基因杂交信号放大技术在子宫颈癌筛查中的应用

目的:了解我市妇女生殖道人乳头瘤病毒(humanpapillomavirsHPV)感染状况,研究13种高危型HPV(16、18、31、33、35、39、45、51、52、56、58、59、68型)感染与宫颈癌的关系。方法:应用第二代基因杂交信号放大技术对1156例23～51岁参加体检的已婚妇女宫颈细胞进行13种高危型HPV脱氧核糖核酸(DNA)的检测。结果:该人群13种高危型HPVDNA总检出率为11.7%。结论:女性生殖道高危型HPV感染是宫颈癌及CIN流行的主要危险因素,提示宫颈癌的防治应重点放在防止HPV感染,而对HPV感染的筛查和密切监测应是已感染高危型HPV的对象。

激光显微切割分选少量胃癌细胞中PSCA基因的SNP分析

随着基因组关联分析方法的应用,越来越多与胃癌相关的易感基因被发现.易感基因的多态性检测已逐步进入胃癌临床诊断和研究.然而,利用少量胃粘膜细胞开展单核苷酸多态性(SNP)分析对胃癌进行早期诊断常遇下述困难,一是少量胃癌细胞混杂在多种细胞中,异常信号常易被淹没,二是细胞量极少,因此获得的基因组DNA量微,进行多位点或全基因组分析存在困难.本文利用激光显微切割技术分选少量胃癌细胞,结合全基因组放大技术,进行胃癌相关的前列腺干细胞抗原基因(PSCA)的SNP分析.通过聚合酶链反应-限制性片段长度多态性(PCR-RFLP)和克隆测序方法分析,在分选的胃癌细胞中检测到PSCA的rs2976392位点胃癌相关的"A"等位与rs2294008位点胃癌相关的"T"等位.研究结果表明,所采用的全基因组放大方法保真性高,经过分选的胃癌细胞中SNP位点的检测灵敏度和可靠性大为提高.所建立的少量细胞基因多位点检测方法将同样应用于其它肿瘤和组织的少量细胞研究中,全基因组放大产物也可进行高通量的基因芯片和第二代测序研究.

组织单腺体内单细胞的基因变异分析方法

从单细胞尺度进行细胞异质性的分析是深度理解细胞群体关系的关键。组织中的单细胞由于细胞类型不同,尺寸往往相差很大,但是目前常用的基于微孔板和Fluidigm公司的微流控的单细胞组学研究方法,需要入口的单细胞大小相近。本研究以胃组织为例,建立了一种组织单细胞的基因变异分析方法,实现了尺寸差异较大的单细胞的基因变异分析。在该方法中,先将胃组织裂解获得单个腺体,再将单个腺体酶解得到不同大小的腺体内单细胞,然后把这些单细胞铺在聚乙烯萘膜载玻片上,进行激光显微切割分选、全基因组放大,最后测其微卫星的长度。利用该方法,成功在肠上皮化生腺体内部检测到微卫星长度的变化,并灵活地对尺寸差异大的组织细胞以及肠化生腺体细胞进行了精细分析。此外,这种单细胞分析方法还可以对带有不同标记的细胞进行低通量和高通量的基因组分析,为单细胞尺度上的组织异质性研究提供了一种高度灵活的分析方法。

金属硫蛋白在辐射照射中的诱导及防护作用

金属硫蛋白(Metallothionein,简称MT)是一类广泛存在于生物体内的低分子量(约6-7 ku),富含半胱氨酸(30％),能被诱导合成的金属结合蛋白。其生物功能广泛,主要参与微量元素的贮存、运输和代谢,有拮抗电离辐射,清除自由基等多种作用。介绍了MT的分类、MT基因结构、基因放大和基因调控,MT合成的辐射诱导及其机理以及MT在辐射照射中所起的保护作用。

聚合酶链反应及其在病毒学工作中的应用

聚合酶链反应(Polymerase Chain reaction,PCR)又称体外基因放大技术,是一种在体外将特异性DNA序列进行高效扩增的方法。DNA或RNA均可作为模板(template),进行扩增。由于逆转录酶的作用,将mRNA转化为cDNA。模板DNA热变性解链,两个寡核苷酸引物(oligonucleotide primers)分别连结在待扩增的DNA片段两侧。

