FISH检测乳腺癌石蜡组织切片的酶消化时间的探究

目的:探讨在FISH实验中,乳腺癌石蜡组织切片的最适酶的消化时间.方法:收集42例手术切除的乳腺癌标本,经过固定、包埋和切片,其中12份标本的组织切片进行胃蛋白酶消化时间的优化实验,分别观察酶的消化时间为10分钟,15分钟,20分钟和25分钟对FISH结果的影响,随后在酶的优化条件对剩余30份组织切片继续杂交实验.结果:酶消化10min提示消化时间不足,酶消化15min提示消化时间适合,结果能很好地判读,而酶消化25min提示消化过度.对剩余的30份组织切片进行了18min的消化,杂交信号基本都能判读.结论:本实验条件下,石蜡包埋切片的最适消化时间在15-20min,不同实验室及不同的FISH处理过程会略有差异.所以建议FISH实验之前,应对酶的消化时间首先进行摸索.     

RBM5荧光原位杂交探针的制备及应用

[目的]研究RBM5(RNA-binding motif protein 5)荧光探针的制备方法,确立RBM5荧光探针用于肺癌组织检测的原位杂交技术体系。[方法]以RP11-493K19菌株为材料,提取含有RBM5的质粒进行PCR验证,采用缺口平移法制备RBM5荧光探针,并与人肺癌组织石蜡切片进行杂交实验建立肺癌荧光原位杂交(fluorescence in situ hybridization,FISH)的检测体系。[结果]RBM5 15℃标记12h可获得合适的探针,探针与样本杂交后样本细胞内出现清晰明亮的绿色荧光信号,通过与呈橘红色荧光信号的CEP-3探针比较,可以判断肺癌细胞是否存在RBM5的缺失。[结论]RBM5探针制备的最佳条件是15℃标记12h,FISH实验的参数为10μg/ml蛋白酶K处理样本100 min、探针与样本37℃杂交16h、2×SSC/0.3%NP-40洗涤杂交样本5min。该实验体系适用于肺癌组织RBM5的FISH检测。     

microRNA原位杂交技术影响因素的探讨

目的探讨miRNA原位杂交技术的多种影响因素,以完善miRNA原位杂交分析方法。方法选择不同组织来源、保存时间、切片厚度的常规病理组织切片进行U。探针的原位杂交分析;选择不同浓度的蛋白酶K对组织切片与MDA-MB-23l爬片细胞进行U。探针的原位杂交分析;实时定量RT-PCR方法和爬片细胞原位杂交对BT549、T47D细胞株进行miR_375的表达分析。结果不同组织来源、保存时间、切片厚度的石蜡标本均能显示细胞核内的u。表达;组织切片原位杂交实验中,乳腺组织切片20μg／ml的蛋白酶K作用组较其他组的原位杂交效果好;肝脏组织切片200μg／ml的蛋白酶K作用组的显色最好;爬片细胞原位杂交实验中,2μg／ml的蛋白酶K作用组的显色更深;U6与miR-375的原位杂交最适浓度分别为lng／ml和l00ng／ml;实时定量RT-PCR和爬片细胞原位杂交结果均显示,BT-549的miR-375表达明显低于T47D。结论miRNA原位杂交操作能在常规固定并长期保存的石蜡组织标本上操作;蛋白酶K与探针的浓度应当依据病理标本类型等多因素预实验摸索;细胞水平的实时定量RT-PCR和爬片细胞原位杂交分析结果的比对为确定miRNA探针的特异性提供了帮助。     

骨髓活检组织EBER原位杂交检测影响因素分析

目的探讨骨髓活检组织行EBV encoded RNA(EBER)原位杂交检测的影响因素,优化检测条件以期提高骨髓活检组织中EB 病毒的检出率。方法收集35 例EB 病毒相关疾病的骨髓活检标本,通过对比实验,比较不同脱钙方法、不同蛋白酶K 消化条件、不同抗体孵育温度下的骨髓活检组织行EBER 原位杂交检测的切片质量、脱片率及染色质量情况。结果脱钙以运用改良EDTA 脱钙液脱钙20~24h 后流水冲洗30min^1h 最佳;蛋白酶K 消化时间为9min 的组织切片脱片率低,杂交效果最好,阳性细胞着色深,定位准确;在37℃条件下进行抗体孵育杂交染色质量为佳。结论选取合适的脱钙方式,延长蛋白酶K 消化时间,选择最佳抗体孵育温度,可降低脱片率,显著提高骨髓活检组织行EBER 原位杂交检测的染色质量,为EBV 相关疾病的诊断和鉴别诊断提供重要依据。     

