用荧光原位杂交技术研究人类未受精卵细胞的染色体非整倍体

目的　探讨人类未受精卵细胞非整倍体的产生机制。方法　取未受精卵细胞固定后行多色荧光原位杂交 ,分析卵细胞中 13、16、18、2 1和 2 2号染色体的核型情况。结果　 4 7%的卵细胞核型正常 ,5 3%的卵细胞为异常核型 ,其中 18%为同源染色体不分离 ,12 %为姐妹染色单体非平衡性过早分离 ,36 %为姐妹染色单体平衡性过早分离 ;在体外培养 >2 4小时的卵细胞中 ,姐妹染色单体平衡性过早分离的发生率明显高于体外培养≤ 2 4小时的卵细胞 ( P<0 .0 1)。结论　同源染色体不分离和姐妹染色单体平衡性及非平衡性过早分离这三种机制均参与了卵细胞非整倍体的产生。姐妹染色单体平衡性过早分离与体外培养时间具有相关性     

Field potentials evoked in the avian optic tectum by diffuse and punctiform luminous stimuli

Summary An investigation was made of the slow field potentials evoked in the pigeon optic tectum by visual stimulation. Diffuse illumination of the retina produces a polyphasic response which is negative-going in the surface layers and reverses polarity at 200–400 m. It is suggested that this response reflects activity in radially disposed nerve cells. Local stimulation, produced by punctiform (small-spot) stimuli, results in a negative-going response in the superficial tectum, paired with a smaller and less widely distributed positive-going wave in the deeper layers. It is suggested that this response involves both radial neurones and the tangential neurones ramifying in layers c and d of the SGFS.     

Testicular microcirculation in the rat studied by videophotometric capillaroscopy, fluorescence microscopy and laser Doppler flowmetry

Testicular capillary blood flow was studied in rats using laser Doppler flowmetry, in vivo fluorescence microscopy and videophotometric capillaroscopy. All the methods revealed rhythmical oscillations in testicular microcirculation with a periodicity of 4-10 c.p.m. In arterioles, capillaries and small post-capillary vessels, periods of continuous blood flow alternated with periods of no or very low flow. No visible leakage of dextran-150 was observed from the testicular blood vessels. Four, 8 and 16 h after an s.c. injection of 200 IU hCG the blood flow was continuous and there was leakage of dextran-150 from the microvessels to the interstitial tissue. Twenty-four and 32 h after hCG the blood flow pattern was again rhythmical, and at 32 h there was no leakage of dextran-150. This suggests that hCG induces changes in blood flow and transvascular fluid exchange in the testis, perhaps by altering smooth muscle activity at the arteriolar-level.     

乳腺癌Her2基因荧光原位杂交及其临床病理关系

目的探讨Her2基因过表达的比例及其与临床病理之间的联系。方法收集乳腺浸润型导管癌标本50例,总结其临床及病理资料,应用石蜡包埋组织切片,以EnVision两步法进行免疫组织化学染色标记ER、PR及HER2,以金菩嘉DNA探针试剂盒行荧光原位杂交检测HER2基因。结果患者平均年龄55.5岁,病理组织分级Ⅰ级11例,Ⅱ级30例,Ⅲ级9例。术后分期Ⅰ期13例,ⅡA期15例,ⅡB期13例,ⅢA期6例,ⅢB期2例,ⅢC期1例。淋巴结中位转移率6.91%。ER阳性33例,PR阳性32例,IHC法HER2阳性40例,FISH阳性33例,阴性17例。FISH法检测HER2基因与HER2蛋白过表达之间相关(P0.05),其与病理分级、术后分期、淋巴结转移率及ER、PR的表达无相关(P0.05),3例脉管内瘤栓及3例原发瘤数大于1者,FISH均为阳性。结论FISH检测HER2基因扩增和免疫组化检测HER2蛋白一致性较好,HER2基因与脉管内瘤栓及多个原发瘤有一定关系,但其作为间接预测预后的指标有待于进一步研究。     