高危型HPV病毒含量与宫颈癌前病变程度的关系研究

目的分析高危型人乳头瘤病毒与子宫颈癌前病变的关系。方法选择来我院宫颈病-宫颈癌筛查防治中心就诊的892例病例,运用第二代基因杂交捕获信号放大技术(Hybrid CaptureⅡ,HC-Ⅱ)检测高危型HPV DNA含量,且同时行阴道镜病理活检。按病理结果将病例分为正常组、CIN-Ⅰ组、CIN-Ⅱ～Ⅲ组。以对数Log10转换原始的RLU/CO结果作为HR-HPV DNA的定量结果。统计各病例组阳性结果的病毒含量分布,验证数据的频数分布规律。应用SPSS 13.0软件一元线性回归分析HR-HPVDNA病毒含量与CIN病变程度的关系。结果 HR-HPV DNA阳性率分别是:正常组32.3%(223/693),CIN-Ⅰ53.7%(66/123)、CIN-Ⅱ为78.7%(37/47)、CIN-Ⅲ为93.1%(27/29)。各组阳性病例HPV DNA定量的均值和95%置信区间分别是:正常组1.56、1.42～1.70;CIN-Ⅰ组1.67、1.43～1.91;CIN-Ⅱ～CIN-Ⅲ组1.76、1.55～1.97,子宫颈癌前病变程度与HPV病毒DNA含量不呈线性关系(P>0.05)。结论高危型HPV感染与宫颈癌前病变的发生相关,并随病变程度的加重,感染率增高;HPV病毒含量的高低,不代表CIN病变的严重程度,然而,病毒含量越高,患高度病变的可能性越大。

应用HC-Ⅱ法筛查宫颈病变的临床评价

目的 探讨高危型HPV病毒和宫颈病变的相关性,分析运用HC-Ⅱ法筛查宫颈病变的临床应用评价.方法 应用非配对病例对照研究方法,统计分析在佛山中医院宫颈病-宫颈癌筛查防治中心同时做病理活体组织检查和HR-HPV DNA检测的893例病例.依据病理组织检查结果,分为无宫颈病变的正常组（对照组）和有宫颈病变的病变组.宫颈病变包括CIN-Ⅰ,CIN-Ⅱ,CIN-Ⅲ和SCC.对照分析全部资料的HR-HPV DNA结果,单因素分析HR-HPV与宫颈病变的相关性,统计HC-Ⅱ法筛查宫颈病变的灵敏度、特异度、阳性预测值和阴性预测值.结果 组病例HR-HPV DNA阳性率分别是：正常组32.18%（223/693）,CIN-Ⅰ 53.66%（66/123）,CIN-Ⅱ 78.72%（37/47）,CIN-Ⅲ 93.10%（27/29）和SCC组100.00%（1/1）.各级病变与HR-HPV感染的比值比（OR）、归因危险度百分比（PAR%）分别是：CIN-Ⅰ级2.44,31.64%;CIN-Ⅱ级7.80,69.12%;CIN-Ⅲ级28.46,90.48%;高度瘤样变（CIN-Ⅱ及CIN-Ⅲ）11.42,72.73%.HC-Ⅱ法筛查宫颈病变的灵敏度为65.51%,特异度为67.83%,阳性预测值为36.81%和阴性预测值为87.19%.结论 高危型HPV感染与宫颈上皮瘤样变的发生密切相关,随宫颈病变程度的加重,相关性增加,感染率也增加;HC-Ⅱ筛查方法的最大价值在于对宫颈病变的阴性预测值,筛选出易患宫颈病变的高危人群.

