胆囊癌p53原位杂交和nm23蛋白表达与淋巴组织增生的关系

研究胆囊癌 p5 3原位杂交和nm2 3蛋白表达淋巴组织增生的关系。采用原位杂交和免疫组织化学技术检测了 5 9例胆囊癌。 5 9例胆囊癌p5 3蛋白表达 44 1% ,p5 3原位杂交 6 4 4% ,二者的检测结果不完全一致 ,但均与胆囊癌的分期有关 ,晚期胆囊癌高于早期胆囊癌 ,差异有显著性 (P <0 0 1)。淋巴结反应性增生比淋巴结伴有癌转移者阳性表达率低 ,差异有显著性 (P <0 0 1)。nm2 3基因蛋白阳性表达 76 3 % ,淋巴结反应性增生者比淋巴结伴有癌转移者阳性表达率高 ,差异有显著性 (P <0 0 1)。结果显示 ,p5 3基因与nm2 3蛋白表达在胆囊癌的分期中呈负相关。p5 3基因和nm2 3蛋白表达在胆囊癌的发生、发展中与淋巴组织增生的程度有关。

荧光原位杂交临床应用进展

荧光原位杂交临床应用进展谢全锦杜传书近年，荧光原位杂交（ＦＩＳＨ）已成为解决细胞遗传学问题，特别是临床细胞遗传学疑难杂症的重要方法。例如分析羊水细胞作产前诊断或对先天发育异常的儿童检测其外周血淋巴细胞的染色体，尤其是那些产后不久就夭折的婴幼儿更应作细...

鼻咽癌Epstein-Barr病毒的原位分子杂交研究

应用 Epstein- Barr病毒编码的小 RNA- 1(Epstein- Barr virus encoded sm all RNA- 1,EBER- 1)分子探针进行原位分子杂交 ,检测沈阳地区鼻咽癌的 Epstein- Barr病毒存在状况及意义。结果发现 :EBER- 1杂交信号定位于鼻咽癌的癌细胞核 ,在正常的鼻咽粘膜上皮细胞、间质细胞、粘液腺及淋巴细胞均为阴性。在 5 3例鼻咽癌病例中 ,37例阳性 ,阳性率为 6 9.8%。EBER- 1的阳性表达与鼻咽癌的分化程度呈负相关 ,与患者的生存期未见明显关系。提示 :应用 EBER- 1分子探针可进行回顾性研究 ;EB病毒的存在与鼻咽癌的分化程度成负相关。

精索静脉曲张对精子非整倍体的影响及手术治疗的变化评估

目的探讨不同程度的精索静脉曲张(VC)对精液质量和精子非整倍体率的影响及手术后的改善情况。方法34例患者经彩色多普勒超声(CDFI)诊断为VC并按不同曲张程度分为亚临床型(SVC)(n=8)、VCⅠ级(n=13)、VCⅡ级(n=7)和VCⅢ级(n=6),分析精液指标,应用X、Y、18号染色体探针对其进行多色荧光原位杂交实验,检测精子染色体非整倍体数目。VCⅡ级和VCⅢ级患者行腹腔镜精索静脉高位结扎术,术后3个月复查精液指标及非整倍体数目并与术前比较。结果 VCⅡ组与VCⅢ组非整倍体率显著高于对照组(P<0.01);VCⅡ组及VCⅢ组患者行手术治疗后,精子密度、活率、活力均较术前改善(P<0.01),非整倍体较术前显著下降(P<0.01);Pearson相关分析显示曲张程度与非整倍体数呈正相关。结论 VC导致精子染色体非整倍体增多,手术对VCⅡ级和VCⅢ级患者精液质量有较明显改善作用,同时在一定程度上降低了非整倍体率。

Physical Mapping of the Sequences Homologous to Disease Resistance Genes myb1 and NDR1 in Maize

Using fluorescence in situ hybridization, the authors investigated the homology between three plant species, maize (Zea mays L.) and tobacco (Nicotiana tabacum L.), maize and Arabidopsis thaliana (L.) Heynh. at cytogenetic level using two probes corresponding to functional disease resistance genes myb1 and NDR1 in Arabidopsis and tobacco respectively. The hybridization signals of the tested probes were detected in maize chromosomes 8 and 5 respectively, and the single location of each of the two probes showed only single copy of them in maize genome. The results provided a valuable insight into searching for genes associated with programmed cell death in plants using heterologous probe with comparative genetic approach. In addition, the improvements of FISH technique using heterologous probes were discussed.

