利用染色体G显带技术鉴定人永生细胞实验条件初探

目的:探索利用染色体G显带技术鉴定人永生淋巴细胞的最佳实验条件。方法:在常规染色体制片技术的方法和手段的基础上,分别观察不同PHA浓度和培养时间对永生淋巴细胞分裂增殖的影响;然后,选择不同浓度秋水仙素处理细胞,观察染色体分散程度和中期分裂相的形态。结果:在永生淋巴细胞培养48h,PHA终浓度为0.1mg/mL,秋水仙素终浓度为0.04μg/mL,且其作用时间为1.5~2h的条件下,所获得的细胞染色体标本分裂相较多、染色体较长、分散较好、利于带型分析。结论:利用染色体G显带技术鉴定人永生淋巴细胞提供了技术保证,降低了实验成本,并且缩短了实验时间。     

半滑舌鳎雌性特异AFLP标记CseF783的克隆及其在遗传性别鉴定中的应用

半滑舌鳎性别控制和全雌育种等研究领域中迫切需要一种能够快速鉴定鱼类个体遗传性别的有效方法。文章采用AFLP技术, 利用选择性引物组合(E-ACT/M-CAA)从半滑舌鳎中筛选到一条雌性特异的AFLP标记。对该标记进行二次PCR扩增、琼脂糖凝胶回收、克隆、测序。分析表明, 序列全长为791 bp, 与GenBank中的序列无同源性。以该雌性特异AFLP标记DNA序列为模板, 设计了一对特异的PCR引物, 成功地将其转化为SCAR(Sequence characterized amplified regions)标记, 并在100尾已知性别的半滑舌鳎个体(雌雄各50尾)中进行验证, 结果表明, 该SCAR标记在所有雌性个体中均扩增得到一条长度为324 bp的DNA条带, 而在49尾雄性个体中均扩增不到该DNA条带(有1尾雄性个体例外), 证明该SCAR标记是雌性特异的, 并可用于半滑舌鳎个体遗传性别鉴定。随后, 利用该SCAR标记检测了3日龄半滑舌鳎幼苗, 结果表明, 雌性个体比例为41.7%。     

半滑舌鳎脑芳香化酶基因cDNA克隆及表达分析

为研究脑型芳香化酶（P450aromB）在半滑舌鳎性别分化中的作用,采用同源克隆策略,从半滑舌鳎脑分离了2184 bp长的脑型芳香化酶的全长cDNA,该基因编码498个氨基酸。氨基酸序列和系统发育分析表明,P450aromB属于脑型P450arom,P450aromB的氨基酸序列与其他鱼类脑型Ｐ450arom的同源性较高（48.3%—66.1%）,与性腺型Ｐ450arom的同源性较低(34.2%—49.9%),与自身的性腺型芳香化酶同源性为45.1％。RT-PCR分析表明：P450aromB mRNA的表达具有明显组织特异性,P450aromB只在性腺、脑、鳃和皮肤中表达,且脑中表达量远高于性腺,而在雌雄鱼的其他组织中都不表达。经过甲基睾酮浸浴处理和高温诱导半滑舌鳎由雌性性反转为雄性后,脑中P450aromB的表达量降低,这些结果表明P450aromB参与了半滑舌鳎的性腺分化和性别决定过程。     

半滑舌鳎脑芳香化酶基因cDNA克隆及表达分析

为研究脑型芳香化酶(P450aromB)在半滑舌鳎性别分化中的作用,采用同源克隆策略,从半滑舌鳎脑分离了2184bp长的脑型芳香化酶的全长cDNA,该基因编码498个氨基酸。氨基酸序列和系统发育分析表明,P450aromB属于脑型P450arom,P450aromB的氨基酸序列与其他鱼类脑型P450arom的同源性较高(48.3%-66.1%),与性腺型P450arom的同源性较低(34.2%-49.9%),与自身的性腺型芳香化酶同源性为45.1%。RT-PCR分析表明:P450aromB mRNA的表达具有明显组织特异性,P450aromB只在性腺、脑、鳃和皮肤中表达,且脑中表达量远高于性腺,而在雌雄鱼的其他组织中都不表达。经过甲基睾酮浸浴处理和高温诱导半滑舌鳎由雌性性反转为雄性后,脑中P450aromB的表达量降低,这些结果表明P450aromB参与了半滑舌鳎的性腺分化和性别决定过程。     

