造血干细胞分化过程中HOXB6基因表达及干预研究

目的探讨脐血造血干细胞(HSC)向粒-单系(CFU-GM)、红系(CFU-E)和淋巴系祖细胞(CFU-TL)增殖分化过程中HOXB6基因的表达情况及全反式维A酸(ATRA)对其的影响。方法1.采用造血干/祖细胞体外培养技术,以ATRA持续干扰人类脐血造血干/祖细胞,观察HSC经人粒细胞-单核细胞集落刺激因子(GM-CSF)、促红细胞生成素(EPO)和植物血凝素(PHA)分别诱导后,在培养第3天、第7天和第12天的CFU-GM、CFU-E及CFU-TL集落生成情况。2.采用实时荧光定量RT-PCR技术(FQ-RT-PCR)检测造血干细胞增殖分化过程中HOXB6基因的表达水平。结果1.人类造血干细胞向CFU-GM、CFU-E和CFU-TL增殖分化过程中,各组细胞HOXB6基因均有表达。2.随时间推移,CFU-GMHOXB6 mRNA在增殖分化的第3天开始表达,第7天表达最强烈,第12天表达明显减弱;CFU-E HOXB6 mRNA表达在第3天、第7天和第12天均持续增强;而在CFU-TL中,HOXB6 mRNA表达在第7天最强烈,第12天表达减弱。3.与对照组比较,ATRA可上调各组HOXB6基因的表达。结论1....     

脐血造血干祖细胞增殖过程中人类巨细胞病毒感染对hoxa9、hoxa10基因表达影响的研究

目的在基因水平探讨人类巨细胞病毒(HCMV)感染导致脐血CFU-G损伤的机制,探讨人类脐血造血干细胞(HSC)向粒系祖细胞(CFU-G)增殖过程中hoxa9、hoxa10基因表达的情况。方法从2006年5月至2007年10月收集12例正常足月顺产新生儿断脐后的胎盘段脐血,采用造血干细胞体外培养技术及荧光定量逆转录聚合酶链反应(FQ-RT-PCR)方法,以HCMV-AD169和(或)全反式维甲酸(ATRA)持续干扰HSC,观察正常对照组、ATRA组、HCMV-AD169组、ATRA+HCMV组的造血干祖细胞经人粒细胞集落刺激因子(G-CSF)诱导后,在增殖分化过程中第3、7和12天检测CFU-Ghoxa9、hoxa10基因表达情况。结果各组hoxa9、hoxa10基因均在CFU-G增殖分化的第7天表达量最高(P0.01),第12天表达量明显减弱(P0.01),与正常对照组比较,CFU-Ghoxa9、hoxa10基因表达受ATRA(6×10-8mol/L)上调,而受HCMV下调(P0.01),与HCMV组比较,ATRA可上调HCMV感染的CFU-Ghoxa9、hoxa10基因的表达。结论 CFU-G的增殖分化与hoxa9、hoxa10基因的表达相关。HCMV可能通过调控hoxa9、hoxa10基因表达异常引起造血功能异常,ATRA不仅能显著上调正常CFU-Ghoxa9、hoxa10基因的表达,而且能上调HCMV感染的CFU-Ghoxa9、hoxa10基因的表达。     

造血干细胞向淋巴系祖细胞增殖分化过程中HOXC6基因表达的研究

目的探讨人脐血造血干细胞(HSC)向淋巴系祖细胞增殖分化过程中HOXC6基因表达的情况及全反式维甲酸(ATRA)对HOXC6基因表达的影响。方法采用造血干/祖细胞体外培养技术,以ATRA持续诱导人脐血造血干细胞,观察人脐血HSC经植物血凝素(PHA-M)诱导后,在培养过程第3、7和12天的淋巴系祖细胞集落生成情况;采用实时荧光定量PCR技术检测人脐血造血干细胞向淋巴系祖细胞增殖分化过程中HOXC6基因的表达水平。结果人脐血造血干细胞向淋巴系祖细胞增殖分化过程中,HOXC6基因表达呈现时间规律性。结论HOXC6基因在增殖分化的第7天表达最强烈,第12天表达明显降低,提示HOXC6基因与人脐血造血干细胞向淋巴系祖细胞增殖分化过程密切相关,可能是其调控基因之一。ATRA能显著上调HOXC6基因的表达。     

