RAEB型骨髓增生异常综合征患者DDP10异常表达的研究

目的:检测难治性贫血伴幼稚细胞增多(RAEB)型骨髓增生异常综合征(MDS)患者外周血二肽基肽酶10(DDP10)mRNA的转录水平,探讨其临床意义。方法:利用实时荧光定量PCR方法,分别检测正常对照、急性髓性白血病及RAEB型MDS患者外周血细胞DDP10mRNA的表达量;采集RAEB患者外周血细胞计数并随访其生存时间资料,分析两者与DDP10表达的相关性。结果:RAEB患者外周血DDP10mRNA的表达量显著高于正常对照组及初诊白血病患者(P<0.05),正常对照组与白血病患者之间DDP10表达无统计学差异(P>0.05);RAEB患者总生存时间及外周血细胞数量与外周血DDP10表达量无显著相关性。结论:DDP10在RAEB型MDS患者外周血中的异常高表达,提示DDP10是RAEB的一个特异性标志,但其与患者外周血细胞数量及生存时间无关。     

骨髓增生异常综合征患者T细胞亚型和CD3zeta表达的分析

免疫介导的骨髓抑制和(或)造血异常被认为是导致MDS患者血细胞减少的重要机制之一。本研究旨在通过分析11例骨髓增生异常综合征患者外周血T淋巴细胞亚群及CD3zeta的表达，以进一步评价MDS患者的免疫状态，探讨MDS患者血细胞减少可能的原因。对11例诊断明确的MDS患者，用流式细胞术计数外周血表达IFNγ^ CD4^ 细胞(Th1)、IL4^ CD4^ 细胞(Th2)、IFNγ^ CD8^ 细胞(Tc1)及IL4^ CD8^ 细胞(Tc2)，同时分析CD3zeta在T细胞亚群中的表达。结果显示：CD8^ 细胞在MDS患者外周血中比例明显升高；T细胞亚群Th1／Th2、Tc1／Tc2比值与正常对照无显著差异；T细胞活化信号传导通路上的功能蛋白CD3zeta在CD8^ 细胞中的表达明显增高。结论：MDS患者外周血T细胞亚群比例异常，以CD8^ 细胞增高为主要表现；CD3zeta在CD8^ 细胞中表达明显增高，在CD4^ 细胞中变化不大，提示MDS患者细胞免疫的过度活化主要与CD8^ 细胞有关；T细胞介导的细胞免疫过度活化是MDS患者外周血细胞减少的重要机制之一。     

WT1基因在骨髓增生异常综合征及急性白血病患者中的表达

为了研究WT1基因在骨髓增生异常综合征 (MDS)和急性白血病 (AL)患者中的表达及其与临床预后的关系 ,采用逆转录 聚合酶链反应 (RT PCR)方法检测 2 2例MDS和 6 9例AL的WT1mRNA的表达。结果表明 ,WT1mRNA在MDS RA及MDS RAS中低表达 ,阳性率为 10 % (1/10 ) ;在MDS RAEB和MDS RAEB t中高表达 ,阳性率为 91.7% (11/12 ) ,两者差异有统计学意义 (P <0 .0 1)。WT1mRNA在各型急性白血病中均有表达 ,初发和复发急性白血病中WT1mRNA的阳性率 (6 9% )高于缓解患者 (12 .5 % ) (P <0 .0 1)。初发与复发AL的WT1mRNA的相对表达量无差异 ,而MDS RAEB和RAEB t的表达量低于初发AL患者。WT1mRNA阳性的AML患者的化疗缓解率 (4 1% )低于阴性患者 (78% ) ;WT1基因相对表达量≥ 1的AML患者的CR率 (18% )低于 <1的患者 (5 5 % )。结论 :WT1基因在MDS RAEB和MDS RAEB t中的表达明显高于MDS RA和MDS RAS ,动态监测WT1基因的表达可能有助于监测MDS的病情变化。WT1基因表达与否及表达高低与初发AL的预后有关 ;WT1基因是急性髓细胞白血病的一个独立的预后因素。     

axl基因在骨髓增生异常综合征患者外周血细胞的表达

为研究axl基因在骨髓增生异常综合征(MDS)及其它几种血液病患者外周血单个核细胞中的表达,用RT-PCR方法测定细胞中axl基因mRNA的表达情况。结果表明:①10/11例(91%)MDS患者和1/2例AML-M_3型患者有axl基因表达。2例再生障碍性贫血患者、2例球形红细胞增多症患者、2例正常人均无axl基因表达。②FAB分型与axl基因表达关系:5例RA有表达,其中强表达2例,弱表达3例;4例RAEB均有表达,其中强表达1例,弱表达3例;1例RAEB-T为强表达。以上结果说明,axl基因在MDS患者中有很高的表达率,提示其可能作为MDS患者的诊断指标之一。     