Taqman探针实时荧光定量PCR检测肝脏疾病患者血清中miR-122的表达水平及其临床意义

 目的:运用Taqman探针实时荧光定量聚合酶链式反应（PCR）检测肝脏疾病患者血清中miR-122的表达水平并探讨其临床意义。方法:设计miR-122及U6 snRNA的茎环引物和Taqman探针,运用Taqman探针实时荧光定量PCR检测27例肝癌术前(HCC)患者、15例乙型肝炎（hepatitis B）患者、15例丙型肝炎（hepatitis C）患者、15例正常对照者（HC）、11例肝癌术后（PHCC）患者及10例肝癌术后复发（recurrence）患者血清中miR-122的表达水平,并分析miR-122与肝脏疾病相关标志物的关系。结果:Taqman探针实时荧光定量PCR方法能检测血清中miR-122的表达。HCC、hepatitis B、hepatitis C及recurrence患者血清中miR-122的表达水平均高于HC和PHCC患者（P<0.05）, hepatitis C患者血清miR-122表达水平高于HCC、hepatitis B和recurrence患者（P<0.05）,但HCC、hepatitis B和recurrence患者血清miR-122的表达水平无明显差异（P>0.05）,PHCC患者血清miR-122的表达水平比HCC和recurrence患者低（P<0.05）。血清乙型肝炎病毒表面抗原（HBsAg）阳性和(或)乙型肝炎病毒e抗原（HBeAg）阳性患者血清miR-122的表达水平高于阴性者（P<0.05）。血清丙型肝炎病毒抗体（HCV-Ab）阳性患者血清miR-122的表达水平高于阴性者（P<0.05）。血清丙氨酸氨基转移酶（ALT）与miR-122的表达水平有正相关性（r=0.34,P<0.05）。血清甲胎蛋白（AFP）≥400 μg/L组血清miR-122的表达高于AFP＜400 μg/L组（P<0.05）。结论: Taqman探针实时荧光定量PCR适用于检测血清miR-122的表达水平。在HCC、hepatitis B、hepatitis C及recurrence患者血清中miR-22均有不同程度的增高,尤其是hepatitis C患者,且PHCC患者血清miR-122表达下降,复发后升高。血清miR-122的表达与肝脏疾病的某些指标有关,提示血清miR-122可作为肝脏疾病,特别是肝癌早期诊断、手术疗效及预后判断的新指标。     

荧光原位杂交检测石蜡包埋间变性大细胞淋巴瘤病例中ALK基因转位及其意义

目的　比较荧光原位杂交 (fluorescence in situ hybridization,FISH)和免疫组织化学在检测间变性大细胞淋巴瘤 (anaplastic large cell lymphoma,AL CL )中间变性淋巴瘤激酶 (anaplastic lymphomakinase,AL K)基因转位及其融合蛋白中的作用 ,并探讨 FISH在石蜡包埋组织中的应用。方法　采用双色FISH和免疫组织化学检测 2 2例石蜡包埋 AL CL病例中 AL K基因转位及其融合蛋白。结果　通过调整组织切片的酶消化时间等优化措施 ,成功地在石蜡切片上进行了双色 FISH实验 ;FISH和免疫组织化学均在 6 0 % (12 /2 0 )系统性 AL CL中检测到 AL K基因转位或融合蛋白 ,在 2例皮肤原发 AL CL中未检测到基因转位或融合蛋白 ,两种方法的符合率为 10 0 %。结论　 (1)在检测 AL CL中有无 AL K基因转位时 ,AL K蛋白免疫组化由于其简单、快捷、价廉成为一般情况下的首选方法 ;在具备 FISH条件时 ,也可以将 FISH作为首选 ;(2 )通过优化实验条件 ,可以在石蜡包埋组织上成功地进行 FISH实验。     