The Distribution of Repetitive DNAs Along Chromosomes in Plants Revealed by Self-genomic in situ Hybridization

The distribution of repetitive DNAs along chromosomes is one of the crucial elements for understanding the organization and the evolution of plant genomes. Using a modified genomic in situ hybridization （GISH） procedure, fluorescence in situ hybridization （FISH） with genomic DNA to their own chromosomes （called self-genomic in situ hybridization, self-GISH） was carried out in six selected plant species with different genome size and amount of repetitive DNA. Nonuniform distribution of the fluorescent labeled probe DNA was observed on the chromosomes of all the species that were tested. The signal patterns varied among species and were related to the genome size. The chromosomes of the small Arabidopsis genome were labeled almost only in the pericentromeric regions and the nucleolus organizer regions （NORs）. The signals in the relatively small genomes, rice, sorghum, and Brassica oleracea var. capitata L., were dispersed along the chromosome lengths, with a predominant distribution in the pericentromeric or proximal regions and some heterochromatic arms. All chromosomes of the large genomes, maize and barley, were densely labeled with strongly labeled regions and weakly labeled or unlabeled regions being arranged alternatively throughout the lengths. In addition, enhanced signal bands were shown in all pericentromeres and the NORs in B. oleracea var. capitata, and in all pericentromeric regions and certain intercalary sites in barley. The enhanced signal band pattern in barley was found consistent with the N-banding pattern of this species. The GISH with self-genomic DNA was compared with FISH with Cot-1 DNA in rice, and their signal patterns are found to be basically consistent. Our results showed that the self-GISH signals actually reflected the hybridization of genomic repetitive DNAs to the chromosomes, thus the self-GISH technique would be useful for revealing the distribution of the regions where repetitive DNAs concentrate along chromosomes and some chromatin differentiation associated with repetitive DNAs in plants.

Identification of Wheat-Barley 2H Alien Substitution Lines

The genetic constitution of fifteen materials derived from the cross wheat (Triticum aestivum L. cv. 'Chinese Spring') X barley (Hordeum vulgare L. cv. 'Betzes') was analyzed, and six disomic alien substitution lines were screened by GISH. The chromosome configurations in pollen mother cells at meiotic metaphase I (PMCs M I) of F, from each disomic substitution line respectively crossed with double ditelocentric lines 2A, 2B and 2D of 'Chinese Spring' were observed, and a set of wheat-barley disomic alien substitution lines 2H(A), 2H(B) and 2H(D) were obtained. The RFLP analysis with the probe psr131 on the short arm of wheat homeologous group 2 combining with four restriction enzymes were carried out. The results indicated that the probe psr131 could be used as molecular marker to tag the barley chromosome 2H. The barley chromosome 2H had good genetic compensation ability for wheat chromosomes 2B and 2D in vitality and other agronomic characters. The result of testing seed was that the wheat appearance starch quality had been changed from the half-farinaceous of 'Chinese Spring' to the half-cutin of substitution lines by transferring the barley chromosome 2H to wheat.

Identification of Plasma Membrane Aquaporin in Guard Cells of Vicia faba and Its Role in Stomatal Movement

Water channels or aquaporins are the main pathways of water transport. Both the existence and function of aquaporins in die guard cells of Vicia faba L. were investigated both by using RD28 cDNA and RD28 antibody as probes, and by controlling stomatal movement as a parameter combined with antibody and inhibitor of aquaporins respectively. The results revealed that RD28 mRNA, encoding a plasma membrane aquaporin, expressed in ale mesophyll cells and vascular tissues of V. faba, especially in guard cells. And the location of RD28-like proteins was mainly on plasma membrane of guard cells. The addition of 25 mumol/L HgCl2, an aquaporin blocker, and antibody of RD28 as well, greatly suppressed the stomatal opening or guardcell protoplast swelling induced by fusicoccin and light, and closing induced by abscisic acid. However, 5 mmol/L, beta-mercaptoethanol, a reverse reagent of aquaporin blocker, reversed the inhibitory effect of HgCl2 Pretreatment oil stomatal opening ( i.e., HgCl2 was removed after HgCl2 pretreatment for 10 min). The results suggest that the aquaporins in V. faba are associated with stomatal movement.

