Preparation of chromosomes from plant leaf meristems for karyotype analysis and in situ hybridization

A reliable method for preparing metaphase chromosomes from plant leaf tissues is described. The chromosomes are suitable for karyotype analysis and gene mapping by fluorescence in situ hybridisation (FISH). The method is based on enzymatic digestion of young leaf tissues (shoot-tips) after which the resulting protoplasts are treated hypotonically before being dropped onto microscopic slides. Compared to root-tip chromosomes, leaf chromosomes tend to be longer, or less condensed, and hence more karyotypically differentiated. Metaphase index in young leaf tissues is also very high. Metaphase spread consists of evenly and well-distributed chromosomes and this allows accurate counting. The plant used to demonstrate this method is birch (Betula L.), a group of tree species that has extremely small chromosomes. Root-tip chromosomes of these plants are difficult to obtain, as cutting does not produce roots readily. Seedling chromosomes do not represent the same genomic constitution as their mother trees due to introgressive hybridisation. Furthermore, sample collection in the field is convenient and actively growing leaf buds are available throughout the growing season. FISH experiments with these leaf chromosomes also give good results comparable to those obtained with root-tip chromosomes or even better as mapping on long or extended chromosomes has high resolution in general. Mapping of the 16S–28S ribosomal genes on birch leaf chromosomes has been shown to differentiate between birch species and therefore can accurately confirm their interspecific hybrids.     

An interactive, microcomputer-based karyotype analysis system for phylogenetic cytotaxonomy

A versatile and practical semi-automatic computerized system of karyotype analysis has been developed for phylogenetic cytotaxonomy for under $4,000. The modular software system is designed for use solely with a small 8-bit microcomputer and requires virtually no custom made hardware. The system interacts with the operator and greatly augments the speed and accuracy of cytotaxonomic investigations.     

中孕期胎儿染色体异常高危孕妇羊水细胞胎儿染色体核型及介入性产前诊断指征的大样本分析

目的探讨中孕期胎儿染色体异常(FCA)高危孕妇羊水细胞胎儿染色体核型(FK)及其介入性产前诊断(IPD)指征。方法选择2014—2018年,在四川大学华西第二医院产前诊断中心进行羊膜腔穿刺术羊水细胞FK检测的63581例FCA高危孕妇(孕龄为19~27+6孕周)为研究对象。回顾性分析孕妇不同IPD指征及其胎儿染色体核型异常(FKA)检测结果。本研究遵循的程序符合四川大学华西第二医院伦理委员会所制定的伦理学标准,并通过该伦理委员会批准[审批文号:医学科研2019伦审批第(077)号]。结果①本组63581例中孕期孕妇的FKA检出率为3.13%(1989/63581)。这63581例孕妇的IPD指征分别为高龄(预产期年龄≥35岁)(25648例)、血清学筛查高风险(29009例)、无创产前筛查(NIPT)高风险(333例)、夫妇一方为染色体异常携带者(603例)、超声筛查发现胎儿结构异常及超声软指标异常(2647例)、异常儿生育史(1546例)、于外院进行羊水细胞FK分析结果异常者(7例)、孕妇智力低下及合并智力低下家族史(149例)、孕妇早孕期有害物质接触史(1400例)、其他(2239例),FKA检出率分别为3.26%(836/25648)、2.04%(593/29009)、65.17%(217/333)、34.33%(207/603)、3.51%(93/2647)、1.68%(26/1546)、85.71%(6/7)、4.03%(6/149)、0.14%(2/1400)、0.13%(3/2239)。②351例孕妇的IPD指征为超声筛查发现胎儿结构异常中,IPD指征为胎儿淋巴水囊瘤、皮肤水肿的FKA检出率最高,为26.09%(6/23)。2296例孕妇的IPD指征为胎儿超声软指标异常中,IPD指征为胎儿颈项透明层(NT)值≥2.5 mm的FKA检出率最高,为7.74%(66/853)。结论孕妇高龄、血清学筛查高风险、NIPT高风险、夫妇一方为染色体异常携带者、超声筛查发现胎儿结构异常及超声软指标异常等,均为中孕期孕妇FCA的IPD指征,孕妇IPD指征类型不同,FCA风险不同。羊膜腔穿刺术羊水细胞FK检测,可预测有IPD指征孕妇的妊娠结局,为降低FKA出生率提供参考。     

