三氧化二砷影响EVI1基因调控造血转录因子的体外研究

目的:观察三氧化二砷(arsenic trioxide,ATO)在体外影响反转录病毒整合位点1基因(ecotropic viral integration site-1,EVI1)基因对造血转录因子的调控作用。方法:实验选取EVI1高表达的急性髓系白血病细胞株THP-1,利用健康成人外周血单个核细胞作为对照,通过反转录实时荧光定量聚合酶链反应(real-time fluorescent quantitative polymerase chain reaction,RFQ-PCR),检测EVI1基因及造血转录因子GATA1、GATA2、RUNX1、MPO、LMO2、CMYB、PU.1和SCL的m RNA相对表达量。分别以1μmol/L、3μmol/L、5μmol/L的ATO溶液处理THP-1细胞株后,再通过反转录RFQ-PCR检测EVI1基因及造血转录因子的m RNA相对表达量。结果:通过反转录RFQ-PCR检测证实,存在EVI1基因的THP-1细胞中GATA2和CMYB基因的表达上调,而GATA1、RUNX1、MPO、LMO2、PU.1及SCL基因的表达水平下调。ATO对EVI1基因的下调作用具有浓度与时间依赖性,并对其他造血转录因子进行调控。结论:通过体外研究发现,高表达EVI1基因的THP-1细胞有GATA2基因的表达上调,同时存在其他造血转录因子的表达异常,与促进原始细胞增殖及髓系、红系的分化成熟受抑密切相关。ATO可特异性地下调EVI1基因的表达,并抑制GATA2转录因子的激活。     

利用斑马鱼模型研究miR-191对髓系造血分化的调控

目的:利用斑马鱼造血发育模型探索microRNA-191(miR-191)对髓系造血分化的调控。方法:将miR-191和对照(cel-miR-67)通过显微注射斑马鱼受精卵,收集发育到10 s、24 hpf、36 hpf和48 hpf的胚胎,分别提取对照及实验组胚胎的RNA,利用荧光定量qRT-PCR比较斑马鱼胚胎红系细胞分化标记基因(gata1,scl,hbbe3,lmo2)和粒-单系细胞分化标记基因(pu.1,L-plastin,mpx,cebpα)的表达水平;选取24 hpf胚胎进行上述基因的整胚原位杂交,比较注射miR-191对髓系分化标记基因的原位时空表达的影响。结果:通过分子克隆及T7 RNA聚合酶体外转录成功制备了斑马鱼红系、粒-单系细胞分化标记基因的RNA探针且特异性良好;与注射对照的受精卵相比较,荧光定量qRT-PCR检测发现,注射miR-191的24 hpf和36 hpf斑马鱼胚胎整体中髓系分化基因的水平均有微弱的上调趋势;而发育10 s的胚胎中髓系基因mpx的mRNA表达出现10-20倍的显著上调;整胚原位杂交结果发现24 hpf的胚胎中髓系特异基因L-plastin的原位表达有所上调。结论:本研究建立了斑马鱼受精卵显微注射技术及分析胚胎髓系分化基因表达的两种方法;miR-191对斑马鱼胚胎髓系分化调控功能的研究结果显示,该miRNA可能参与调控粒-单核系和红系分化,其对髓系基因mpx表达调控的分子机制及相应功能有待进一步的研究。     

MYCN转基因型斑马鱼系岩藻糖转移酶基因谱表达的研究

目的研究岩藻糖转移酶在髓系白血病的发生与发展中的作用。方法应用实时定量逆转录-聚合酶链反应检测具髓系白血病表型的MYCN转基因型斑马鱼系岩藻糖转移酶基因（DFs）表达。结果 MYCN转基因型斑马鱼系岩藻糖转移酶7/8/9/10/11（DF7/8/9/10/11）表达量与野生型斑马鱼相比显著改变（t=-32.756、8.405、12.719、34.673、27.113,P〈0.05）;岩藻糖转移酶1/2（DF1、2）表达量的差异无统计学意义（t=-1.651、-0.244,P〉0.05）。结论在MYCN转基因型斑马鱼系中,岩藻糖转移酶的表达与髓系白血病的发生与发展存在相关性。     