Isolation of CA/GT Microsatellites from the Paralichthys olivaceus Genome

A library rich in CA/GT microsatellites was constructed from the Paralichthys olivaceus genome by combining biotin capture method and radioactive labeling hybridization. Five hundred and twenty six positive clones were obtained through twice screens. Sequencing confirmed 133 microsatellite loci （number of repeats t〉 5） in 119 positive clones. Of these microsatellites, two （1.5%） had compound repeat motifs, 63 （47.37%） had perfect motifs and 68 （51.13%） had imperfect motifs. Primer pairs were designed in the flanking regions of 22 microsatelites and subjected to PCR amplification. In 8 artificial gynogenesis families, four pairs failed to amplification, one pair was monomorphic, and the rest were polymorphic with an average of 5.2 alleles per locus. Heterozygosities ranged between 0. 375 and 0. 846, PIC ranged between 0. 305 and 0. 823. The results suggested that most of the microsatellites we isolated were qualified to be applied to the population genetic studies of P. olivaceus.

Microdissection of Haynaldia villosa Telosome 6VS and Cloning of Species-specific DNA Sequences

The material T240_6 derived from SC 2 young embryo of the combination CA9211/RW15 (6D/6V alien substitution) was telosomic substitution line of 6VS identified by GISH (genomic in situ hybridization) analysis. The 6VS was microdissected with a needle and transferred into a 0.5 mL Ep tube. In the 'single tube', all the subsequence steps were conducted. After two round of LA (Linker adaptor)_PCR amplification, the size of PCR bands ranged from 100 to 3 000 bp, with predominate bands 600-1 500 bp. The products were confirmed by Southern blotting analysis using Haynaldia villosa (L.) Schur. genomic DNA labeled with 32 P as probe. The PCR products were purified and ligated into clone vector-pGEM_T easy vector. Then, the plasmids were transformed into competence E. coli JM109 with cool CaCl 2. It was estimated that there were more than 17 000 white clones in the library. The size of insert fragments distributed from 100-1 500 bp, with average of 600 bp. Using H. villosa genomic DNA as probe, dot blotting results showed that 37% clones displayed strong and medium positive signals, and 63% clones had faint or no signals. It is demonstrated that there were about 37% repeat sequence clones and 67% single/unique sequence clones in the library. Eight H. villosa_specific clones were screened from the library, and two clones pHVMK22 and pHVMK134 were used for RFLP analysis and sequencing. Both of them were H. villosa specific clones. The pHVMK22 was a unique sequence clone, and the pHVMK134 was a repeat sequence clone. When the pHVMK22 was used as a probe for Southern hybridization, all the powdery mildew resistance materials showed a special band of 2 kb, while all the susceptible ones not. The pHVMK22 may be applied to detect the existence of Pm21.

Cytological and Molecular Identification of Alien Chromatin in Giant Spike Wheat Germplasm

Alien chromosomes of twelve giant spike wheat germplasm lines were identified by C-banding, genomic in situ hybridization (GISH), sequence characterized amplified region (SCAR), and random amplified polymorphic DNA (RAPD). All lines showed a chromosome number of 2n = 42, five of them carried both a pair of wheat-rye (Triticum aestivum-Secale cereal) 1BL/1RS translocation chromosomes and a pair of Agropyron intermedium (Ai) chromosomes, three carried a pair of Ai chromosomes only, three others carried a pair of 1BL/1RS chromosomes only, and one carried neither 1BL/1BS nor Ai chromosome. Further identification revealed that the identical Ai chromosome in these germplasm lines substituted the chromosome 2D of common wheat (T aestivum L.), designated as 2Ai. The genetic implication and further utilization of 2Ai in wheat improvement were also discussed.