半滑舌鳎外周血T淋巴细胞的分离、鉴定及TCRβ基因免疫应答分析

为开展半滑舌鳎(Cynoglossus semilaevis)免疫学研究提供细胞平台, 利用密度梯度离心法分离半滑舌鳎外周血淋巴细胞, 采用短期细胞培养法分离悬浮淋巴细胞, 悬浮淋巴细胞在含有0.3 μg/mL的PHA的DMEM完全培养基, 于24℃条件下可连续培养3—4d左右, 采用自制的尼龙毛柱可将悬浮淋巴细胞中的非黏附淋巴细胞和黏附淋巴细胞成功分离; 利用流式细胞仪结合特异抗体检测对非黏附淋巴细胞和黏附淋巴细胞进行鉴定, 结果表明, 非黏附细胞与鼠抗人FTIC-CD3单克隆抗体特异结合, 为T样淋巴细胞; 黏附细胞和鼠抗人FTIC-CD19单抗特异结合, 为B样淋巴细胞。T细胞表面抗原受体TCRβ基因可特异性的在非黏膜细胞中表达, 而在黏附细胞中不表达, 证明分离获得的非黏膜细胞为T淋巴细胞, 采用qRT-PCR (Quantitative Real-Time PCR)方法检测TCRβ基因表达, 结果表明, TCRβ基因在半滑舌鳎肝、脾、头肾、后肾、小肠、胃、血液、鳃、皮肤、肌肉、心脏、脑、卵巢组织中均有表达, 其中在肠、胃、脾、头肾中表达量较高; 鳗弧菌感染后TCRβ基因在肝、脾、鳃中呈现明显的上调表达, 且表达峰值出现在感染后72—96h, 表明TCRβ基因在获得性免疫应答中起重要作用。     

半滑舌鳎AKT-ineracting protein基因的克隆及免疫应答表达分析

研究克隆了半滑舌鳎Cynoglossus semilaevis膜蛋白AKT-interacting protein (AKTIP)基因, 研究了其在健康组织中的表达模式、鳗弧菌(Vibrio anguillarum)感染后免疫组织和不同病原物刺激外周血淋巴细胞中的表达特征。通过常规克隆和RACE技术, 获得的半滑舌鳎AKTIP基因全长cDNA序列为1224 bp, 其中包括5'-UTR为116 bp, 3'-UTR为117 bp和完整的ORF序列891 bp, 编码296个氨基酸, 预测蛋白质的等电点(PI)为9.12,分子量是33.74 kD; 同源比对发现半滑舌鳎AKTIP的氨基酸序列在不同物种之间具有较高的保守性; 荧光实时定量PCR (qRT-PCR)检测到半滑舌鳎各个组织中均有AKTIP基因的表达, 在卵巢中表达量最高; 鳗弧菌感染半滑舌鳎后, AKTIP基因在肝、鳃、血液、肠、头肾和脾中均上调表达; 病原模拟物PGN、LPS、poly I:C和WGP刺激半滑舌鳎外周血淋巴细胞均诱导AKTIP基因下调表达。研究表明半滑舌鳎AKTIP基因参与了机体免疫反应为给半滑舌鳎免疫防御技术的研究提供理论依据。     

半滑舌鳎FTZ-F1cDNA克隆及表达分析

为研究性别相关基因FTZ-F1在半滑舌鳎鱼中的表达特征,采用同源克隆策略,从其精巢分离了3143bp长的半滑舌鳎FTZ-F1(hsFTZ-F1)的全长cDNA,该序列包含1458bp开放阅读框,66bp长的5'末端非编码区(UTR),1619bp长的3'末端UTR。mRNA的组织分布、氨基酸序列和系统发生分析表明:hsFTZ-F1属于SF-1/Ad4BP类群。RT-PCR分析表明:hsFTZ-F1mRNA的分布广泛,几乎在所有组织都有表达,但在性腺、肾脏、脑和头肾组织中表达最强,其他组织表达较弱,雌鱼脑和头肾中的表达量明显高于雄性。胚胎发育过程中表达量都高于孵化后仔鱼的表达量,表明hsFTZ-F1可能参与了半滑舌鳎的器官形成过程。     