Clec2在骨髓增生异常综合征患者中的表达及其与TPO的相关性

目的:探讨clec2在骨髓增生异常综合征(MDS)患者中的表达,并分析clec2与TPO的相关性。方法:应用ELISA方法检测47例MDS患者及20例正常人血浆中clec2的表达,分析MDS患者临床特征与clec2表达的相关性;同时检测42例MDS患者血浆TPO的表达水平,并分析clec2与TPO的相关性。结果:Clec2在正常人中表达水平为171.5±57.6 ng/ml,在MDS患者中的表达水平为347.9±121.5 ng/ml(P0.01)。其中初治MDS患者clec2表达水平为375.4±124.3 ng/ml,复治患者的表达水平为288.6±98.7 ng/ml(P0.05)。Clec2与患者性别、年龄、外周血细胞数、骨髓原始细胞数均无相关性。进一步分析发现,clec2与TPO存在正相关关系(r=0.419)。结论:Clec2在MDS患者中表达增高,且与TPO呈正相关,2者相互作用可能在MDS疾病的发生、发展中起着重要作用,其可能是该病的新的分子机制及靶向治疗机制。     

U2AF1突变对骨髓增生异常综合征预后及治疗影响的研究现状

骨髓增生异常综合征(MDS)是一组起源于骨髓造血干/祖细胞的恶性克隆性疾病, 其主要特征是骨髓一系或多系病态造血, 外周血细胞减少, 并且向急性髓细胞白血病(AML)转化风险极高。MDS发病机制与体细胞基因突变、表观遗传学改变、免疫系统与骨髓微环境调节异常等因素密切相关。多项研究表明, 基因突变是MDS患者预后的影响因素, 但是目前单基因突变并未纳入MDS预后评分系统。MDS患者的U2AF1突变率较高, 其作用机制主要为影响RNA剪接体对前体信使RNA(pre-mRNA)的3′剪接位点(SS)识别过程, 进而生成异常mRNA, 造成染色体不稳定。笔者拟就近年U2AF1突变对MDS的预后影响和治疗进展进行阐述, 为探索伴U2AF1突变MDS患者的新治疗方向提供理论依据。     

MDS患者端粒相关蛋白TRF1基因的表达

目的探讨骨髓增生异常综合征(myelodysplastic syndrome,MDS)患者骨髓端粒相关蛋白TRF1 mRNA的表达水平,以研究其在MDS发生发展中的作用。方法以实时定量RT-PCR方法,检测24例MDS患者和8例对照骨髓TRF1 mRNA表达水平,以SPSS11.5软件进行统计分析。结果MDS患者骨髓均表达TRF1,初诊低危组和高危组TRF1 mRNA的表达均升高(P<0.005)。随着病情的进展,TRF1表达升高(P<0.005);随着病情的改善,TRF1表达降低(P<0.01)。结论TRF1 mRNA随着病情变化在MDS患者骨髓的表达改变,提示TRF1作为端粒长度的负性调节因子,是揭示MDS预后的不良因素之一。     

Evi-1反义寡核苷酸对人红白血病细胞系HEL的抑制作用

Evi 1(ecotropicvirusintegrationsite 1)定位于染色体3q2 6 ,为一原癌基因 ,与髓系恶性肿瘤的发生密切相关[1 ] 。Evi 1基因编码含两个锌指区的蛋白 ,能特异地结合基因启动子DNA序列 ,发挥转录调节作用[2 ] 。正常人骨髓和外周血细胞Evi 1mRNA表达阴性[1 ] 。我们的初步研究结果表明 ,近 5 0 %的骨髓增生异常综合征 (MDS)患者Evi 1基因表达阳性 ,Evi 1的表达水平与MDS向急性髓系白血病 (AML)演变有关[3,4 ] 。为进一步探讨Evi 1在AML发生和发展中可能的作用 ,我们观察了Evi 1反义寡核苷酸 (Evi 1 AS)对人红白血病细胞系HEL增…     