全反式维甲酸对脐血粒-单系祖细胞HOXB6基因表达的影响

目的探讨脐血造血干祖细胞(HSPC)向粒-单系(CFU-GM)增殖过程中HOXB6mRNA表达及全反式维甲酸(ATRA)对HOXB6mRNA表达的影响。方法1.采用造血祖细胞体外培养技术,以ATRA持续干扰人类造血干祖细胞,观察人脐血HSPC经人粒细胞-单核细胞集落刺激因子(GM-CSF)诱导后,在培养过程第3天,7天和12天的CFU-GM集落生成情况。2.采用实时荧光定量PCR技术(FQ-RT-PCR)检测造血祖细胞增殖分化过程中HOXB6基因的表达水平。3.结果用DNA相对拷贝数和RNA表达相对量(2-△△Ct)表示HOXB6基因相对表达量。结果1.人类造血干祖细胞向粒单系增殖分化过程中,各组细胞HOXB6基因均表达;2.随时间延长,CFU-GM-HOXB6-mRNA在增殖分化的第7天表达最强烈,第12天表达明显减弱;3.与正常对照组比较,ATRA可上调HOXB6基因的表达。结论1.在脐血CFU-GM祖细胞的不同增殖阶段,HOXB6基因呈现持续稳定的表达,提示HOXB6是人类造血干祖细胞向粒单系正常增殖分化过程中的调控基因之一;2.ATRA能显著上调HOXB6基因的表达。     

人类巨细胞病毒对粒单系祖细胞分化过程中HOXB6、A5基因的影响

目的探讨人类造血干细胞(HSPC)向粒单系(CFU-GM)增殖过程中HOXB6、A5基因的表达情况,及人类巨细胞病毒(HCMV)对其的影响。方法采用real-timePCR技术测定正常对照组与HCMV组HSPC向CFU-GM增殖过程中HOXB6、A5基因的表达水平。结果①HOXA5基因表达多为阴性,HOXB6基因在增殖过程的第3天开始表达,第7天增高,第12天显著降低(P<0.05)。②相对于正常组,HCMV感染组HOXB6基因表达下调(P<0.05)。结论HOXA5可能不是人类HSPC向CFU-GM增殖分化过程的主控基因;HOXB6是人类HSPC向CFU-GM定向分化的主控基因之一,其表达呈现时间规律性;HCMV干扰的细胞组HOXB6表达下调的同时,其细胞集落生成较正常组差。提示HCMV可能通过调控HOXB6基因表达异常,引起造血干细胞增殖分化异常。     

巨细胞病毒感染对淋巴系祖细胞增殖的影响及其干预措施

目的探讨人类巨细胞病毒（HCMV）对淋巴系祖细胞（CFU-TL）体外增殖的影响、作用机制及其干预措施。方法本实验共分为空白对照组、灭活对照组、HCMV感染组、更昔洛韦（GCV）干预组、黄芪注射液（AMI）干预组5组。采用甲基纤维素半固体培养CFU-TL集落，不同浓度HCMV-AD169持续攻击CFU-TL，用AMI、GCV粉剂干预，观察CFU-TL集落形成：用四甲基偶氮唑盐法测HCMV对宿主细胞增殖活性的影响；用PCR检测CFU-TL集落细胞内HCMV-AD169DNA。结果1．与空白对照组及灭活对照组比较，各病毒感染组CFU-TL集落产率明显减少、维持时间明显缩短（P均〈0．01），抑制程度与病毒感染滴度呈剂量依赖关系；2．HCMV感染48、72、96、120h对宿主细胞增殖活性的影响与对照组比较均有显著差异（F分别为11．412，18．058，13．259，56．360 P均〈0．01）。3．PCR检测到HCMV感染组CFU-TL集落细胞内HCMV-AD169DNA。4．与HCMV感染组比较，HCMV＋AMI组、HCMV＋GCV组CFU-TL集落产生率明显增加，集落维持时间均显著延长（P均〈0．01）。结论HCMV在体外能抑制CFU-TL集落的增殖和分化；HCMV感染患儿免疫功能低下可能与HCMV直接抑制CFU-TL有关；GCV、AMI在体外能有效干预HCMV感染所致的CFU-TL增殖抑制的发生。     