Expression Patterns of a Vernalization-related Genes Responding to Jasmonate

利用RNA原位杂交技术分析了春化相关基因ver2 0 3F在冬小麦 (TriticumaestivumL .cv .JingdongNo .1 )胚芽组织内的表达模式。结果表明 ,ver2 0 3F基因的转录本在春化处理的冬小麦胚芽幼叶中有明显的积累 ,而在胚芽鞘以及生长点处却未见杂交信号。茉莉酸诱导冬小麦胚芽后的基因表达模式与春化处理后的模式相似。实验结果暗示感受春化作用的信号部位可能是胚芽的幼叶 ,细胞对春化的应答与茉莉酸介导的信号转导途径有关。

Primary Identification of Alien Chromatin in T911289,a Maintainer of Wheat Male Sterile Line with Cytoplasm of Aegilops kotschyi

The genomic composition of 1911289, a wheat ( Tritium aestivum L.) maintainer of K-CMS, was examined by several methods, such as genomic in situ hybridization (GISH), biochemical marking, and DNA molecular marking. The results got by GISH and PCR amplification of dispersed rye-specific repetitive DNA sequence suggested that the alien chromatin in T911289 derived from rye. Specifically PCR amplification of the rye-specific microsatellite primers (SCM9) and seed storage protein analysis indicated that the alien chromatin in T911289 had developed from the short arm of 1R chromosome of rye (1RS). PCR amplification by using microsatellite primers locating on 1BS and seed storage protein analysis also revealed that 1911289 had lost the arm of 1BS or a small distal segment of it. We conclude that T911289 is a heterogeneous population which displays two distinct different types of translocation, i.e. the Robertsonian translocation and small segment translocation. The Robertsonian translocation type observed in our study is different from the 1BL/1RS translocation which is widely used in wheat production; it may be a novel and complex translocation form. Though the linkage between the desirable agronomic traits and the deleterious genes expressed as sticky dough has not got broken in T911289, the recovery of small segment translocation will still benefit the genetic study of wheat and rye.

Isolation and Chromosomal Mapping of a Corn B Chromosome Specific RAPDs

B染色体存在于多种动植物中 ,具有很多独特的性状。B染色体与正常染色体在DNA组成方面十分相似 ,寻找B染色体特异序列一直是B染色体研究的难点和热点。通过对含有和不含有B染色体的两种玉米 (ZeamaysL .)基因组进行了RAPD分析 ,筛选到一个B染色体特异性分子标记B480。该标记与玉米的自主复制起始序列ARS1和ARS2同源 ,特别是该序列中的 2 5bp出现在多种模式生物基因组中。FISH的结果显示 。

Using Isolated Embryo Sacs and Early Proembryos for Localization of Calmodulin mRNA Before and After Fertilization in Nicotiana

An in situ hybridization technique for localization of calmodulin(CaM) mRNA in isolated entire embryo sacs and proembryos in Nicotiana tabacum L.cv.W38 has been developed. This technique can be applied to small amounts of materials in which a whole view of CaM mRNA distribution can be obtained. The authors revealed that CaM mRNA expression changes dramatically before and after fertilization. Especially interesting is that a prominent CaM mRNA band appears between the egg apparatus and polar nuclei temporarily during the period of pollination and fertilization. The band disappears just prior to fertilization and expands to a fan_shaped region that occupies the micropylar portion of the embryo sac. After fertilization, CaM mRNA accumulates in the elongated zygotes with higher concentration in their chalazal portion than in the micropylar portion. Such an asymmetrical pattern continues to manifest in the early proembryos. It is supposed that CaM mRNA may be involved in the early events and signaling steps associated with double fertilization and zygote polarization in higher plants.