Activation of PI 3-kinase/Akt/NF-κB and Stat3 signaling by avian reovirus S1133 in the early stages of infection results in an inflammatory response and delayed apoptosis

Avian reovirus (ARV) strain S1133 causes apoptosis in host cells in the middle to late stages of infection. This study investigated the early-stage biological response and intracellular signaling in ARV S1133-infected Vero and chicken cells. Treatment with conditioned medium from ARV S1133-infected cells increased the chemotactic activity of U937 cells. Neutralizing antibodies against IL-1β and IL-6 showed that both cytokines contribute to viral-induced inflammation but neither affect cell survival. Inhibition of Akt, NF-κB, and Stat3 released the chemotactic activity and anti-apoptotic effect elicited by ARV S1133. ARV S1133 activated PI 3-kinase-dependent Akt/NF-κB and p70 S6 kinase, as well as Stat3; however, p70 S6 kinase was not involved in ARV S1133-mediated effects. DF1 cells over-expressing constitutively active PI 3-kinase and Stat3 showed association with enhancement of anti-apoptotic activity. In conclusion, in the early stages of ARV S1133 infection, activation of cell survival signals contributes to virus-induced inflammation and anti-apoptotic response.     

Recombinant avian adenovirus CELO expressing the human interleukin-2: characterization in vitro, in ovo and in vivo

In our study, a recombinant adenovirus based on the avian adenovirus CELO genome, has been constructed that contains the human interleukin-2 gene. We have shown the production of biologically active recombinant interleukin-2 in vitro (LMH and 293 cells) and in ovo (chicken embryos) infected with recombinant virus CELO-IL2. An increase in the median survival time of C57BL/6 mice carrying B16 melanoma tumors has been demonstrated after multiple intra-tumors injections of the recombinant adenovirus CELO-IL2.     

Chromosome Missegregation and Aneuploidy Induction in Human Peripheral Blood Lymphocytes In vitro by Low Concentrations of Chlorpyrifos,Imidacloprid and α-Cypermethrin

Chlorpyrifos, imidacloprid, and α-cypermethrin are some of the most widely used insecticides in contemporary agriculture. However, their low-dose, nontarget genotoxic effects have not been extensively assayed. As one of the most relevant cancer biomarkers, we aimed to assess the aneuploidy due to chromosome missegregation during mitosis. To aim it we treated human lymphocytes in vitro with three concentrations of insecticides equivalents relevant for real scenario exposure assessed by regulatory agencies. We focused on chlorpyrifos as conventional and imidacloprid and α-cypermethrin as sustainable use insecticides. Cytokinesis-blocked micronucleus assay was performed coupled with fluorescence in situ hybridization (FISH) with directly labeled pancentromeric probes for chromosomes 9, 18, X and Y. None of the insecticides induced significant secondary DNA damage in terms of micronuclei (MN), nuclear buds (NB), or nucleoplasmic bridges (NPB). However, significant disbalances in chromosomes 9, 18, X and Y, and in insecticide-treated cells has been observed. According to recent studies, these disbalances in chromosome numbers may be atributted to defect sister chromatid cohesion which contribute to the increase of chromosome missegregation but not to micronuclei incidence. We conclude that tested insecticidal active substances exert chromosome missegregation effects at low concentrations, possibly by mechanism of sister chromatid cohesion. These findings may contribute to future risk assesments and understanding of insecticide mode of action on human genome. Environ. Mol. Mutagen. 60:72–84, 2019. © 2018 Wiley Periodicals, Inc.     

Complex chromosome rearrangement involving chromosomes 1, 4 and 16 revealed by fluorescence in situ hybridization

A 7-year-old boy with mental retardation had apparently balanced reciprocal translocations, involving the telomeric regions of chromosomes 1p and 4q, which was detected by routine chromosome analysis. Fluorescence in situ hybridization (FISH) was used and also revealed the telomeric region of chromosome 16p to be involved in a still apparently balanced translocation-complex, impossible to discover with classical cytogenetic analysis. We want to emphasize the importance of FISH in detecting small chromosomal aberrations. We discuss whether the abnormal phenotype is caused by unbalanced karyotype with cryptic undetected translocations or small deletions or mutations in the translocation-breakpoints.     