几种变性剂对无花果蛋白酶分子去折叠与活力的影响

用不同浓度的变性剂盐酸胍、脲、十二烷基硫酸锂（ＬＤＳ）对无花果蛋白酶（Ｆｉｃｉｎ）变性,用荧光光谱及圆二色谱（ＣＤ谱）监测无花果蛋白酶去折叠过程中的构象变化并与活力变化比较,发现在１－２ｍｏｌ／Ｌ胍浓度及９．２×１０－４ｍｏｌ／ＬＬＤＳ浓度条件下,ＣＤ谱显示的二级结构含量较高,荧光谱的发射峰位刚开始红移,活力的变化则较为显著,表现为胍溶液中激活,ＬＤＳ溶液中失活,揭示酶的这二种变性剂的这二个浓度范围内,可能存在变性中间态。     

嗜热脂肪土芽孢杆菌CHB1产蛋白酶特性

目的：研究嗜热脂肪土芽孢杆菌CHBl产酶特性。方法：以蛋白酶活力为主要指标,考察温度、pH、接种量、装量等条件对CHB1产蛋白酶的影响。结果：CHB1适宜的产蛋白酶条件为：装量40mLt250mL,pH8．0,接种量5％,温度58℃,转速180r／min,时间36～44h,Tween-80对CHB1产蛋白酶具有抑制作用。结论：优化后蛋白酶产量有较大提高,最高酶活力达48U／mL,是所报道多数嗜热细菌产蛋白酶量的2—12倍。CHB1蛋白酶特性的研究及产蛋白酶量的提高,有利于揭示CHB1在堆肥化中的作用机理,提高堆肥效果。     

一种新颖的抗原信号增强方法─BT法在免疫组织化学中的应用

本文报道了一种新颖的抗原信号增强方法在免疫组织化学中的应用。该方法的特点是利用生物素标记的酪氨（ＢＴ）结合到ＨＲＰ的催化位点上来达到增强信号的目的。本研究以冰冻切片及石蜡切片中ＡＢＣ法为对照,结合微波和蛋白酶消化预处理,采用ＢＴ法检测了人扁桃体石蜡切片标本中淋巴细胞ＩｇＤ的表达。结果表明该方法检测敏感性高,当一抗稀释至１：５０００时仍能测得ＩｇＤ的表达,与对照的ＡＢＣ法相比,检测敏感性成百倍地提高。该方法能将原先只能用于冰冻切片的单抗ＩｇＤ应用于石蜡切片中。本文还对该方法的可能机制进行了探讨。本研究提示ＢＴ法在病理检验和医学研究中有良好的应用前景。     

番茄随机扩增DNA多态性体系的条件优化

利用改进的SDS法提取代号为03748的栽培番茄叶片基因组DNA。对影响番茄随机扩增DNA多态性（RAPD）扩增结果的因素进行了分析,确定了模板、Mg^2＋、dNTPs、引物和Tap DNA聚合酶的适宜浓度及反应的最佳循环次数。实验结果表明,在以下条件下,番茄的RAPD扩增效果较好：20μL反应体系中使用20-40ng的模板、1．5-2．0mmol／L的Mg^2＋、0．15＋0．20μmol／L的dNTPs、0．15-2．0μmol／L的引物、1．0U的Taq DNA聚合酶;94℃预变性5min,然后经94℃变性1min、360℃ 1min、720℃ 1．5min,进行35个循环,最后在72℃时再延伸10min。     

质粒浓度对氯化钙转化法转化效率的影响

在保证其他条件一致的情况下．设置质粒浓度梯度并建立阴性对照进行研究来确定大肠杆菌DH50t氯化钙转化法时的最佳质粒浓度,结果表明质粒浓度为10^2ng／μL时的转化效率最高。与其他质粒浓度相比,质粒浓度为10^2ng／μL时的转化效率最高,实验重复性好,成本低廉,操作简便,而且对目的基因的克隆也具有重要意义,因此值得推广使用。     

响应面法优化重组毕赤酵母表达米曲霉碱性蛋白酶诱导条件的研究

为探索重组米曲霉碱性蛋白酶（rAlp）在毕赤酵母中表达的最佳诱导条件,本研究在单因子实验的基础上,运用Box-Behnken设计的响应面试验对诱导条件进行了优化。根据回归分析确定了影响rAlp表达的因子,求得最佳诱导条件为：诱导温度28．53℃,诱导pH6．63,甲醇浓度1．28％。此工艺条件下碱性蛋白酶活力实测值为49．78U／mL,回归模型的预测值与实测值的相对误差〈1％,说明该回归方程与实际情况拟合很好。     

Chromosome CPD(PI/DAPI)- and CMA/DAPI-Banding Patterns in Allium cepa L.