脐血粒系祖细胞发育过程同源盒基因B7的表达及干预

目的 探讨人类脐血造血干细胞向粒系祖细胞发育过程中同源盒(HOX)B7 mRNA和蛋白的表达及全反式维A酸(ATRA)和(或)三氧化二砷(As2O3)对脐血粒系祖细胞发育过程中HOXB7表达的影响.方法 采用实时荧光定量PCR(RT-PCR)、Western-blot技术测定空白对照组、ATRA组、As2O3组、ATRA+As2O3组造血干细胞向粒系祖细胞增殖过程中HOXB7 mRNA及蛋白表达水平.结果 1.琼脂糖凝胶电泳显示RNA电泳图的5 S,18 S,28 S 3条带型整齐,无明显拖尾及弥散,说明RNA结构完整,无明显降解.2.反转录PCR扩增各组均有目的 基因HOXB7和内参照ACTB基因的cDNA产物,与DNA分子Marker相比扩增产物大小分别如下:HOXB7为129 bp,ACTB基因为111 bp,与预定理论值大小符合.3.人类造血干细胞向粒系祖细胞增殖分化过程中,空白对照组、ATRA组、As2O3组及ATRA+As2O3组的HOXB7 mRNA第3天少量表达,第7天表达较强烈,第12天表达减弱;空白对照组、ATRA组的HOXB7蛋白在增殖分化的第3天少量表达,第7天表达较高,第12天表达减弱,As2O3组及ATRA+As2O3组的HOXB7蛋白的表达在各时间点无明显差异.4.与空白对照组比较,ATRA组HOXB7 mRNA及蛋白的表达上调(P<0.05),而As2O3组HOXB7的表达无明显差异(P>0.05).结论 HOXB7基因可能是人类造血干祖细胞向粒系祖细胞增殖分化过程的调控基因之一,HOXB7基因与粒系造血有相关性.ATRA治疗白血病的作用可能与调节HOX基因的表达有关.As2O3对HOXB7 mRNA及蛋白的表达无明显调节作用.     

人巨细胞病毒感染对脐血巨核系祖细胞体外增殖的影响

目的　探讨人巨细胞病毒 (HCMV)对脐血巨核系祖细胞 (CFU Mk)体外增殖的影响及其机制。方法　采用造血祖细胞体外培养技术 ,观察、计数HCMV AD169感染的CFU Mk集落产率、抑制率、集落峰值及维持时间 ;并采用聚合酶链反应 (PCR)技术检测集落细胞内HCMV AD169DNA。结果　 1.HCMV AD169感染组集落产率较对照组明显减少 ,HCMV对CFU Mk集落的抑制程度与病毒滴度有关 ,集落产率随病毒感染滴度增高而减少 ,抑制率随病毒感染滴度增高而渐增加 ;各组集落峰值出现时间无明显差异 (P均 >0 .0 5 ) ,但各感染组集落维持时间明显短于对照组 (P <0 .0 1) ;2 .用PCR在集落细胞内检测到HCMV AD169DNA。结论　CFU Mk是HCMV的宿主细胞之一 ,HCMV能直接感染CFU Mk ;在体外HCMV AD169对CFU Mk的增殖和集落形成有明显抑制作用 ,此作用可能与临床HCMV感染后患者血小板减少有关     