半滑舌鳎病原菌轮虫弧菌(Vibrio rotiferianus)的分离与鉴定

从患有严重皮肤溃疡病的半滑舌鳎(Cynoglossus semilaevisGünther)病灶处分离出4株优势菌,经人工感染证实其中一株菌株BV1为养殖半滑舌鳎皮肤溃疡病的病原菌。通过Biolog系统、细菌常规形态特征和生理生化反应指标测定以及16S rRNA和gyrB序列分析对菌株BV1进行了综合鉴定,结果表明该致病菌为轮虫弧菌(Vibrio rotiferianus),其半致死量LD50为6.7×103CFU/mL。进一步的药敏试验表明其对链霉素、四环素等敏感,而对其他用于试验的抗生素敏感度低或具有一定的抗性。     

半滑舌鳎基因组的提取及ISSR反应体系的建立

目的:比较CTAB法和STE法提取半滑舌鳎基因组DNA的效果,通过优化半滑舌鳎ISSR-PCR反应体系,将新型分子标记简单重复间序列(Inter-simple sequence repeat,ISSR),引用到半滑舌鳎遗传多样性研究中。方法:以95%酒精固定的半滑舌鳎鳍条为材料,运用CTAB法和STE法提取基因组DNA,同时分析了模板DNA、Mg2 、dNTPs、引物浓度,以及退火温度对ISSR-PCR扩增结果的影响。结果:CTAB法提取的基因组DNA质量好于STE法提取的结果,同时确立了稳定性强、重复性好的半滑舌鳎ISSR-PCR最佳反应体系和扩增参数。在25lμPCR反应体系中包括:1×PCR缓冲液,2mmol/L MgCl2,0.2mmol/L dNTP,0.2μmol/L ISSR引物,20ng模板DNA,1.5U Taq DNA聚合酶。反应程序为:94℃预变性5min,然后进入PCR循环,即94℃变性45s,52℃退火45s,72℃延伸90s,共进行40个循环。最后72℃延伸10min。结论:ISSR-PCR反应体系稳定可靠,该新型分子标记可应用于半滑舌鳎遗传多样性研究中。     

半滑舌鳎miR-200a和miR-200b前体克隆及其免疫应答分析

为了探究半滑舌鳎（Cynoglossus semilaevis）miR-200a和miR-200b在免疫应答中的作用,采用PCR方法克隆了半滑舌鳎miR-200家族的miR-200a和miR-200b的前体序列,长度分别为82和88 bp;用The mfold Web Server和Clustalx1.83软件对其前体序列进行了二级结构和同源性分析,miR-200a和miR-200b都具有典型的颈环结构,与其他物种具有较高的同源性。qRT-PCR分析结果显示,miR-200a和miR-200b在健康半滑舌鳎13种组织（肝脏、肠、脾脏、头肾、后肾、鳃、血液、脑、皮肤、肌肉、胃、心脏和卵巢）中均有表达,miR-200a在头肾中表达量最高,在血液中表达量最低,miR-200b在肝脏中表达量最高,在肌肉中表达量最低;miR-200a和miR-200b在鳗弧菌（Vibrio anguillarum）感染半滑舌鳎后不同时间点的4种免疫相关组织（肝脏、肠、脾脏和头肾）中的表达呈现出先上调后下降的规律,但表达达到峰值的时间点有所不同。miR-200a在肝脏和脾中的表达峰值出现在鳗弧菌感染后6h,在肠和头肾中则是鳗弧菌感染后12h,miR-200b在肠、脾和头肾中均在鳗弧菌感染后12h达到表达高峰;miR-200a和miR-200b在脂多糖（LPS）、肽聚糖（PGN）、葡聚糖（WGP）、聚肌胞苷酸（poly I:C）4种病原模拟物刺激后的半滑舌鳎肝脏细胞系中呈现出上调表达趋势,其中Poly I:C刺激半滑舌鳎肝脏细胞系后miR-200a上调表达趋势明显,6h的表达量为0h的9倍,在WGP刺激半滑舌鳎肝脏细胞后miR-200b上调表达趋势明显,2h的表达量为0的9倍。研究结果为揭示miRNA在半滑舌鳎免疫应答中的作用提供了科学依据。     

Generating sexually differentiated songs

The past year has witnessed increased confusion as to the role of gonadal hormones in the development of neuroeffectors for sexually differentiated vocalizations in several species. Are sex differences in levels of circulating gonadal hormones robust enough to account for the full spectrum of male/female differences? Understanding how vocal behaviors are generated has improved, permitting greater insights into how differences in cell number and type contribute to male- and female-specific songs in frogs and birds.     