Disseminated intravascular coagulation induced by Liquoid in the rat. II. Effect of heparin on hematologic and complement abnormalities and renal lesions studied by light, fluorescence, and electron microscopy.

A single intravenous injection (12.5 mg.) of Liquoid (polyanethol sulfonate) was given to anticoagulated (heparinized) rats. Fibrinogen concentrations, platelet counts, total serum complement (CH50),C3 protein, and terminal components (C3 to C9) were measured. Histopathology was assessed by light, fluorescence, and electron microscopy. Heparin given before Liquoid remarkably diminished the seferity of the histologic lesions, with good correlation among light, fluorescence, and electron microscopy. Levels of clotting factors, CH50, C3 and C3 to C9, however, were not statistically different in the heparinized rats injected with Liquoid from those of animals receiving Liquoid alone. Actually C3 protein concentration was lower in the anticoagulated (Liquoid-heparin) rats. It is postulated that under the present experimental conditions, heparin did not antagonize the procoagulant and precipitating or complement-activating Liquoid effects. The attenuated histopathology observed was perhaps the result of either local or systemic, as yet undefined, heparin effects other than anticoagulation.     

Pallister-Killian syndrome: A mild case diagnosed by fluorescence in situ hybridization. Review of the literature and expansion of the phenotype

Pallister-Killian syndrome (PKS) is a rare disorder characterized by a specific combination of anomalies, mental retardation and mosaic presence of a supernumerary isochromosome 12p which is tissue-limited. We report an atypical case of PKS with a mild phenotype. Fluorescence in situ hybridization (FISH) was used to demonstrate that the supernumerary marker chromosome identified in the patient's fibroblasts was an isochromosome 12p. This study broadens the spectrum of PKS phenotype. It also illustrates the usefulness of fluorescence in situ hybridization in diagnosis of patients with chromosomal abnormalities and mild or atypical clinical findings. © 1996 Wiley-Liss, Inc.     

Demonstration of the Philadelphia translocation by fluorescence in situ hybridization (FISH) in paraffin sections and identification of aberrant cells by a combined FISH/immunophenotyping approach

The Philadelphia translocation was demonstrated by two-colour fluorescence in situ hybridization (FISH) in decalcified paraffin sections of bone marrow from patients with chronic myelogenous leukaemia. FISH was combined with immunocytochemical detection of different membrane-bound or cytoplasmic antigens. With this new technique, the cells bearing the 9;22 translocation can be identified morphologically, as well as immunocyto-chemically, in tissue sections.     

Pharmacological characterization, anatomical distribution and sex differences of the non-NMDA excitatory amino acid receptors in the quail brain as identified by CNQX binding