62例妇女高危型HPVDNA检测临界值结果的分析

目的分析运用分子杂交信号放大法检测高危型人乳头瘤病毒(HPV)DNA临界值结果的形成原因,探讨对临界值结果的合理处理方法。方法运用美国Digene公司的第二代基因杂交捕获信号放大技术(HybridCapture,HCII),检查妇女宫颈标本中的高危型HPV病毒的DNA含量。结果62例临界值结果来源于65次日常检查工作中的1676例标本,临界值结果产生的比例为3.70%(62/1676)。62例临界值标本的重复检测中,阳性结果14例(22.58%),阴性结果48例(77.42%)。临界值结果的形成原因主要在以下三个方面:交叉杂交、检测的灵敏度、实验的操作质量。结论对HCII法检测的宫颈标本的高危型HPVDNA的临界值结果,做好每次实验的质量控制前提下,进行重复检测,不能简单报告为阳性或阴性。

Chromosomal Localization of Genes bz1,bz2 in Maize by Using Ultra-sensitive FISH with Tyramide Signal Amplification(TSA-FISH)

It has been reported that endosperm undergoes programmed cell death (PCD) during maize kernel development.Both bz1 (bronze ) and bz2 are anthocyanin biosynthetic genes,and related to development of aleuronic layer of maize seeds.Tyramide signal amplification fluorescence in situ hybridization (TSA FISH) is a novel and high sensitive FISH technique,which is suitable for routine application in plant cytogenetic research.Using this technique,we physically mapped the bz1 gene onto the short arm of chromosome 9 and the long arm of chromosome 1;the percentage distances from centromere to hybridization site were 40.2,75.4 respectively,and the bz2 onto the long arm of chromosome 1 and the short arm of chromosome 5;the percentage distances from centromere to hybridization site were 21.6,15.3 separately.The TSA FISH techniques of small low copy DNA sequences for plants are discussed.

利用DGGE分析无龋和有龋儿童牙菌斑细菌组成

目的:通过变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)技术分析无龋(caries free,CF)儿童和重型早期婴幼儿龋(severe early childhood caries,SECC)儿童集合牙菌斑内细菌多样性的差异。方法:无龋儿童和SECC儿童各34例,牙菌斑基因组DNA等量混合后,制备无龋和SECC儿童牙菌斑基因组库,进行全基因组放大。分别以放大前后的基因组为模板,PCR扩增16S rDNA的V2-V3区,DGGE分析,切取SECC样本的特异条带进行克隆、测序、核酸序列比对。结果:基因组在放大前后DGGE图谱一致,SECC组样本的条带数多于CF组的条带数。切取的SECC样本的4条特异条带测序后证实为3种未培养微生物和嗜沫嗜血杆菌。结论:利用DGGE技术发现了SECC儿童菌斑与CF儿童菌斑细菌组成的差异,并在SECC样本中发现了区别于CF样本的未培养微生物,这些微生物在致龋过程中发挥的作用还有待研究。

组织中少量细胞的微卫星长度精细分析

微卫星是基因组上的特殊短重复序列,微卫星不稳定的程度与一些肿瘤的分型、治疗和预后相关。目前,微卫星长度变化的检测往往是基于大量细胞,检测灵敏度低。本文整合激光显微切割技术,基于多次退火环状循环扩增(multiple annealing and looping-based amplification cycles,MALBAC)的单细胞全基因组放大技术和毛细管电泳长度测量方法,经过关键技术点的改进,建立了一套针对组织中少量细胞的多微卫星位点检测方法。研究结果表明,基于技术改进,HE染色的组织细胞经激光显微切割分选后,可成功用MALBAC技术进行全基因组放大,并在多微卫星位点的检测上,获得了高度的准确性和重复性。利用所建立的方法,发现肠型胃癌早期病变组织-肠上皮化生(intestinal metaplasia,IM)的单个腺体内部出现多种长度变化的微卫星改变,说明该癌前病变组织中DNA错配修复系统已经出现问题。本方法所获得的全基因组放大产物,也可以用于外显子组测序和全基因组测序。另外,该方法适用于任何组织的少量细胞,甚至单细胞的多微卫星位点检测,以及全基因组的研究,为精细研究少量病变细胞的基因组特征以及组织异质性提供了有效的方法。

Distribution and generic composition of culturable marine actinomycetes from the sediments of Indian continental slope of Bay of Bengal