Chromosome banding patterns of Allium cepa L. were obtained by using fluorescent dye combinations chromomycin A₃ (CMA) + 4",6-diamidino-2-phenylindole (DAPI), DAPI + actinomycin D (AMD) and propidium iodide (PI) + DAPI. In A. cepa,telomeric heterochromatin displayed dull fluorescence after staining with DAPI and DAPI/AMD. After joint staining with the GC-specific CMA and AT-specific DAPI, the CMA-positive fluorescence of the NOR region and the telomeric bands of C-heterochromatin was observed. In combination with DAPI, PI, a dye with low AT/GC specificity, produced almost uniform fluorescence of chromosomal arms and heterochromatin, whereas the NOR-adjoining regions displayed bright fluorescence. Denaturation of chromosomal DNA (2 × SSC, 95°C for 1–3 min) followed by renaturation (2 × SSC, 37°C, 12 h) altered the chromosome fluorescence patterns: specific PI-positive bands appeared and the contrast of CMA-banding increased. Bright fluorescence of NOR and adjoining regions was also observed in the case. Three-minute denaturation led also to a bright PI-positive fluorescence of telomeric heterochromatin. The denaturation of chromosomal DNA before staining results in changes of the DAPI fluorescence pattern and in the appearance of bright DAPI fluorescence in GC-rich NOP regions. The mechanisms underlying the effects of denaturation/renaturation procedures on chromosome banding patterns obtained with different fluorochromes are discussed.     

Chromosome CPD(PI/DAPI)- and CMA/DAPI-banding patterns in Allium cepa L

Chromosome banding patterns of Allium cepa L. were obtained by using fluorochrome combinations chromomycin A3 (CMA) + 4',6-diamidino-2-phenylindole (DAPI), DAPI + actinomycin D (AMD) and propidium iodide (PI) + DAPI. In A. cepa, telomeric heterochromatin displayed dull fluorescence after staining with DAPI and DAPI/AMD. After staining with the GC-specific CMA and AT-specific DAPI, the CMA-positive fluorescence of the NOR region and the telomeric bands of C-heterochromatin was observed. In combination with DAPI, PI, a dye with low AT/GC specificity, produced almost uniform fluorescence of chromosomal arms and heterochromatin, whereas the NOR-adjoining regions displayed bright fluorescence. Denaturation of chromosomal DNA (95 degrees C for 1-3 min) followed by renaturation in the 2 x SSC buffer (37 degrees C, 12 h) altered the chromosome fluorescence patterns: specific PI-positive bands appeared and the contrast of CMA-banding increased. Bright fluorescence of the NOR and adjoining regions was also observed in the case. Three-minute denaturation led also to a bright PI-positive fluorescence of telomeric heterochromatin. The denaturation of chromosomal DNA before staining results in changes of the DAPI fluorescence pattern and in the appearance of DAPI fluorescence in GR-rich NOP regions. The mechanisms underlying the effects of denaturation/renaturation procedures on chromosome banding patterns obtained with different fluorochromes are discussed.     

一株植物乳酸杆菌的高密度发酵

目的研究一株植物乳酸杆菌的高密度发酵。方法通过单因素实验来确定此植物乳酸杆菌的最适生长温度、pH、碳源、氮源、缓冲盐等。结果最适生长温度是37℃、起始pH6.4、最适碳源为葡萄糖、最适氮源为牛肉膏,通过对缓冲盐的选择最终确定NaAc：CaCO3比为0.5：1.5;控制发酵条件,结合优化培养基,可使发酵后的培养液达到2.75×10^10CFU/ml。结论该缓冲盐能够有效的缓解发酵液pH的下降,延长对数生长期,使活菌数提高1倍,适合高密度发酵培养。     

