than single components, which indicates that more than one RNA component was necessary to establish infection.     

The detection and characterization of viral-related double-stranded RNAs in tobacco mosaic virus-infected plants

The 2 M LiCl-soluble RNA fraction extracted from tobacco mosaic virus (TMV)-infected tobacco plants contains, in addition to the viral replicative form of 4 x 10(6) MW, three smaller double-stranded (ds) RNA species with apparent molecular weights (estimated by polyacrylamide gel electrophoresis, using ds RNAs as markers) of 2.25, 1.1, and 0.23 x 10(6). The synthesis of all four ds RNAs is insensitive to actinomycin D. They are completely RNase insensitive at high salt concentrations and are found both in directly inoculated and in apical tissues. In tissues incubated in the presence of 3H-uridine and actinomycin D, the three small ds RNAs accounted for 6 to 11.5% of the total radioactivity incorporated into viral ds RNA. On a molar basis, however, in apical leaves the smallest ds RNA was synthesized to almost the same level as the replicative form. By molecular hybridization, the three small ds RNAs have been shown to be of viral origin, and each contains sequences represented in the 5' end of complementary (negative strand) TMV RNA. Based on molecular weight data, none of the ds RNAs can be considered to be a ds form of the subgenomic TMV coat protein mRNA (the LMC), suggesting that it is not replicated independently. None of the small ds RNAs was found to be an endogenous product of the bound TMV RNA replicase.     

QIAamp及Biomed粪便细菌基因组DNA提取试剂盒提取人肠道细菌基因组DNA质量的差异*

背景：有研究表明,不同试剂盒提取的粪便细菌基因组DNA质量有差别,选择一种操作简便、质量优良的粪便细菌基因组DNA提取试剂盒成为研究者们关注和急需解决的问题。 目的：比较QIAamp及Biomed粪便细菌基因组DNA提取试剂盒提取的人肠道细菌基因组DNA质量的差异。 方法：收集健康成年人新鲜粪便标本30例,用两种试剂盒分别提取细菌基因组DNA,检测其浓度、吸光度A260/280 nm值及提取率,设计乳酸菌属16S rDNA基因特异性引物,以各自所提DNA为模板,进行常规聚合酶链反应,凝胶电泳后比较条带数量、明暗度及密度,并进行实时荧光定量聚合酶链反应定量检测各自所含乳酸菌属的数量。 结果与结论：QIAamp试剂盒提取的正常人肠道细菌基因组DNA的浓度、提取率、表达量及乳酸菌属的数量均高于Biomed试剂盒(P < 0.05或P < 0.01)。说明QIAamp试剂盒提取的粪便细菌基因组DNA质量优于Biomed试剂盒。     

Apolipoprotein E deficiency associated with type III hyperlipoproteinemia--analysis of apolipoprotein E

Case report: The patient was a 50-year old male who was found to have a high cholesterol level during a routine health check up at work 5 years before and was examined at Keio University Hospital. Lipoprotein electrophoresis on agarose gel revealed type III hyperlipidemia, and a screening test yielded the following values (mg/dl): total cholesterol, 420; TG, 138; and HDL-cholesterol, 105. Turbidimetric immunoassay showed that the apolipoprotein E (apoE) level was below the limit of detection. Since he was 25 years old, the patient had sometimes noticed xanthomas on his knees and eyelids, and for that reason we made a diagnosis of apoE deficiency associated with type III hyperlipidemia. We tried using SDS-polyacrylamide gel electrophoresis, Western blot, and the protein chip method to detect apoE in this case, but the level was below the limit of detection by the first two methods, and it was so low that it was detected near the sensitivity limit of the protein chip method. Diet therapy, statin therapy, and fibrate therapy have been continued, and the latest data are: total cholesterol, 373; TG, 95; and HDL-cholesterol, 83. No manifestations associated with arteriosclerotic disease other than mild xanthomas have been observed.     