BAMBI基因过表达人结肠癌细胞移植斑马鱼致肝转移模型的构建

目的构建BAMBI基因过表达人结肠癌细胞移植斑马鱼肝转移模型,分析斑马鱼肝内转移性结肠癌细胞BAMBI基因表达水平与肝转移发生率的关联性。方法分别构建BAMBI过表达稳转结肠癌细胞株SW620(BAMBI+组)、空载体红色荧光蛋白基因转染结肠癌细胞株SW620(EV组),分别显微注射到受精后第2天转基因斑马鱼Tg(Apo14∶GFP)胚胎卵黄囊(每组各680条),激光共聚焦荧光显微镜观察、拍摄,采用Image J软件测量转移至斑马鱼肝脏内结肠癌细胞红色荧光表达强度,计算肝转移发生率;采用t检验比较BAMBI+组及EV组斑马鱼肝脏内结肠癌细胞红色荧光表达强度及肝转移发生率的差异。结果与EV组相比,激光共聚焦荧光显微观察显示BAMBI+组斑马鱼肝内结肠癌细胞红色荧光表达量显著增加(t=6.247,P=0.003 3);BAMBI+组斑马鱼肝转移发生率与EV组相比显著增加(t=4.276,P=0.012 9)。结论构建了基因修饰人结肠癌细胞移植斑马鱼致肝转移模型,可较好地模拟活体状态的结肠癌肝转移;BAMBI基因可促进异种移植入斑马鱼的结肠癌细胞发生肝转移。     

原癌基因c-myb在巨核-红系造血发育中的研究进展

细胞核内原癌基因c-myb是造血系统的重要调控因子,参与造血细胞周期调控并调节造血细胞增殖和分化.近年来通过对不同细胞系及转基因鼠模型的研究发现,c-myb对巨核-红系祖细胞的定向分化起决定作用,该原癌基因的缺失导致红系定向严重受损,而对巨核系定向影响较小.瞬时转染及免疫沉淀实验已证实c-myb通过与造血调控的多个转录因子相互作用来实现其在造血系统的生理功能.对c-myb的结构、功能及相关分子调控机制的研究,有助于进一步阐明其在巨核-红系造血发育中的作用,并为血小板疾病、红细胞疾病的分子靶向治疗提供新的思路.本文就c-myb结构、功能、参与巨核红系造血调控的有关作用及相关分子机制作一综述.     

原癌基因c-myb在巨核-红系造血发育中的研究进展

细胞核内原癌基因c-myb是造血系统的重要调控因子,参与造血细胞周期调控并调节造血细胞增殖和分化。近年来通过对不同细胞系及转基因鼠模型的研究发现,c-myb对巨核-红系祖细胞的定向分化起决定作用,该原癌基因的缺失导致红系定向严重受损,而对巨核系定向影响较小。瞬时转染及免疫沉淀实验已证实c-myb通过与造血调控的多个转录因子相互作用来实现其在造血系统的生理功能。对c-myb的结构、功能及相关分子调控机制的研究,有助于进一步阐明其在巨核-红系造血发育中的作用,并为血小板疾病、红细胞疾病的分子靶向治疗提供新的思路。本文就c-myb结构、功能、参与巨核红系造血调控的有关作用及相关分子机制作一综述。     