Actinomycetes population from continental slope sediment of the Bay of Bengal was studied. Samples were collected during two voyages of FORV Sagar Sampada in 2004 （May-June） and 2005 （July） respectively from 11 transects （each transect had ca. 200 m, 500 m, and 1 000 m depth stations）. The physicochemical parameters of overlying water, and sediment samples were also recorded. The actinomycete population ranged from 5.17 to 51.94 CFU/g dry sediment weight and 9.38 to 45.22 CFU/g dry sediment weight during the two cruises respectively. No actinomycete colony was isolated from stations in 1 000 m depth. Two-way analysis of variance showed significant variation among stations （ANOVA two-way, P〈0.05）, but no significance was found between the two cruises （ANOVA two-way, P〈0.05）. Populations in stations in 500 m depth in both cruises were higher than that of 200 m depth stations with statistically insignificant difference （ANOVA two-way, P〉0.05）. Three actinomycetes genera were identified. Streptomyces was found to be the dominating one in both the cruises, followed by Micromonospora, and Actinomyces. The spore of Streptomyces isolates showed the abundance in spiral spore chain. Spore surface was smooth. Multiple regression analysis revealed that the influencing physico-chemical factors were sediment pH, sediment temperature, TOC, porosity, salinity, and pressure. The media used in the present study was prepared with seawater. Thus, they may represent an autochthonous marine flora and deny the theory of land runoff carriage into the sea for adaptation to the salinity of the seawater and sediments.

Study on the protein expression and amplification of HER2 gene in gastric cancer

Objective: The aim of the study was to investigate the human epidermal growth factor receptor 2(HER2) gene amplification and protein expression and interpretation points in the stomach mixed carcinomas. Methods: Immunohistochemistry(IHC) and fluorescence in situ hybridization(FISH) technique were used to detect HER2 gene amplification and expression of HER2 protein in 442 cases of gastric mixed carcinoma. Results: The expression rate of HER2 protein was 41.2%(182/442): the HER2 protein expression IHC 3+ extensive type in 18 cases, partial type in 21 cases, focal type in 8 cases, accounting for 10.6%(47/442); the HER2 protein expression IHC 2+ extensive type in 23 cases, partial type in 28 cases, focal type in 11 cases, accounting for 14.0%(62/442); the HER2 protein expression IHC 1+ extensive type in 27 cases, partial type in 31 cases, focal type in 15 cases, accounting for 16.5%(73/442). HER2 gene amplification rate of 442 cases was 16.1%(71/442). In 182 cases of HER2 protein positive expression, the HER2 gene cluster amplification rate was 14.8%(27/182), large granular amplification rate 11.0%(20/182), punctate amplification rate 6.0%(11/182) and high polysomy 7.1%(13/182). In 71 cases of HER2 gene amplification, there was 42 cases of HER2 protein expression IHC 3+, 22 cases of HER2 protein expression IHC 2+, and 7 cases of IHC 1+. Conclusion: HER2 detection of gastric mixed carcinoma has great heterogeneity, HER2 protein positive expression is divided into extensive type, partial type and focal type, and HER2 gene positive amplification is divided into cluster amplification, large granular amplification, punctate amplification and high polysomy. These typing of HER2 protein expression and HER2 gene amplification provide reference index to quantify for targeted therapeutic effect of anticancer drugs.

Genetic study of Kelp “901” strain

Based on DNA extraction and optimization of random amplified reaction (RAPD) to the gametophytes and sporophytes of Kelp “901” strain, genetic study on variation was conducted to its parents and offsprings of F6, F7, F8, and F9 generation. RAPD results have shown that among 30 selected primers for gametophytes, 297 loci ranging from 200 to 3 000 bp were obtained in the average of 9.9 loci for each primer. This indicated a high polymorphic rate with RAPD detection. UPGMA (unweighted pair-group method arithmetic average) analysis showed that each male and female gametophyte of a generation could be clustered into one pair separately. The genetic distances of the Kelp 901 generation were 0.321 2–0.476 7, and the maximum was between F7 and F8 (0.476 7). Identity analysis showed that F6 generation was more close to the female parent (0.659 3), and F7 generation was more close to the male parent (0.578 8). To the sporophytes study in 24 selected primers for RAPD amplification, 191 loci ranging from 230-2 800 bp were obtained, in the average to each primer of 8.0 loci. The heterozygosity to six populations were male parent (0.223 9), female parent (0.107 2), F6 (0.216 4), F7 (0.228 6), F8 (0.229 6) and F9 (0.317 2). The nearest genetic distance was 0.083 5 (F8, F9). Total heterozygosity (HT) of F6, F7, F8 and F9 generations was 0.318 6, the average heterozygosity (HS) for F6, F7, F8 and F9 generations was 0.248 0, and deduced coefficient of population differentiation (Gst ) was 22.2%. Six sequence characterized amplified regions (SCAR) were preliminary screened through RAPD analysis. It needed to be verified in detail as they are significant for molecular marker assistance in breeding and selecting Laminaria.