Isolation and in vitro translation of messenger RNA from the American cockroach

Total RNA was extracted from the whole body of American cockroach Periplaneta americana) using chaotropic salt guanidine isothiocyanate in the presence of 2-mercaptoethanol (2-ME). Polyadenylated mRNA was isolated by oligothymidylic acidcellulose chromatography and mRNA was translated using a rabbit reticulocyte lysate system. The translation, as judged by the incorporation of-S-tnethionine, was obtained with poly(A)‘*’RNA, where an approximately 9-5-fold increase in label incorporation over control was achieved. Analysis of translation products by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) in combination with autoradiography showed that many proteins with apparent molecular weights ranging from 12 to 200 kD were synthesized, and no labelled proteins were found with negative RNA control and poly(A) RNA. Immunoprecipitation studies performed using polyclonal and monoclonal antibodies revealed that synthesized proteins of MW 90, 78, 72, 49, 45, and 26 kD corresponded with previously identified principal and major allergens of American cockroach from our laboratory. In addition, the allergenicity of the translation mixtures was also confirmed by fluoroallergosorbent test (FAST) inhibition studies with IgE antibodies of human reaginic serum pool.     

Assessment of Circulating Agglutinating Anti-Sperm Antibodies in Buffalo Cows With Unexplained Infertility and an Attempt to Identify Buffalo Sperm Isoantigens

The presence of circulating agglutinating anti-sperm antibodies as cause for unexplained infertility of artificially inseminated buffalo cows was assessed. An attempt to identify buffalo sperm isoantigens was also made. The following methods were applied for that purpose: the tray agglutination test (TAT), SDS-polyacrylamide gel electrophoresis, and immunoblotting. The results obtained showed that three of 90 sera from buffalo cows with unexplained infertility were positive in TAT (3.3%) and their titers were low. A total of 27 spermatozoal polypeptides reacted positively with the IgG-isoantibodies of one of the sperm agglutinating sera in immunoblotting. Of the control sera 12 also revealed individual variations in the number of positive fractions on the blots. On the basis of comparing the blot with the positive sperm agglutinating serum to the blots of the positive controls, two buffalo sperm isoantigens were identified, with the respective molecular weights of 40 kDa and 120kDa. In conclusion, circulating agglutinating anti-sperm antibodies are very rarely detected in buffalo cows with unexplained infertility after several artificial inseminations.     

Human rotavirus RNA prepared from stool samples by a simple procedure suitable for the determination of electropherotypes

A simple procedure has been developed for the determination of human rotavirus electropherotypes. Human rotavirus particles were concentrated from stool samples by differential centrifugation. Genomic RNAs were extracted from virus particles by protease K digestion and analysed by polyacrylamide gel electrophoresis. Using this procedure variations in the electrophoretic mobilities of the RNA segments were detected among human rotavirus isolates collected in Scotland.     

Comparisons of Three Automated Systems for Genomic DNA Extraction in a Clinical Diagnostic Laboratory

Purpose

The extraction of nucleic acid is initially a limiting step for successful molecular-based diagnostic workup. This study aims to compare the effectiveness of three automated DNA extraction systems for clinical laboratory use.

Materials and Methods

Venous blood samples from 22 healthy volunteers were analyzed using QIAamp® Blood Mini Kit (Qiagen), MagNA Pure LC Nucleic Acid Isolation Kit I (Roche), and Magtration-Magnazorb DNA common kit-200N (PSS). The concentration of extracted DNAs was measured by NanoDrop ND-1000 (PeqLab). Also, extracted DNAs were confirmed by applying in direct agarose gel electrophoresis and were amplified by polymerase chain reaction (PCR) for human beta-globin gene.

Results

The corrected concentrations of extracted DNAs were 25.42 ± 8.82 ng/µL (13.49-52.85 ng/µL) by QIAamp® Blood Mini Kit (Qiagen), and 22.65 ± 14.49 ng/µL (19.18-93.39 ng/µL) by MagNA Pure LC Nucleic Acid Isolation Kit I, and 22.35 ± 6.47 ng/µL (12.57-35.08 ng/µL) by Magtration-Magnazorb DNA common kit-200N (PSS). No statistically significant difference was noticed among the three commercial kits (p > 0.05). Only the mean value of DNA purity through PSS was slightly lower than others. All the extracted DNAs were successfully identified in direct agarose gel electrophoresis. And all the product of beta-globin gene PCR showed a reproducible pattern of bands.

Conclusion

The effectiveness of the three automated extraction systems is of an equivalent level and good enough to produce reasonable results. Each laboratory could select the automated system according to its clinical and laboratory conditions.