骨髓增生异常综合征向急性白血病转化相关影响因素的研究

目的探讨影响骨髓增生异常综合征(MDS)向急性白血病转化的相关因素。方法动态观察129例MDS患者的转白情况,并对其临床特征及实验室检查进行分析以探讨相关影响因素。结果 129例MDS患者中33例转化为急性白血病,中位转白时间7(3~26)个月。外周血三系血细胞减少组的转白率明显高于非三系血细胞减少组(P<0.05)。骨髓原始细胞比例越高转白率明显升高(P<0.05)。染色体核型预后好、中、差组转白率依次升高(P<0.05)。有AUER小体明显高于无AUER小体组(P<0.05)。病态造血累及系数越多转白率越高(P<0.05)。MDS分型与转白有相关性(P<0.05)。结论 MDS是一类高风险向急性白血病转化的疾病,MDS分型、外周血细胞减少、骨髓原始细胞比例高低、染色体核型异常、病态造血及AUER小体影响MDS转化为白血病。     

伴骨髓纤维化的骨髓增生异常综合征(MDS)作为急性骨髓纤维化一种主要的原发性疾患

为了阐明MDS 合并骨髓纤维化(MF)的意义和说明该病与同类疾病特别是急性MF 与巨核细胞白血病的关系,在87例MDS 患者中作者就7例(8%)符合伴MF 的MDS 患者作了分析。伴MF 的MDS(MF-MDS)诊断标准为:(1)全血细胞减少,外周血原始细胞<5%;(2)轻度或无脾大;(3)骨髓有核细胞增生正常的骨髓纤维化;(4)骨髓内无原始细胞的弥漫性增生;(5)骨髓象和外周血细胞存在增生异常的特征。7例MF-MDS 患者年龄54～82岁,男性3例,女性4例。多数患者主诉贫血,7例均无脾大。1例有肝大,1例淋巴结稍肿大。7例患者中6例全血细胞减低,外周血原始细胞<1%,1例贫血和血小板减少.外周血原始细胞2.5%。3例外周血可见有核红细胞。骨髓中原始细胞1例为0.2～     

初发免疫性血小板减少症患者外周血中维生素D及其受体mRNA的表达水平和意义

为了探讨维生素D（VD）对初发免疫性血小板减少症（ITP）发病与治疗的作用,采用酶联免疫吸附试验（ELISA）检测45例初发ITP患者和30例健康体检者外周血中25羟基维生素D3[25（OH）D3]和1,25-二羟基D3[1,25（OH）2D3]的浓度,利用逆转录一聚合酶链反应（RT-PCR）法检测患者外周血VD受体（VDR）mRNA表达水平。研究结果表明,ITP患者外周血中25（OH）D3（10.6±7.7 ng/ml）和1,25（OH）2D3（69.9±29.0 pg/ml）的浓度明显低于健康对照组（13.7±9.1 ng/ml,87.3±19.9 pg/ml）（P〈0.05）,而VDR mRNA的表达（1.588±0.127）明显高于健康对照组（1.055±0.734）（P〈0.05）。结论：VD水平及其受体的表达异常可能参与了ITP的发病,VD及类似物可用于ITP的治疗。     

维生素D及其受体在强直性脊柱炎患者中的表达及其临床意义

目的：研究As患者PBMC维生素D受体（VDR）mRNA的表达及血清25-羟维生素D,和1,25-二羟维生素D3的水平,探讨其与AS疾病活动性（BASDAI、CRP、ESR）的相关性。方法：选取26例AS患者（As组）和年龄、性别与之相匹配的13名健康志愿者（健康对照组）。采用SYBRGreenI实时荧光定量PCR检测两组受检者PBMC的VDRmRNA表达水平,应用ELISA法检测两组受检者血清25-羟维生素D3和1,25-二羟维生素D3水平,分析VDRmRNA表达水平、血清25-羟维生素D3和1,25-二羟维生素D3水平与临床相关指标（BASDAI、CRP、ESR）的关系。结果：As患者PBMC的VDRmRNA表达水平明显高于健康对照组（P〈0．01）,VDRmRNA表达水平与临床相关指标（BASDAI、CRP、ESR）无关（P〉0．05）。AS患者血清25．羟维生素D3、1,25-二羟维生素D3水平分别为（5．3±2．6）μg／L、（12．8±6．0）ng／L,明显低于健康对照组（14．7±3．5）μg/L、（32．6±18．5）ng／L（P均〈0．01）。AS患者血清1,25-二羟维生素D3的水平与BASDAI（r=-0．481,P〈0．05）、ESR（r=-0．535,P〈0．01）、CRP（r=-0．674,P〈0．01）均呈负相关。血清25-羟维生素D,水平与BASDAI、CRP、ESR无关（P〉0．05）。结论：As患者VDRmRNA表达水平升高,但与As的疾病活动无关。As患者血清1,25-二羟维生素D3水平下降,与疾病活动呈负相关,可作为AS疾病活动的指标之一。AS患者PBMC的VDR活化可能与1,25-二羟维生素D,的作用无关。     