MOLECULAR ANALYSIS OF RADIATION-INDUCED MUTATION IN EXON 7/8 OF RAT HPRT GENE

Objective To investigate the relationship between the radiation dose and the HPRT gene lo-cus mutation in rat smooth muscle cells, and provide the molecular basis for prevention of restenosis after percutaneous transluminal coronary angioplasty (PTC4). Methods The smooth muscle cells cultured in vitro were irradiated by radionuclide 188Re in different doses. HPRT gene mutation colonies were selected and isolatedby 6-thioguanine. Analysis of mutation in exon 7/8 of HPRT gene were accomplished by polymerase chain reaction and single-strand conformation polymorphism. Results The HPRT gene mutation frequency of rat smooth muscle cells that were irradiated by radionuclide 188Re ranged from 5.5× 10-6 to 13 ×10-6. Of 91 HPRT gene mutation colonies, 13 (14.3%) contained exon 7/8 deletion and 15(16.5%) had point mutation.The exon 7/8 mutation frequency was 30.8% . There were significant relationships between radiation dose and mutation frequency of HPRT gene and exon 7/8 . Conclusion The DNA damage and gene mutation inducedby radiation has positive relationship with radiation dose, and is a basis of proliferation inhibition and apopto-sis of smooth muscle cells.

高危型人乳头瘤病毒DNA载量与子宫颈病变程度的关系

目前已知与人类致病相关的人乳头瘤病毒（HPV）亚型有100多种，与生殖道感染和子宫颈癌有关。HPV病毒分为高危型（HR）和低危型。高危型HPV病毒的持续感染是导致宫颈癌及癌前病变——子宫颈上皮内瘤样病（CIN）的首要致病因素。

On-chip detection of a single nucleotide polymorphism without polymerase amplification

A nanoparticle-assembled photonic crystal （PC） array was used to detect single nucleotide polymorphism （SNP）. The assay platform with PC nanostructure enhanced the fluorescent signal from nanoparticle-hybridized DNA complexes due to phase matching of excitation and emission. Nanoparticles coupled with probe DNA were trapped into nanowells in an array by using an electrophoretic particle entrapment system. The PC/DNA assay platform was able to identify a 1 base pair （bp） difference in synthesized nucleotide sequences that mimicked the mutation seen in a feline model of human autosomal dominant polycystic kidney disease （PKD） with a sensitivity of 0.9 fg/mL （50 aM）-sensitivity, which corresponds to 30 oligos/array. The reliability of the PC/DNA assay platform to detect SNP in a real sample was demonstrated by using genomic DNA （gDNA） extracted from the urine and blood of two PKD-wild type and three PKD positive cats. The standard curves for PKD positive （PKD＋） and negative （PKD-） DNA were created using two feline-urine samples. An additional three urine samples were analyzed in a similar fashion and showed satisfactory agreement with the standard curve, confirming the presence of the mutation in affected urine. The limit of detection （LOD） was 0.005 ng/mL which corresponds to 6 fg per array for gDNA in urine and blood. The PC system demonstrated the ability to detect a number of genome equivalents for the PKD SNP that was very similar to the results reported with real time polymerase chain reaction （PCR）. The favorable comparison with quantitative PCR suggests that the PC technology may find application well beyond the detection of the PKD SNP, into areas where a simple, cheap and portable nucleic acid analvsis is desirable.