黄鳝生殖干细胞在性逆转生殖发育过程中的变化

应用组织学方法及免疫组织化学技术显示,黄鳝性逆转生殖发育过程中,生殖干细胞(GSCs)定位分布于生殖褶中,黄鳝雌性发育阶段的GSCs分散或成团存在,间性及雄性发育阶段GSCs均区分为A、B两种不同类型,雌性发育阶段GSCs与A、B两类GSCs在超微结构上存在差异。结果表明,生殖褶中GSCs是黄鳝分化生殖腺中唯一具有有丝分裂能力的生殖细胞群,雌性发育阶段GSCs表现出卵原干细胞特征,间性及雄性发育阶段GSCs为精原干细胞。CD49整合素是黄鳝雌性发育阶段GSCs和A类GSCs的表征分子。     

Simple method for culture of peripheral blood lymphocytes of Testudinidae

We developed and optimized a simple, efficient and inexpensive method for in vitro culture of peripheral blood lymphocytes from the Brazilian tortoise Chelonoidis carbonaria (Testudinidae), testing various parameters, including culture medium, mitogen concentration, mitotic index, culture volume, incubation time, and mitotic arrest. Peripheral blood samples were obtained from the costal vein of four couples. The conditions that gave a good mitotic index were lymphocytes cultured at 37°C in minimum essential medium (7.5 mL), with phytohemagglutinin as a mitogen (0.375 mL), plus streptomycin/penicillin (0.1 mL), and an incubation period of 72 h. Mitotic arrest was induced by 2-h exposure to colchicine (0.1 mL), 70 h after establishing the culture. After mitotic arrest, the cells were hypotonized with 0.075 M KCl for 2 h and fixed with methanol/acetic acid (3:1). The non-banded mitotic chromosomes were visualized by Giemsa staining. The diploid chromosome number of C. carbonaria was found to be 52 in females and males, and sex chromosomes were not observed. We were able to culture peripheral blood lymphocytes of a Brazilian tortoise in vitro, for the preparation of mitotic chromosomes.     

A novel sex-specific DNA marker in Columbidae birds

That most Columbidae birds have no conspicuous sexual dimorphism often makes it difficult to identify their sex on the basis of external morphology. In the present study, we report a novel sex-specific DNA marker in Columbidae birds. DNA was extracted from one member of this bird group, Streptopelia orientalis (S. orientalis, oriental turtle dove), and used to identify a female-specific DNA marker using a random amplified polymorphic DNA (RAPD) fingerprinting. One hundred and sixty random primers were used for the RAPD-PCR reactions. When using the OPAV17 primer, a novel 902 bp sex-specific PCR product was amplified from known female birds. This fragment of DNA was cloned and sequenced. Two primers, TurSexOPAV17-F and TurSexOPAV17-R, were designed from the cloned sex-specific sequence, and were successfully used to amplify a 777 bp female-specific fragment using PCR from S. orientalis DNA. This sex-specific marker was also amplified from genomic DNA samples of two other female Columbidae, S. chinensis and Columba livia. Sequence analysis showed that this novel sex-specific marker was highly conserved amongst these three bird species. In contrast, the PCR product was not amplified from male DNA of these species, nor from either sex of the S. chinensis formosa birds. Therefore, we concluded that our novel marker can be used to rapidly and accurately identify the sex of birds from three species of Columbidae.     

First small supernumerary ring chromosome carrying 10q euchromatin in a patient with mild phenotype characterized by molecular cytogenetic techniques and review of the literature

We report the identification and characterization of the first supernumerary ring chromosome 10 containing a considerable proportion of 10q euchromatin by microdissection and reverse painting in a female patient presenting with short stature. Fluorescence in situ hybridization studies showed that the marker chromosome originates from chromosome 10 and includes the euchromatic bands p11.2 and q11.2. The supernumerary marker chromosome 10 was found in 14% of the peripheral blood lymphocytes analyzed. This constitutional mosaic could be confirmed in oral mucosa cells as a second cell system (16%) by interphase FISH using an alphoid centromeric probe for chromosome 10. Parental karyotypes were normal, uniparental disomy for the normal chromosomes 10 could be excluded by microsatellite analysis. The karyotype of the patient detected in peripheral blood cells can be described as mos 47,XX,+mar.rev ish r(10)(p11.2q11.2)(wcp10+,cep10+)/46,XX.     

Identification of Y-chromosomal DNA in a Turner syndrome mosaic by polymerase chain reaction.