应用双色荧光原位杂交技术检测克氏综合征

探讨用双色荧光原位杂交技术（dual-color fluorescence in situ hybridization,D-FISH）检测性染色体数目异常克氏综合征的应用价值,建立常规分裂期染色体和间期细胞FISH技术的实验方法。以Biotin标记的X染色体α-卫星DNA（pBamX7）探针和以Digoxigenin标记的Y染色体长臂末端重复序列（pY3.4）探针对19例克氏综合征标本同时进行外周血染色体及其间期细胞核的原位杂交,分别用Avidin-FITC和Rhodamine-FITC及其Anti-avidin进行信号的检测与放大,DAPI复染。于Olympus AX-70型荧光显微镜下,分别通过WIB、WIG及其WU滤光镜观察杂交信号及其染色体或间期核背景,并统计外周血中期染色体及其间期细胞核的杂交信号颗粒数量。在显微镜下可见以Biotin标记的pBamX7探针显示2个绿色杂交信号,以Digoxigenin标记的pY3.4探针显示1个红色杂交信号,染色体或间期核背景经DAPI复染显示蓝色;18例出现XXY杂交信号的细胞,染色体及其间期细胞核杂交平均出现率分别为95.89%和95%,明显大于正常对照标准值2.75%,证实核型为47,XXY,与染色体检测的结果一致;其余1例染色体核型检测为嵌合体,XXY杂交信号细胞出现率为92%,同时检出6.7%的XY杂交信号细胞（>正常对照标准值4.17%）。用FISH 技术检测性染色体数目异常克氏综合征具有快速、敏感度高、信号强、背景低、多色等优点,故FISH 技术在产前诊断检测领域中显示其重要的应用价值和发展前景。 Abstract:The objective of the work is to study the technique of dual-color fluorescence in situ hybridization(D-FISH) and its application value in the diagnosis of sex chromosomal count abnormality Klinefelter syndrome and establish an experimental approach to metaphase chromosome and interphase nucleus FISH technique.Biotin labeled alpha satellite X-chromosome DNA(pBamX7) probe and Digoxigenin labeled Y-chromosome long arm terminal repetitive sequence (pY3.4) probe were hybridized with pre-treated slides of peripheral blood chromosome and interphase nucleus in 19 cases of Klinefelter syndrome specimens.After being washed,the slides were treated with Avidin-FITC,Rhodamine-FITC and Anti-avidin,amplified with an additional layer and counter-stained with DAPI in an antifade solution.The hybridization signals,chromosomal or interphase nucleus settings were observed respectively with WIB,WIG and WU filters under fluorescence microscope Olympus AX-70,and the number of metaphase chromosome and interphase nucleus in the peripheral blood was counted.It was observed under the microscope that the Biotin labeled pBamX7 probe showed 2 green hybridization signals and that the Digoxigenin labeled pY3.4 probe showed 1 red hybridization signal.Chromosome or interphase nucleus counter-stained with DAPI showed blue.The average signal rate of chromosome and interphase nucleus hybridization was 95.89% and 95% respectively,significantly higher than the normal control (2.75%).Karyotype 47,XXY was confirmed,which agrees with the chromosomal findings.One case showed mosaic nuclei.XXY chromosome hybridization signal rate was 92% and XY hybridization signal rate was 6.7%,higher than the normal control rate of 4.17%.FISH is a valuable technique in diagnosing sex chromosomal count abnormality Klinefelter syndrome with the merits of fast speed,high sensitivity,strong signal,low background and multiple color.Therefore,FISH technique can find wide application and potential in prenatal diagnosis.     

荧光定量PCR检测人巨细胞病毒的方法学建立

目的建立人巨细胞病毒（HCMV）的TaqMan MGB探针荧光定量PCR（FQ—PCR）检测方法。方法选取HCMV MIE exon4为PCR扩增靶序列,经TA克隆构建重组质粒作为定量标准品,经FQ—PCR反应条件的优化及方法学评价,再将其应用于临床检测。结果FQ—PCR最适循环参数为：95℃ 5 min;95℃ 20 s,60℃ 60 s（40 cycles）,20μl最适反应体系为：2．0mmol／L Mg^2＋、0．5μmol／L引物、1．5μmol／L探针、200μmol／L dNTP、2110×buffer、1．0 U Taq酶、2．0μl DNA模板。检测批内CV（变异系数）值为1．32％,批间CV值为1．96％;特异性较好;线性范围为10^2-10^8copies／μl。结论成功地建立了检测HCMV的FQ—PCR法,完全适用于临床检测。     

Human bone marrow colony formation in diffusion chambers. Normal values.

In situ hybridization techniques have previously employed a series of manipulations to effect denaturation of chromosomal DNA and reannealing of DNA-RNA hybrids. This report presents a new protocol which combines the denaturation and reannealing processes. DNA is heated in a solution of 50% formamide, 50% 4 × SSC containing the RNA to be hybridized. After l h at 70 °C the preparation is slowly cooled to 37 °C over a period of 6 h and incubated at 37 °C for an additional 10 h. This technique eliminates the possibility of premature reannealing of the DNA while employing hybridization conditions which, in vitro, lead to accurate base pairing.     