The distribution of non-N-methyl-d-aspartate binding sites was studied in coronal and sagittal sections through the brain of adult Japanese quail by quantitative autoradiography, using tritiated 6-cyano-7-nitroquinoxaline-2,3-dione as a radioligand. Saturation binding experiments were, in addition, carried out in areas showing high levels of binding (cerebellar molecular layer, nucleus anterior medialis and nucleus infundibularis) and demonstrated that the binding of tritiated ligand was specific and saturable. Competition studies with α-amino-3-hydroxy-methyl-4-isoxazole propionic acid and kainic acid indicated that kainic acid strongly inhibited ligand binding in all brain areas. α-Amino-3-hydroxy-methyl-4-isoxazole propionic acid was only a weak inhibitor in the hypothalamic nuclei whereas in the cerebellar molecular layer both high and low affinity inhibitions were detected. The highest binding levels of tritiated ligand were observed in the molecular layer of the cerebellum. Very high levels of binding were detected in various preoptic/hypothalamic sites including the nucleus suprachiasmaticus pars medialis, nucleus anterior medialis hypothalami, nucleus infundibularis, nucleus mammillaris medialis, nucleus posteromediale hypothalami and nucleus hypothalami ventromedialis. High levels of binding were also detected in the bulbus olfactorius, bed nucleus commissuralis anterior, bed nucleus commissuralis pallii, nucleus accumbens, bed nucleus striae terminalis and nucleus interpeduncularis. In the preoptic area/hypothalamus, high levels of binding were clearly present in all areas that contain gonadotropin releasing hormone cells or fibers. In the pons and mesencephalon, moderate levels of binding were associated with catecholaminergic areas such as the area ventralis tegmentalis (area ventralis of Tsai) and the locus coeruleus. Saturation analysis demonstrated the presence of a higher number of binding sites in females than in males in the cerebellar molecular layer, nucleus infundibularis and nucleus anterior medialis. This latter difference was confirmed in the one point assays that also identified higher levels of specific binding in the nucleus suprachiasmaticus pars medialis of males as compared with females. These anatomical data suggest a possible implication of non-N-methyl-d-aspartate receptors in the synthesis and/or release of both gonadotropin releasing hormone and catecholaminergic neurotransmitters that should now be tested by pharmacological experiments.     

Application of fluorescence in situ hybridisation to study the relationship between cytotoxicity,chromosome aberrations,and changes in chromosome number after treatment with the topoisomerase II inhibitor amsacrine

Amsacrine (4′-(9-acridinylamino)methanesulphon-m-anisidide) is an antileukemic drug which inhibits topoisomerase II (topo II) enzymes. We studied effects of two concentrations of amsacrine on the GM10115A cell line. This is a Chinese hamster line containing a single human chromosome 4, which can be readily visualised using fluorescence in situ hybridisation (FISH). The low amsacrine concentration slowed cell growth but did not cause significant arrest in the G2 phase of the cell cycle, while a higher concentration caused more long-term effects on the growth of the cells and caused G2 arrest. Either concentration led to chromosomal fragments which were lost with increasing time after treatment, and chromosomal translocations which appeared stable for at least 8 days after treatment. At the low concentration, the loss or gain of a single chromosome was a common event. The higher concentration led to polyploid cells, usually containing an uneven number of chromosome 4. We propose two mechanisms for aneuploidy by amsacrine (or related topo II poisons), either of which can be readily detected using FISH. At low drug concentrations, aneuploidy may occur directly through, for example, a failure to resolve catenated chromatids prior to anaphase. However, there has been considerable interest in the role of the cell division control (cdc) kinase and cyclins in regulating the mammalian cell cycle, and these may also be involved in the response of cells to high concentrations of topo II poisons. Cdc2 proteins and cyclins are involved in coordinating diverse activities during the M phase of the cell cycle, including catalysis of chromosome condensation and reorganisation of microtubules to allow chromosome separation during mitosis. Chromosome damage by topo II poisons will lead to G2 arrest, which allows the cells time to repair the damage. During this time, cyclin A and cdc2 levels will fall, preventing the cell from entering mitosis and effectively resetting the clock to G1 and the ploidy to tetraploid. Aneuploid cells will derive from polyploid cells through loss of extra chromosomes. © 1996 Wiley-Liss, Inc.     

Dose-response studies of the induction of hyperdiploidy and polyploidy by diethylstilbestrol and 17β-estradiol in cultured human lymphocytes using multicolor fluorescence in situ hybridization