Identification and regulation of 1,25-dihydroxyvitamin D3 receptor activity and biosynthesis of 1,25-dihydroxyvitamin D3. Studies in cultured bovine aortic endothelial cells and human dermal capillaries

Because 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has been shown to play roles in both proliferation and differentiation of novel target cells, the potential expression of 1,25(OH)2D3 receptor (VDR) activity was investigated in cultured bovine aortic endothelial cells (BAEC). Receptor binding assays performed on nuclear extracts of BAEC revealed a single class of specific, high-affinity VDR that displayed a 4.5-fold increase in maximal ligand binding (Nmax) in rapidly proliferating BAEC compared with confluent, density-arrested cells. When confluent BAEC were incubated with activators of protein kinase C (PKC), Nmax increased 2.5-fold within 6-24 h and this upregulation was prevented by sphingosine, an inhibitor of PKC, as well as by actinomycin D or cycloheximide. Immunohistochemical visualization using a specific MAb disclosed nuclear localized VDR in venular and capillary endothelial cells of human skin biopsies, documenting the expression of VDR, in vivo, and validating the BAEC model. Finally, additional experiments indicated that BAEC formed the 1,25(OH)2D3 hormonal metabolite from 25(OH)D3 substrate, in vitro, and growth curves of BAEC maintained in the presence of 10(-8) M 1,25(OH)2D3 showed a 36% decrease in saturation density. These data provide evidence for the presence of a vitamin D microendocrine system in endothelial cells, consisting of the VDR and a 1 alpha-hydroxylase enzyme capable of producing 1,25(OH)2D3. That both components of this system are coordinately regulated, and that BAEC respond to the 1,25(OH)2D3 hormone by modulating growth kinetics, suggests the existence of a vitamin D autocrine loop in endothelium that may play a role in the development and/or functions of this pathophysiologically significant cell population.     

骨髓增生异常综合征淋巴细胞亚群及其激活状态的分析

本研究探讨骨髓增生异常综合征（MDS）患者外周血T细胞亚群，B细胞，NK细胞数量及激活状态的临床意义．采用流式细胞术对30例MDS患者外周血T细胞，B细胞，NK细胞及其表面活化分子CD28，CD45RA，CD45RO，CD69，HLA—DR的表达进行检测和分析。30例MDS患者中低危组（RA＋RAS）22例，高危组（RAEB＋RAEBT）8例。结果表明：MDS组T细胞（CD3^＋细胞）的百分率低于正常对照组，CD4^＋CD45RA^＋细胞（未致敏CD4^＋细胞）的数值低于正常对照组。MSD组早期活化T细胞（CD3^＋CD69^＋细胞）和晚期活化T细胞（CD3^＋HLA—DR^＋细胞）的数值均显著高于正常对照组。低危组（RA和RAS）主要表现为T细胞活化功能的改变：早期活化T细胞（CD3^＋CD69^＋细胞）和晚期活化T细胞（CD3^＋HLA—DR^＋细胞）比例均增高，以及B细胞数量的减少。高危组（RAEB和RAEBT）主要表现为T细胞亚群数量的改变：CD3^＋细胞，CD3^＋CD4^＋CD8^-细胞（T辅助细胞）数量的减少，CD3^＋细胞HLA—DR和CD69的表达并不增高。NK细胞数量减少。结论：MDS病人存在有T细胞数量和功能的异常，且随着病情的进展而发生改变，所以T细胞亚群及活化功能的检测对于判断疾病的进程和指导治疗具有重要的意义。     

Vitamin D receptor binding to DNA is altered without the change in its expression in human renal clear cell cancer