骨髓增生异常综合征和再生障碍性贫血等细胞遗传学研究

为了探讨骨髓细胞遗传学分析在鉴别诊断骨髓增生异常综合征（ＭＤＳ）和再生障碍性贫血（ＡＡ）等不同血液病中的意义，我们联合应用骨髓细胞Ｒ－带核型分析和姐妹染色单体分化（ＳＣＤ）检测，对３３４例ＭＤＳ、ＡＡ等不同血液病进行分析并随访。结果表明：（１）ＲＡ／ＡＡ、ＲＡ／ＩＴＰ和ＲＡ／ＨＡ等可疑ＲＡ即为不典型ＲＡ或早期ＲＡ；（２）骨髓细胞从ＳＣＤ阳性转为ＳＣＤ阴性是骨髓细胞癌变过程；（３）骨髓细胞ＳＣＤ检测和核型分析具有肯定的诊断和预后价值；（４）ＦＡＢ分类法同细胞遗传学技术相结合可明显地提高ＭＤＳ诊断率或确诊率；（５）ＭＤＳ核型异常检出率６４．４％，与国外（４０％～７０％）相仿。本系列ＭＤＳ常见染色体异常为＋８，２ｑ￣－，一５／５ｑ￣－，一７／７ｑ￣－，７ｐ￣＋，以及一１１，一１４等，也与国外相似。（６）ＭＤＳ发病最初染色体异常看来是染色体数目异常即单体性为主。     

273例曾生育非整倍体患儿父母染色体核型分析

目的：探讨曾生育非整倍体患儿父母染色体核型异常的检出情况。方法：利用G显带技术对273例曾生育非整倍体患儿父母以及116例健康体检者进行染色体核型分析,必要时加做C显带。结果：273例样本中共检出72例染色体变异,其中包括4例染色体结构异常,检出率为1.46%;68例染色体多态变异,检出率为24.91%;剔除高龄女性样本后,多态变异检出率为64/255（25.10%）。116例对照组染色体核型共检出1例染色体结构异常,5例染色体多态变异,多态变异检出率为4.31%,2组多态变异检出率差异有统计学意义（P＜0.05）。结论：曾生育非整倍体患儿父母染色体多态变异具有较高检出率,应引起临床医生的重视。     

1193例慢性粒细胞白血病细胞及分子遗传学分析

目的了解国内慢性粒细胞白血病（CML）不同病期染色体核型及其演变和ber—abl融合基因特点。方法对1193例CML患者的染色体核型及524例CML患者的ber-abl融合基因资料进行回顾性分析。染色体的检测采用R显带技术，融合基因检测采用RT-PCR技术。结果在所有病例中，Ph染色体阳性（Ph^＋）患者占98．07％，Ph^-患者占1．93％。在Ph^＋患者中，95．64％为典型Ph染色体阳性，4．36％为变异型Ph染色体阳性。11．88％的典型Ph^＋患者发生染色体核型演变。急变期、加速期和慢性期染色体核型演变的发生率分别为49．04％、27．78％及7．94％。在所有典型Ph^＋患者中，发生染色体核型演变后最常见的附加染色体异常类型为＋Ph（14．62％），＋8（10．77％）和-21（7．69％）。急变期和加速期最常见的附加染色体异常类型为＋Ph（28．57％），＋8（16．67％）和＋19（7．14％）；慢性期最常见的附加染色体异常为-21（10．26％），＋Ph（8．97％）和＋8（8．97％）。附加染色体异常联合出现类型以＋Ph与＋8的联合最多（3．60％）。在所有524例检测过融合基因的标本中，h3a2（＋）为54．01％，b2a2（＋）为27．67％。结论国内CML患者染色体的核型及演变可能有着自己的特点。     

Percutaneous umbilical blood sampling

In the present day, fetal blood is sampled from the umbilical vein via a needle guided by dynamic ultrasound from the maternal abdomen (percutaneous umbilical blood sampling, PUBS). PUBS has become important in the fields of fetal physiology, diagnosis and therapy. Indications for PUBS are fetal karyotyping, prenatal diagnosis of blood disorders and congenital infection, and evaluation of fetal hypoxemia. Although risk of fetal loss following PUBS depends on the indication for the procedure and the experience of the operator, the rate of fetal loss after PUBS is assumed to be around 1%. Recent advances in molecular and cytogenetic techniques have restricted the number of reasons for using PUBS. However, since this technique offers a considerable advantage in enabling access directly to the fetal circulation, PUBS is likely to be used more frequently for fetal therapy than for prenatal diagnosis in the future.     