Diethylstilbestrol (DES) and 17β-estradiol (E₂) are known inducers of aneuploidy and polyploidy in vivo and in vitro. Isolated human lymphocytes were treated with the stilbene estrogen DES (0.05–50 μM) and the steroid estrogen E₂ (0.05–75 μM) in culture. Multicolor fluorescence in situ hybridization (FISH) with DNA probes for the centromere and adjacent heterochromatin regions of chromosomes 1, 9, and 16 was used to detect hyperdiploidy, polyploidy, and chromosomal breakage affecting these chromosomes. Using this FISH technique, significant nonlinear increases in hyperdiploidy were observed with both compounds, whereas no induction of chromosomal breakage affecting the pericentric heterochromatin regions of chromosomes 1, 9, and 16 could be detected. DES induced a maximum of approximately 13% hyperdiploid cells at 30 μM, whereas E₂ showed its highest induction at 75 μM with 7% hyperdiploid cells. To distinguish hyperdiploidy from polyploidy, a FISH labeling strategy to detect multiple chromosomes simultaneously was established. Using this approach, we could show that most of the cells showing multiple hybridization regions after treatment with both chemicals were most likely the result of polyploidy rather than true hyperdiploidy. These results indicate that the induction of hyperdiploidy/polyploidy with DES and E₂ show sublinear dose-response relationships with likely threshold concentrations in human lymphocytes and that FISH with multiple probes targeting different chromosomes can be used to estimate hyperdiploidy and polyploidy frequencies. Environ. Mol. Mutagen. 31:263–273, 1998 © 1998 Wiley-Liss, Inc.     

Dynamic changes in cortical NADH fluorescence in rat focal ischemia: evaluation of the effects of hypothermia on propagation of peri-infarct depolarization by temporal and spatial analysis

Suppression of peri-infarct depolarizations (PIDs) is one of the major mechanisms of hypothermic protection against transient focal cerebral ischemia. Previous studies have shown the lack of hypothermic protection against permanent focal ischemia. We hypothesized the lack of hypothermic protection was due to the poor efficacy in suppression of PIDs. To examine the hypothesis, we elucidated the effects of hypothermia on the manner of propagation of PIDs with temporal and spatial resolutions using NADH (reduced nicotinamide adenine dinucleotide) fluorescence images by illuminating the parietal-temporal cortex with ultraviolet light. Spontaneously hypertensive rats (n=14) were subjected to permanent focal ischemia by occlusion of the middle cerebral and left common carotid arteries. 2-h hypothermia (30 degrees C) was initiated before ischemia. Although hypothermia delayed the appearance of PIDs, it did not suppress their appearance. Furthermore, 54% of the PIDs enlarged the high-intensity area of NADH fluorescence in the hypothermia group, similar to the normothermia group (53%). The high-intensity area of NADH fluorescence widened by each PID was larger in the hypothermia group than in the normothermia group. These findings suggest that PIDs even in hypothermia are one of the major factors causing growth of infarction, emphasizing the importance of therapy that targets suppression of PIDs even during hypothermia.     

HER2 status in pure ductal carcinoma in situ and in the intraductal and invasive components of invasive ductal carcinoma determined by fluorescence in situ hybridization and immunohistochemistry

AIM: To determine the HER2 status of ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) of the breast. The increased prevalence of HER2 amplification and overexpression in DCIS is considered to be maintained in the intraductal component of IDC; however, HER2 amplification and overexpression are detected much less in IDC. METHODS AND RESULTS: Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) were performed to detect HER2 in 270 IDCs with an intraductal component and in 50 pure DCIS samples; IHC was also performed in 116 metastatic nodes. HER2 was found to be amplified in 77 cases (28.5%) and overexpressed in 79 (29.3%) of the 270 IDCs. HER2 amplification was similar between intraductal and invasive components of the same tumour. The concordance for HER2 status between invasive and intraductal components of individual tumours was 98.5% and 99.3% by FISH and IHC, respectively. HER2 was amplified in 25 (50%) of the 50 pure DCIS samples. HER2 overexpression in metastatic nodes resembled the HER2 status in the primary tumour for 108 (93.1%) of 116 cases (kappa =0.831). CONCLUSION: Our study indicates that the intraductal component of IDC may differ biologically when compared with pure DCIS. HER2 appears to lack a critical role in the progression from DCIS to IDC and HER2 status is maintained in metastatic lesions.