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目的研究脐血红系祖细胞的HOXB6基因的表达情况以及人类巨细胞病毒(HCMV)感染对红系祖细胞的HOXB6基因表达的影响,以探讨HCMV导致脐血红系祖细胞损伤的机制。方法在泸州医学院附属医院儿科以实时荧光定量PCR技术(FQ-RT-PCR)检测人类巨细胞病毒感染和(或)用全反式维甲酸(ATRA)处理后HOXB6基因的表达水平。结果人脐血红系祖细胞可表达HOXB6基因;HCMV感染后,红系祖细胞HOXB6基因的表达明显下调;ATRA能显著上调红系祖细胞HOXB6基因的表达水平,且能够在一定时间内上调HCMV感染后的红系祖细胞HOXB6基因的表达。结论HCMV感染能诱导脐血红系祖细胞HOXB6的表达下调,HC-MV有可能通过调控HOXB6基因表达异常而引起红系祖细胞的增殖障碍。     

人巨细胞病毒对造血系统的影响

目的 探讨人巨细胞病毒(HCMV)对造血系统的影响及其机制。方法 采用造血祖细胞体外半固体培养技术,研究HCMV AD169株对粒-巨噬系祖细胞(CFU-GM)、红系祖细胞(CFU—E)、多向造血祖细胞(CFU—Mix)及巨核系祖细胞(CFU-MK)集落生长的影响;分别用原位聚合酶链反应(IS-PCR)和聚合酶链反应(PCR)检测CFU-GM、CFU-Mix、CFU-MK及CFU-E集落细胞中的HCMV DNA;用逆转录聚合酶链反应(RT-PCR)检测CFU-E集落细胞中的晚期抗原(late antigen,LA)mRNA及CFU-MK集落细胞中的前早期抗原(immediate early antigen,IEA))mRNA;用免疫组化方法检测CFU—Mix集落细胞中HCMV早期蛋白P52。结果 HCMV AD169株在体外能明显抑制CFU—GM、CFU—E,CFU—Mix及CFU—MK的生长,抑制程度与病毒感染滴度呈剂量依赖关系;经IS-PCR或PCR检测发现病毒感染组CFU-GM,CFU-E,CFU-Mix及CFU-MK细胞中有HCMV DNA存在;RT—PCR检测发现病毒感染组CFU-MK细胞中有IEA mRNA的表达,而CFU-E细胞中的LA mRNA为阴性;免疫组化(IHC)检测发现病毒感染组CFU-Mix集落细胞中有HCMV P52的表达。结论 HCMV AD169可抑制CFU-GM,CFU-E,CFU-Mix及CFU-MK的分化和增殖;HCMV AD169对造血系统有直接损害作用。此作用可能是HCMV感染所致的粒细胞和血小板减少及贫血的原因之一。     

Expression of homeotic genes Hoxa3, Hoxb3, Hoxd3 and Hoxc4 is decreased in the lungs but not in the hearts of adriamycin-exposed mice

Exposure of rat and mouse embryos to adriamycin (doxorubicin chlorhydrate) induces esophageal atresia (EA) and VACTERL association. Sonic hedgehog (Shh) and Gli2/Gli3 pathways are involved in these conditions and knockout mice for homeotic Hox genes Hoxa3, Hoxb3, Hoxc3, Hoxc4 and Hoxa5 show phenotypes with some of the associated VACTERL features. This study aims at evaluating the possible influence of Hoxa3, Hoxb3, Hoxd3 and Hoxc4 as upstream regulators of this complex signalling. Pregnant mice were exposed either to 4 mg/kg of adriamycin (EA group) or vehicle (controls) on embryonic days 7.5 and 8.5. Embryos were recovered at four endpoints (E12.5–E15.5) and randomly assigned for immunohistochemical or molecular biology studies. Lungs and hearts were separately harvested and processed for Hoxa3, Hoxb3, Hoxd3 and Hoxc4 quantitative RT-PCR measurements. Antibodies for Hoxa3, Hoxb3 and Hoxd3 proteins were used for immunohistochemical studies. RT-PCR studies showed a drastic and statistically significant decrease of the four genes in the lungs of EA mice when compared to controls, with a slight recovery from E15.5. Hearts of both groups showed a similar expression of all the genes throughout gestation. Control embryos expressed the hox3 paralogous genes in heart, skin, foregut derivatives and their surrounding mesoderm through E12.5–E15.5 whereas adriamycin-exposed embryos showed a severe decrease in expression of these three proteins in the same tissues but not in the heart. Adriamycin drastically reduced the expression of Hoxa3, Hoxb3, Hoxd3 and Hoxc4 in mice embryonic lungs. Their expression in the heart did not seem to be influenced by adriamycin in this experimental setting. Supported by #03/0892 FIS (Fondo de Investigación Sanitaria) and FMMM grants and research fellowship from the Hospital Universitario La Paz.     