A 15.5-year-old female was referred for primary amenorrhea and slow development of secondary sex characteristics. The karyotype revealed 45,X/46,X,+mar (75%/25%). The small marker chromosome was C-band and Q-band negative. It appeared to be primarily centromeric with some light G-band staining material on either side. Females with Y-chromosomal material are at an increased risk for gonadal neoplasia and this patient was studied further to investigate the possibility that the marker was a deleted Y chromosome. Polymerase chain reaction (PCR) analysis of this patient's DNA revealed the presence of Y-chromosomal material presumably derived from the marker chromosome. These results indicate that the PCR technique, in conjunction with cytogenetic analysis, can identify possible Y-chromosomal material. This testing provides critical information necessary for correct medical followup of Turner syndrome mosaic patients.     

The effect of estrogen on the incorporation of 3H-thymidine by PHA-stimulated human peripheral blood lymphocytes

The effect of estrogen on the incorporation of tritiated thymidine by phytohemagglutinin (PHA)-stimulated human peripheral blood lymphocytes was evaluated in 15 adult males. Varying concentrations of diethylstilbestrol diphosphate (DEP-S) were added to peripheral blood lymphocytes with and without PHA to study the effects of estrogen on blastogenesis. Maximum suppression of blastogenesis occurred after the addition of 500 mcg/ml culture of DEP-S. The absence and presence of DEP-S 500 mcg/ml culture resulted in a 52% reduction in lymphocytic reactivity (p.002). It was concluded that this reduction or suppression of lymphocytic blastogenesis in the presence of estrogen suggests that the palliative effects of estrogenic therapy in treating patients with hormone-dependent tumors may be countered by its adverse effect on the host's immunologic responsiveness to malignancy.     

The number of x chromosomes causes sex differences in adiposity in mice

Sexual dimorphism in body weight, fat distribution, and metabolic disease has been attributed largely to differential effects of male and female gonadal hormones. Here, we report that the number of X chromosomes within cells also contributes to these sex differences. We employed a unique mouse model, known as the "four core genotypes," to distinguish between effects of gonadal sex (testes or ovaries) and sex chromosomes (XX or XY). With this model, we produced gonadal male and female mice carrying XX or XY sex chromosome complements. Mice were gonadectomized to remove the acute effects of gonadal hormones and to uncover effects of sex chromosome complement on obesity. Mice with XX sex chromosomes (relative to XY), regardless of their type of gonad, had up to 2-fold increased adiposity and greater food intake during daylight hours, when mice are normally inactive. Mice with two X chromosomes also had accelerated weight gain on a high fat diet and developed fatty liver and elevated lipid and insulin levels. Further genetic studies with mice carrying XO and XXY chromosome complements revealed that the differences between XX and XY mice are attributable to dosage of the X chromosome, rather than effects of the Y chromosome. A subset of genes that escape X chromosome inactivation exhibited higher expression levels in adipose tissue and liver of XX compared to XY mice, and may contribute to the sex differences in obesity. Overall, our study is the first to identify sex chromosome complement, a factor distinguishing all male and female cells, as a cause of sex differences in obesity and metabolism.     

Blast crisis of Philadelphia chromosome-positive chronic myelocytic leukemia (CML). Treatment results of 69 patients

We have studied the clinical courses of 69 patients with blastic crises of Philadelphia chromosome positive CML to identify parameters that were associated with an increased response rate or survival. Cytogenetic analysis at the time of blastic transformation revealed additional chromosome changes in 70% of the patients tested. Bone marrow fibrosis was detected in 58% of evaluable patients. Lymphoblastic transformation was seen in 28% of the patients tested with cell surface marker analysis. The value of 5'-nucleotidase as a marker for distinguishing lymphoid from non-lymphoid blast crisis was confirmed. Of 57 evaluable patients, 23 (40%) responded to therapy (CR/PR longer than 14 days). Median survival was 75 days. Longer survival was related to the following factors: Ph1-chromosome as the only detectable cytogenetic abnormality; lymphoblastic transformation; no bone marrow fibrosis; high percentage of blasts and promyelocytes in the bone marrow, and response to therapy. No prognostic significance was associated with age, sex, Tdt, LDH, spleen size, duration of the chronic phase of the disease, white blood cell count, Hb, platelet count and percentages of basophils, eosinophils, erythroblasts and blasts and promyelocytes in the peripheral blood. These data confirm the poor prognosis of patients with blastic crisis of CML treated by conventional chemotherapy.