Comparative Physical Localization of Rice Pib Gene and Its Linked RFLP Markers in Oryza sativa, O. officinalis and Zea mays

Comparative genetic studies have shown that there are widespread synteny and colinearity of the genes among different species within grass family. Rice ( Oryza sativa L.) is a model plant, and analysis of its genome allows us to reveal the common features and the evolutionary rules of the gramineous genomes and accumulate the data for establishment of a common genetic system in the Poaceae. In this study, a rice gene Pib ( 10.3 kb), a map-based cloned gene, and RFLP markers linked with it are used as the tested probes to investigate their homology and physical location among the tested species. Southern blotting analysis showed that there were sequences homologous to Pib in maize genome. Further, Pib was localized onto the chromosomes of O. sativa ssp. indica cv. Guangluai 4, O. officinalis Wall ex Watt and the inbred line of Zea mays cv. Huangzao 4. The results of fluorescence in situ hybridization (FISH) and double-color FISH indicated that a synteny of Pib and RFLP markers linked with Pib existed among the genomes of the three tested species.

Establishment of 6VS Telocentric Lines of Haynaldia villosa Resistant to Powdery Mildew Induced by Immature Embryo Culture

The line of T240-6 was selected from 32 SC 2 lines of immature embryo culture of T240 (common wheat (Triticum aestivum L.)× Wheat-Haynaldia villosa (L.) Schur. 6D/6V substitution line) through powdery mildew characterization, glutamate oxaloacetate transaminase (GOT) enzyme and gliadin (Gli) analyses and in situ hybridization. All of the individual plants resistant to powdery mildew lacked the locus of GOT at 6VL arm (GOT-V 2) and had gliadin locus at 6VS arm (Gli-V 2) of Haynaldia villosa. All the plants resistant to powdery mildew had one or two telocentric chromosomes that did not pair with wheat chromosomes but paired between themselves. T240-6 was identified as a telocentric line through in situ hybridization.

Detection of Differentiation Among BB, CC and EE Genomes in the Genus Oryza by Two-probe Genomic in situ Hybridization (GISH)

The genus Oryza consists of two cultivated species (O. sativa L. and O. glaberrima Steud.) and approximately 20 wild relative species widely distributed in the pan-tropics. These species have been classified into four complexes following the Vaughan's taxonomic system([1]). The O. officinalis complex is the largest complex in the genus, which includes ten species, having BE, CC, on, and EE genomes in the diploids as well as BBCC and CCDD genomes in the tetraploids. The relationships among the BE, CC, and EE genomes still remain unclear, although previous studies have indicated certain affinities of these genomes([2-4]). Genomic in situ hybridization (GISH) is a powerful technique to detect the relationships among the related genomes at chromosome and DNA levels. The objective of the present study was to investigate the relationships among the BE, CC and EE genomes in the genus Oryza by the two-probe GISH.

Expression Profiles of TRAIL Receptors and Their Clinical Significance in Human Hepatocellular Carcinoma

Objective To investigate the expression profiles and their clinical significance of TRAIL receptors (TRAILR) in human hepatocellular carcinoma (HCC). Methods The expression profiles of TRAILR were determined in 60 samples from hepatocellular carcinoma, 20 from normal liver tissue and two HCC cell lines HepG2, SMMC-7721 by in situ hybridization. Results Both DR4 and DR5 were present in all HCC tissues as well as normal hepatic tissues. In contrast, 54 HCC tissues did not express DcR1 and 25 did not express DcR2. But both DcR were detectable in all of the normal liver tissues. The expression patterns of DR and DcR in HCC samples (higher DR expression level and lower DcR expression level) were quite different from those in normal tissue. DR5, DR4, and DcR2 expressed in both cell lines, while no DcR1 expression was detected. The expression level of DR was correlated with HCC differentiation and stage. The weaker expression was more commonly found in HCC with poor differentiation and late stage, while the stronger expression was more common in HCC with middle to high-differentiation and early stage. No relationship was found between DR and gender, age, negative or positive HBsAg, tumor size, grade or metastasis. Multidrug resistance cell lines expressed lower level DR. Conclusion TRAILR expression was prevalent and discrepancy of receptor types was exited in HCC. Loss of DcR1 may contribute for TRAIL therapy for HCC. Key words TRAILR - apoptosis - hepatocellular carcinoma Supported by the Major Fundation of Ministry of Health, NO. 2001–2003