Specifics of histopathological and genetical diagnosis and classification of lymphomas in children and adolescents

Malignant lymphoma along with leukemias account for nearly half of all malignancies arising in childhood and adolescence. The correct tissue-based histopathological diagnosis of lymphomas results from a close interdisciplinary exchange between pediatric oncologists and hematopathologists. We describe here relevant features of lymphoma subtypes arising in the young age group, Burkitt lymphoma, precursor/lymphoblastic lymphomas, anaplastic large cell lymphoma and diffuse large B-cell lymphoma as well as primary mediastinal B-cell lymphoma and the rare pediatric follicular lymphomas. Special focus is put on specific diagnostic difficulties as well as new insights into biological features of pediatric lymphomas in comparison with their adult counterpart. In addition the relevance of newly defined lymphoma entities of the WHO-classification 2008, e.g. greyzone lymphomas, will be discussed for the young age group.     

Age-dependency of alpha- and beta-adrenoceptors on thrombocytes and lymphocytes of asthmatic and nonasthmatic children

Among the possible mechanisms which may cause wheezing or asthmatic episodes a genetically determined -adrenoceptor blockade and a hyperresponsiveness of -andrenoceptors has been postulated. Evidence to support this hypothesis stems from an increased bronchial sensitivity to -blockers, a reduced formation of cyclic AMP in response to -adrenergic stimulation and enhanced -adrenergic responses in asthmatic subjects. The recent development of techniques for measuring the specific, high-affinity binding of radiolabeled -and -adrenergic antagonists made it possible to study - and -adrenoceptors in vitro. Based upon the assumption that a change in the number and/or affinity of adrenergic receptors might be a general phenomenon, we have performed - and -receptor binding studies on lymphocytes and platelets from wheezing infants and asthmatic children as well as of infants, children, and adults not suffering from these diseases.Using ¹²⁵[I]-cyanopindolol (ICYP) and ³[H]-yohimbine (HYOH) as highly specific ligands for - and -adrenoceptors, the following results were obtained: (1) Lymphocytes and platelets from control subjects and asthamatics bound similar amounts of ICYP and HYOH and thus showed no differences either in the number or the affinity of - and -adrenoceptors. Lymphocytes and platelets of wheezing and nonwheezing infants also bound the same amounts of the radioligands. (2) In asthmatic children receiving 4×2 puffs salbutamol -adrenoceptor were down-regulated and this may mimic -adrenoceptor blockade. (3) When subjects were divided into four categories according to age (0–5, 5–10, 10–20 years, adults) the number of -adrenoceptor binding sites showed an age-dependent increase. The number and affinity of -adreneceptor binding sites on platelets was neither influenced by age nor disease.It is concluded that the - and -adrenoceptors of wheezing infants and asthmatic children at least on blood cells are normal. However the -adrenoceptors show an age-dependent maturation process, which may account for an unresponsiveness to -adrenoceptor agonists in wheezing infants.Supported by a grant from the Ministerium für Wissenschaft und Forschung, NRWPresented at the 19th Workshop for Pediatric Research, University of Göttingen, March 10–11, 1983     

小阴茎、小睾丸的诊断与治疗

小阴茎、小睾丸的病因复杂,临床诊断较为困难。下丘脑-垂体-性腺轴及雄激素合成和转化过程中任何一个环节出现异常,皆会影响阴茎和睾丸的发育。治疗应在明确诊断的基础上予以个体化方案。提倡重视小青春期内的早期诊断,以期早期治疗,使病变的危害降至最低。