葡萄胎恶性趋势探讨

对97例葡萄胎进行了细胞遗传学分析、病理学检查和临床分析。97例中15例属部分性葡萄胎,核型为三倍体,82例属完全性葡萄胎,核型为二倍体。82例中62例是纯合子,核型为46,XX,20例是杂合子,其中11例核型是46,XX,9例是46,XY。完全性葡萄胎中18例是恶性病例,恶变率为22%(18/82),其中8例纯合子,恶变率12.9%(8/62),10例杂合子,恶变率50%(10/20)。均为46,XX。因此,杂合子46,XX完全性葡萄胎恶变率为90.9%(10/11)。而9例46,XY和15例三倍体中无一例发生恶变。可见完全性葡萄胎比部分性者恶变率高,杂合子完全性葡萄胎比纯合子者恶变率高。     

Morphological and Cytogenetic Characterization and N-myc Oncogene Analysis of a Newly Established Neuroblastoma Cell Line

A permanent cell line established from a xenograft of neuroblastoma which occurred in a 5 year old girl was investigated for its morphological and biological characteristics. The cultured cells were tumorigenic in nude mice. Microscopically, each tumor consisted of small round to polygonal cells with irregular nuclei and prominent nucleoli, corresponding to the features of the primary and xenografted tumor cells. Electron microscopic examination revealed that both the transplanted tumor cells and the cultured cells contained scanty microtubules and dense-core neurosecretory granules. Chromosome analysis of this cell line showed monosomy for chromosomes 1,10,19 and X, and structural rearrangements involving chromosomes 8, 17 and 20, in addition to numerous double minutes. The N- myc oncogene was found to be amplified 40 to 80 fold in the transplanted and cultured tumor cells, as well as in the primary tumor cells. In situ hybridization with a digoxigenin labeled uridine-triphosphate N- myc RNA probe detected abundant mRNA in the tumor cells. This neuroblastoma line may become a valuable in vitro experimental model system for studies aimed at better characterization of neuroblastoma. Acta Pathol Jpn 41: 507 515, 1991.     

HA为基础的三药方案治疗急性髓系白血病疗效分析及与染色体核型的关系

目的对12年来用HA（高三尖杉酯碱、阿糖胞苷）为基础的三药联合方案诱导化疗的243例急性髓系白血病（AML）患者的完全缓解（CR）率、无病生存（DFS）以及总生存（OS）情况进行总结分析,同时探讨不同染色体核型分组对治疗缓解率及预后的影响.方法用HA为基础的三药联合方案诱导治疗初治、原发AML 243例,计算CR率、DFS期、OS期及3年和5年DFS率、OS率.对其中有染色体核型结果的184例患者根据SWOG或MRC核型分级标准进行分组,比较不同核型组之间CR率、DFS期（率）及OS期（率）.采用SPSS软件分析所得数据.结果243例原发、初治AML患者CR率达77.4%,188例CR患者中位DFS期为28.5（1.0～153.0）个月,3年DFS率为45.4%,5年DFS率为40.2%.243例患者中位OS期为18.4（0.5～154.0）个月,3年OS率为36.9%,5年OS率为31.4%.按SWOG标准将184例患者根据染色体核型分析结果分为良好、中等、差、未知四组,四组的CR率、中位DFS期及OS期分别为良好核型组97.8%、87.4个月、89.1个月,中等核型组81.9%、17.6个月、22.3个月,差核型组61.5%、9.0个月、11.5个月,未知核型组79.3%、29.0个月、19.9个月.以上各组之间的差异均具有统计学意义（P＜0.01）.按MRC标准将184例患者分为良好、中、差三组,三组的CR率、中位DFS期及OS期分别为良好核型组96.1%、79.9个月、72.2个月,中等核型组79.5%、17.6个月、19.7个月,差核型组43.8%、16.5个月、12.0个月.中等核型组与差核型组的DFS期差异无统计学意义（P＞0.05）,其余指标各组之间有统计学意义（P＜0.01）.结论采用HA为基础的三药联合方案诱导治疗AML,CR率较二药方案高,有利于患者的长期生存.染色体核型同样是本组患者重要的预后相关因素,SWOG染色体核型分组标准更适合患者的疗效和预后分析,而且可将未知组与预后中等组合并分析.三组不同核型患者的预后差异具有统计学意义,良好核型组的预后明显比其他两组好,中等核型组比差核型组好.