微小RNA-221在宣威肺癌细胞中对靶基因p53正向凋亡调控因子调控机制的研究

目的探讨宣威肺癌细胞中微小RNA-221(miR-221)对靶基因p53正向凋亡调控因子(PUMA)的调控机制。方法在宣威肺癌细胞株(XWLC-05)中转染miR-221过表达/抑制表达载体后,将实验分为4组,Control组:未转染任何质粒;Scramble组:转染PUMA质粒;Over-miR-221+PUMA组:转染miR-221过表达载体、PUMA质粒;In-miR-221+PUMA组:转染miR-221抑制表达载体、PUMA质粒。应用实时荧光定量PCR技术检测各组PUMA在mRNA水平的表达差异。荧光素酶报告实验验证PUMA是否为miR221的靶基因。四甲基偶氮唑蓝(MTT)比色法分析miR221对细胞增殖的影响。检测Caspase3/7、9活性分析miR221对细胞凋亡的影响。结果 miR-221可以直接作用于PUMA基因的3’UTR端(t=-8.662,12.761,P<0.001);过表达转染组能够有效地促进宣威细胞增殖,抑制表达转染组能够有效地抑制宣威细胞增殖(24 h:F=98.181,P<0.001;48 h:F=109.561,P<0.001;72 h F=143.782,P<0.001)。在抑制表达转染组中Caspase3/7、9的酶活性是升高的(t=12.851,15.491,P<0.001);而过表达转染组中Caspase 3/7、9酶活性与Control组及Scramble组差异无统计学意义(t=-2.181,P=0.061;t=-2.056,P=0.074);各组PUMA基因的表达差异无统计学意义(H=1.262,P=0.532)。结论 PUMA为miR-221的靶基因之一,miR-221可能通过负向调控靶基因PUMA参与宣威肺癌的发生、发展。     

AML1-ETO转基因小鼠的建立及初步表型分析

目的:建立AM L1-ETO融合基因转基因小鼠,在整体动物水平研究AM L1-ETO融合蛋白在白血病发病中所起的作用。方法:构建hCG/AM L1-ETO转基因质粒,通过显微注射将该质粒转入小鼠受精卵中,植入假孕母鼠输卵管,获得G0代转基因小鼠;用聚合酶链反应(PCR)方法检测融合基因的整合情况,逆转录-聚合酶链反应(RT-PCR)方法检测融合基因的表达情况和组织表达谱。结果:PCR法共检出10只G0代转基因阳性小鼠,其中8个系均产下F1代小鼠,由此建立了8个AM L1-ETO转基因小鼠系。RT-PCR法证实AM L1-ETO融合基因在22系中稳定表达,但血常规、肝脏、脾脏等组织未见异常变化;对该系小鼠的组织表达谱检测表明,融合基因在肝脏、脾脏、心脏和肌肉组织存在不同程度的表达,但在脑组织中不表达。结论:AM L1-ETO融合基因能在转基因小鼠的骨髓等组织中表达,但尚不足以引发白血病,推测其他致病基因在小鼠白血病的发生中也起一定作用。     

造血干细胞向淋巴系祖细胞增殖HOXB4基因的表达

背景:HOXB4基因不仅能促进造血干细胞的扩增及其功能的活化与表达,而且体内试验表明它不会诱发白血病.因此更好地研究HOXB4在造血细胞增殖分化中的变化及作用对进一步研究造血干细胞的扩增可以提供更多的理论基础.目的:观察人类脐血造血干细胞向淋巴系祖细胞增殖分化过程中HOXB4基因表达的情况及全反式维甲酸对HOXB4基因表达的影响.方法:将培养的淋巴系造血祖细胞按干预方式不同分为2组,全反式维甲酸组:在培养体系中加入全反式维甲酸,终浓度6×10-8mol/L.正常组:不加全反式维甲酸,代之以等量的1640培养液.观察人类脐血造血干细胞经植物血凝素诱导后,在培养第3,7,12天的淋巴细胞集落形成单位生成情况.采用实时荧光定量PCR技术检测脐血造血干细胞向淋巴系祖细胞增殖分化过程中HOXB4基因的表达水平.结果与结论:人脐血造血干细胞向淋巴系祖细胞增殖分化过程中,HOXB4基因晕规律的表达.随培养时间推移,正常组和全反式维甲酸组HOXB4基因的表达均逐渐降低.与正常组比较,全反式维甲酸可上调HOXB4基因的表达.     