Vitamin D co-regulates cell proliferation, differentiation and apoptosis, the processes that are disturbed in cancer tissues. It acts through the vitamin D nuclear receptor (VDR) that binds to DNA in the regulatory sequences of the target genes. As the kidney is one of the key organs for vitamin D metabolism and action, we analyzed VDR expression and its DNA binding activity in human renal clear cell cancer. 24 tumors, 24 controls that were excised from the opposite pole of the same kidney and 7 controls originating from kidneys without cancer were examined. Independently of tumor grading neither Northern blots nor immunoblotting demonstrated statistically significant differences of the mean VDR mRNA and protein amounts, respectively, in the cancer as compared to both control types. In contrast, the amount of VDR-DNA complexes was lower in 52.2% of the tumors in comparison to their corresponding controls. After normalization against VDR receptor protein amount in 34.8% of the tumors VDR-DNA binding was at least 3-4 times weaker than in the controls. However, the expression of vitamin D-dependent P21 gene on the mRNA level was not decreased in these cancers. It remains to be elucidated if altered VDR function due to its impaired binding to DNA contributes to the process of tumorigenesis, and what potential vitamin D-dependent mechanisms are involved in this process.     

Mineralization of three‐dimensional osteoblast cultures is enhanced by the interaction of 1α,25‐dihydroxyvitamin D3 and BMP2 via two specific vitamin D receptors

1α,25‐Dihydroxyvitamin D3 [1α,25(OH)2D3] and bone morphogenetic protein‐2 (BMP2) are both used to stimulate osteoblastic differentiation. 1α,25(OH)2D3 regulates osteoblasts through classical steroid hormone receptor mechanisms and through rapid responses that are mediated by two receptors, the traditional vitamin D receptor (VDR) and protein disulphide isomerase family A member 3 (Pdia3). The interaction between 1α,25(OH)2D3 and BMP2, especially in three‐dimensional (3D) culture, and the roles of the two vitamin D receptors in this interaction are not well understood. We treated wild‐type (WT), Pdia3‐silenced (Sh‐Pdia3) and VDR‐silenced (Sh‐VDR) pre‐osteoblastic MC3T3‐E1 cells with either 1α,25(OH)2D3, or BMP2, or with 1α,25(OH)2D3 and BMP2 together, and measured osteoblast marker expression in 2D culture and mineralization in a 3D poly(ε‐caprolactone)–collagen scaffold model. Quantitative PCR showed that silencing Pdia3 or VDR had a differential effect on baseline expression of osteoblast markers. 1α,25(OH)2D3 + BMP2 caused a synergistic increase in osteoblast marker expression in WT cells, while silencing either Pdia3 or VDR attenuated this effect. 1α,25(OH)2D3 + BMP2 also caused a synergistic increase in Dlx5 in both silenced cell lines. Micro‐computed tomography (μCT) showed that the mineralized volume of untreated Sh‐Pdia3 and Sh‐VDR 3D cultures was greater than that of WT. 1α,25(OH)2D3 reduced mineral in WT and Sh‐VDR cultures; BMP2 increased mineralization; and 1α,25(OH)2D3 + BMP2 caused a synergistic increase, but only in WT cultures. SEM showed that mineralized matrix morphology in 3D cultures differed for silenced cells compared to WT cells. These data indicate a synergistic crosstalk between 1α,25(OH)2D3 and BMP2 toward osteogenesis and mineral deposition, involving both VDR and Pdia3. Copyright © 2013 John Wiley & Sons, Ltd.     

Steroid and xenobiotic receptor and vitamin D receptor crosstalk mediates CYP24 expression and drug-induced osteomalacia

The balance between bioactivation and degradation of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] is critical for ensuring appropriate biological effects of vitamin D. Cytochrome P450, family 24-mediated (CYP24-mediated) 24-hydroxylation of 1,25(OH)2D3 is an important step in the catabolism of 1,25(OH)2D3. The enzyme is directly regulated by vitamin D receptor (VDR), and it is expressed mainly in the kidney, where VDR is also abundant. A recent report suggests that activation of steroid and xenobiotic receptor (SXR) also enhances the expression of CYP24, providing a new molecular mechanism of drug-induced osteomalacia. However, here we showed that activation of SXR did not induce CYP24 expression in vitro and in vivo, nor did it transactivate the CYP24 promoter. Instead, SXR inhibited VDR-mediated CYP24 promoter activity, and CYP24 expression was very low in tissues containing high levels of SXR, including the small intestine. Moreover, 1,25(OH)2D3-induced CYP24 expression was enhanced in mice lacking the SXR ortholog pregnane X receptor, and treatment of humans with the SXR agonist rifampicin had no effect on intestinal CYP24 expression, despite demonstration of marked CYP3A4 induction. Combined with our previous findings that CYP3A4, not CYP24, plays the dominant role in hydroxylation of 1,25(OH)2D3 in human liver and intestine, our results indicate that SXR has a dual role in mediating vitamin D catabolism and drug-induced osteomalacia.