Comparisons between C-banding Patterns on Root Tip Chromosomes of Different Cultivars in Maize( Zea mays)

The technique of Giemsa banding, C-banding patterns on the root tip chromosomes and the chromocentres of interphase nuclei with three eultivars (Dai Zi Bai, Qun Dan 105 and 2×6) of maize were studied. The results are as follows: 1. After fixation and treatment in a saturated solution of barium hydroxide the preparations were incubated in 0.5×SSC, I×SSC, 3×SSC, 4×SSC or distilled water respectively forⅠ h at 60 ℃ and the other steps in C-banding procedure were not changed so as to find the optimum saline concentration for Giemsa banding in maize. The experimental results shown that 0.5×SSC was the best. But bands could not produced very well by treating samples in distilled water. 2. There were terminal, subterminal and centric bands in Dai Zi Bai and Qun Dan 105. The C-banding patterns on the root tip chromosomes of these two cultivars were different from each other. Qun Dan 105 had 10 prominent bands in total, while Daf Zi Bai had 7. The banding patterns of each chromosome were described in detail. 3. The average chromocentres per cell in Dai Zi, Qun Dan 105 and cultivar 2×6 were 7.1, 10.9 and 7.2 respectively. Their prominent band numbers on the chromosomes were 7, 10 and 8 correspondingly. It seems that the number of C-bands on the chromosomes is close to that of chromocentres.     

Influence Factors of in Situ Hybridization in Triticeae

In order to increase the efficiency, accuracy, fidelity and reliability of in situ hybridization to identify the alien chromosomes and chromosome fragments in triticeae, major steps including probe labelling, chromosome denaturation, DNA concentration for blocking and post-hybridization washing in in situ hybridization were optimized. The results are as fel-lows. (1) The cloned repetitive DNA sequence could be biotin labelled more efficiently by nick translation than by random oligonucleotide labelling method: whereas the random oligonucleotide labelling is more suitable for genomic DNA probe and the labelling efficiency could be increased by prolonging the labelling time appropriately. (2) Denaturation of the biotinylated probe and chromosomes together in oven at 75 ℃ showed the satisfactory results of in situ hybridization, but the contour of treated rye chromosomes often became blurred when the temperature of denaturation was higher than 85℃. When 70% formamide (in 2 × SSC) was used to denature the chromosome DNA, rye chromosomes often swelled although the biotinylated signals could be detected. (3) The unlabeled DNA concentrations for blocking were tested in genomic in situ hybridization to detect the Haynaldia villosa chromosomes with biotin labelled H. villosa genomic DNA as probe. The best contrast between H. villosa and wheat chromosomes was obtained without using the blocking DNA (unlabeled wheat genomic DNA). (4) Post-hybridization washes were carried out in 50% formamide (in 2 × SSC) or in 2 × SSC at different temperature. When the post-hybridization washing temperature were increased gradually from room temperature to 42℃ in 50% formamide (in 2 × SSC). specific in situ hybridization signals on chromosome in triticeae were observed using both biotinylated repetitive DNA and genomic DNA as probe. With the improved resolution of this protocol, in situ hybridization would be widely applied to wheat breeding and genetics researches.     

Oligotrophic bacterioplankton with a novel single-cell life strategy

A large fraction of the marine bacterioplankton community is unable to form colonies on agar surfaces, which so far no experimental evidence can explain. Here we describe a previously undescribed growth behavior of three non-colony-forming oligotrophic bacterioplankton, including a SAR11 cluster representative, the world's most abundant organism. We found that these bacteria exhibit a behavior that promotes growth and dispersal instead of colony formation. Although these bacteria do not form colonies on agar, it was possible to monitor growth on the surface of seawater agar slides containing a fluorescent stain, 4',6'-diamidino-2-phenylindole (DAPI). Agar slides were prepared by pouring a solution containing 0.7% agar and 0.5 micro g of DAPI per ml in seawater onto glass slides. Prompt dispersal of newly divided cells explained the inability to form colonies since immobilized cells (cells immersed in agar) formed microcolonies. The behavior observed suggests a life strategy intended to optimize access of individual cells to substrates. Thus, the inability to form colonies or biofilms appears to be part of a K-selected population strategy in which oligotrophic bacteria explore dissolved organic matter in seawater as single cells.