调控红细胞生成的转录因子及其作用

哺乳类红细胞生成分为胚胎型 (或称原始型 )和成年型(或称定型 )两个不同的发育时期 ,前者主要在卵黄囊的血岛中进行 ,产生有核红细胞 ;后者则集中于胚胎 ,骨髓及脾脏 ,产生无核红细胞。如果从红系分化角度出发 ,则可分为两个阶段 :红系早期分化 (由造血干细胞到红系祖细胞 )和终末分化 (由红系祖细胞到成熟红细胞 )。在整个分化过程中 ,红系特异基因相继开启并呈优势表达 ,非红系基因则逐渐关闭 ,细胞最终呈现红系特有表型。调控基因特异表达的主要方式之一是由公用转录因子与红系特异转录因子共同参与的基因转录水平的调控。红系特异转录…     

A new variant of MYCN gene as a cause of Feingold syndrome

Feingold syndrome 1 (FS1) is a rare disorder that is inherited in autosomal dominant manner with full penetrance but with variable expressivity. The most common phenotypical features described are finger and toe anomalies, microcephaly, short stature, and intestinal atresia. Dysmorphic features, intellectual disability and other organ anomalies are less frequently described. Here, we present a 7‐year‐old boy with severe intellectual disability who is diagnosed with FS1 syndrome caused by a new heterozygous variant of MYCN gene     

Fast detection of MYCN copy number alterations in brain neuronal tumors by real-time PCR

Increased MYCN gene copy number is a characteristic property of neurogenic tumors. Fluorescence in situ hybridization (FISH) and array-based comparative genomic hybridization (array-CGH) are traditionally used to determine MYCN amplification for tumor stratification. A unique ability of real-time quantitative polymerase chain reaction (qPCR) to determine gene copy number, even within a small percent of observed tumor cells, and can be more appropriate. MYCN genomic copy number from 44 human brain tumors (22 medulloblastomas and 22 neurocytomas) was determined by means of FISH, array-CGH, and qPCR. By qPCR, with the original set of oligonucleotides, 17 out of 44 (38.6%) tumors were found to contain a 1.3- to 2.9-fold increase of MYCN defined as low-level gain. An absolute qPCR method was used to get high accuracy of results. Strong correlation was observed between the three methods: for medulloblastomas, r=1 (P<0.01) between FISH and array-CGH and r=0.92 (P<0.01) between qPCR and FISH/array-CGH. For neurocytomas, r=0.9 (P<0.01) between FISH and array-CGH and r=0.34/0.43 (P<0.01) between qPCR and FISH/array-CGH. Absolute qPCR assays possess high precision compared to other conventional methods and can be used for accurate and quickness detection of MYCN status (low-level gene gain and amplification).     

The aurora kinase inhibitor CCT137690 downregulates MYCN and sensitizes MYCN-amplified neuroblastoma in vivo

Aurora kinases regulate key stages of mitosis including centrosome maturation, spindle assembly, chromosome segregation, and cytokinesis. Aurora A and B kinase overexpression has also been associated with various human cancers, and as such, they have been extensively studied as novel antimitotic drug targets. Here, we characterize the Aurora kinase inhibitor CCT137690, a highly selective, orally bioavailable imidazo[4,5-b]pyridine derivative that inhibits Aurora A and B kinases with low nanomolar IC(50) values in both biochemical and cellular assays and exhibits antiproliferative activity against a wide range of human solid tumor cell lines. CCT137690 efficiently inhibits histone H3 and transforming acidic coiled-coil 3 phosphorylation (Aurora B and Aurora A substrates, respectively) in HCT116 and HeLa cells. Continuous exposure of tumor cells to the inhibitor causes multipolar spindle formation, chromosome misalignment, polyploidy, and apoptosis. This is accompanied by p53/p21/BAX induction, thymidine kinase 1 downregulation, and PARP cleavage. Furthermore, CCT137690 treatment of MYCN-amplified neuroblastoma cell lines inhibits cell proliferation and decreases MYCN protein expression. Importantly, in a transgenic mouse model of neuroblastoma that overexpresses MYCN protein and is predisposed to spontaneous neuroblastoma formation, this compound significantly inhibits tumor growth. The potent preclinical activity of CCT137690 suggests that this inhibitor may benefit patients with MYCN-amplified neuroblastoma.     

Real-time quantitative PCR for the measurement of MYCN amplification in human neuroblastoma with the TaqMan detection system.

BACKGROUND: Neuroblastoma is the most common extracranial malignant solid tumor in children under 5 years and is characterized by a wide clinical and biological heterogeneity, from spontaneously regressive forms to cancers with a rapid and fatal progression. MYCN oncogene amplification is considered the most important prognostic factor to evaluate survival and therapeutic choices in these patients. METHODS: Here we present a new assay for rapid and accurate measurement of MYCN amplification, based on real-time quantitative PCR with the TaqMan(TM) reaction. The degree of MYCN amplification was derived from the ratio of the MYCN oncogene and the single-copy reference gene, beta-actin. The absolute abundance of these two genes in tumor sample DNA was obtained by extrapolation on external calibration curves generated with reference DNA. RESULTS: We found a variable degree of MYCN amplification, from 2 to 29, in 26 of 49 (53%) neuroblastomas. These results were well correlated to those obtained with a competitive PCR assay in the same samples (r = 0. 987). MYCN amplification was associated mainly with advanced cancer stages, and the analysis of overall survival confirmed that the measurement of MYCN amplification is a predictor of patient outcome in neuroblastoma. Patients without MYCN amplification had a cumulative survival significantly higher than patients with low (<9; P = 0.02) and high (>/=9; P = 0.03) oncogene amplification. CONCLUSION: The assay is rapid and reproducible and does not require any post-PCR analytical procedure.     

Detection of MYCN amplification and chromosome 1p36 loss in neuroblastoma by cDNA microarray comparative genomic hybridization.

BACKGROUND: In the last decade, microarray technology has been extensively used to evaluate gene expression profiles and genome imbalances. We have developed a microarray-based comparative genomic hybridization (CGH) approach to identify MYCN gene amplification and 1p36 chromosome loss, two markers of tumor aggressiveness in neuroblastoma. AIM: The aim was to use microarray CGH technology to detect the two major prognostic markers for neuroblastoma, MYCN amplification and 1p36 chromosome deletion, in neuroblastoma patients and, therefore, confirm the usefulness of this approach in this cancer. METHODS: DNA was purified from 16 tumors containing at least 90% malignant neuroblasts and collected at the onset of disease. Pooled fluorescent-labeled reference and neuroblastoma tumor genomic DNA was hybridized to epoxide-coated glass slides on laboratory-made complementary DNA microarray. The microarray contained cDNA mapped at the 1p36.33-36.1 chromosomal region and MYCN gene. cDNA from the 2q33-q34 and 12p13 chromosomes was used as a control and Arabidopsis thaliana DNA was spotted to control unspecific hybridization. Fluorescence in situ hybridization analysis was also performed to validate results from the microarray CGH. RESULTS: Both MYCN amplification and 1p36 chromosome deletion were detected by microarray CGH. The sensitivity and specificity for 1p36 loss detection were 66.7% and 90.0%, respectively. The method had a sensitivity of 66.7% and specificity of 90.9% to detect MYCN amplification. DISCUSSION: Our results demonstrated that the microarray CGH can be efficiently applied to study DNA gain and loss of specific chromosome regions.     

Determination of creatine kinase isoenzyme MB activity in serum using immunological inhibition of creatine kinase M subunit activity. Activity kinetics and diagnostic significance in myocardial infarction.

This is a new method for the determination of creatine kinase isoenzyme MB activity in serum. The method uses direct activity measurement of creatine kinase B subunit activity after blocking of CK-M subunit activity by inhibiting antibodies. The test takes no longer than 15 min. The method yields an intra-serial C.V. of 2.0-12.9%, and a C.V. from day to day of 5.5%. The detection limit is 3.4 U/l creatine kinase MB. In the 95 cases with proven myocardial infarction several types of creatine kinase MB activity kinetics could be determined. The percentage of creatine kinase MB of peak CK-total is 6-25%, with a mean of 11.1%. The amount of creatine kinase MB with respect to total CK activity after reinfarction is higher than the amount after initial infarction.