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目的探讨糖皮质激素受体α(GRα)和糖皮质激素受体β(GRβ)在原发性血小板减少性紫癜(ITP)糖皮质激素抵抗中的分子生物学机制。方法选择2003-01—2005-12在深圳市儿童医院血液内科住院的ITP患儿30例,将患儿分为2组:激素敏感组(18例)和激素抵抗组(12例),另选10例正常儿童作对照。应用RT-PCR法检测激素敏感组、激素抵抗组ITP患儿及10例正常儿童外周血单个核细胞GRα和GRβmRNA的表达水平,以探讨GRα和GRβ与ITP糖皮质激素抵抗的关系。结果(1)对照组、激素敏感组和激素抵抗组3组之间GRαmRNA的表达[分别为(0.75±0.17)、(0.70±0.15)、(0.73±0.16)]差异无显著性,P>0.05;(2)对照组和激素敏感组的GRβmRNA[分别为(0.13±0.03)、(0.15±0.04)]差异亦无显著性,P>0.05,但激素抵抗组的GRβmRNA水平[为(0.39±0.12)]显著高于对照组和激素敏感组,P<0.01,差异具有统计学意义。结论ITP患儿糖皮质激素抵抗可能与GRβmRNA的表达亢进有关。     

糖皮质激素受体表达与特发性血小板减少性紫癜激素抵抗关系

目的:通过研究原发性糖皮质激素抵抗型特发性血小板减少性紫癜（ITP）患儿糖皮质激素受体（GR）亚型GRα和GRβ表达水平,以揭示ITP糖皮质激素抵抗的可能机制。方法:应用实时荧光定量-PCR法检测18 例糖皮质激素敏感型、12例糖皮质激素抵抗型ITP患儿及10例正常儿童外周血单个核细胞GRα和GRβ mRNA 的表达水平,同时采用酶免疫分析法测定血清总皮质醇水平。结果:①对照组、糖皮质激素敏感组和抵抗组3组之间GRα mRNA的表达水平差异无显著性;对照组和糖皮质激素敏感组的GRβ mRNA水平差异无显著性,但糖皮质激素抵抗组的GRβ mRNA水平显著高于对照组和糖皮质激素敏感组,差异有显著性（P<0.01）。②总皮质醇水平在对照组与糖皮质激素敏感组之间差异无显著性,但糖皮质激素抵抗组总皮质醇水平明显高于糖皮质激素敏感组,差异有显著性（P<0.01）。结论: ITP患儿糖皮质激素抵抗可能与GRβ mRNA的表达增高有关。［中国当代儿科杂志,2009,11（9）：714-716］     

GRβ在原发性肾病综合征患儿糖皮质激素耐药中的作用

目的　研究原发性肾病综合征患儿PBMCsGRβ表达水平与肾组织中原位GRβ表达水平的关系及其在PNS患儿糖皮质激素耐药中的作用。方法　40例原发性肾病综合征患儿分成激素敏感组与激素耐药组,每组各 20例,应用免疫组织化学技术,对其肾活检组织与PBMCs中GRα、GRβ进行检测,应用半定量评分法对各组肾病患儿肾小球与肾小管间质病理改变进行半定量分析。结果　激素耐药组肾小球病理积分与肾小管病理积分高于激素敏感组 (P均 <0.01);激素敏感组与激素耐药组原发性肾病综合征患儿肾活检组织GRα与PBMCs中GRα表达低于正常对照组 (P均 <0.01);激素敏感组与激素耐药组原发性肾病综合征患儿肾活检组织GRβ与PBMCs中GRβ表达高于正常对照组(P均<0.01);中度损害组肾活检组织与PBMCsGRβ表达高于轻度损害组,重度损害组高于中度损害组(P均<0.01);相关分析表明:原发性肾病综合征患儿PBMCs和肾固有细胞GRβ表达明显正相关(r=0. 651; P<0.01), PBMCs和肾固有细胞中GRβ表达与肾脏病理积分正相关(r=0.579, 0.623,P均<0.01)。结论　原发性肾病综合征患儿肾局部、PBMCs中GRβ过表达与PNS患儿糖皮质激素耐药发生有关,与PBMCs和肾局部GRα数目的多少无关,过表达的GRβ可能是通过拮抗GRα的活性影响激素效应的发挥。     

糖皮质激素受体表达水平与急性淋巴细胞白血病诱导治疗疗效的相关性

目的探讨在儿童急性淋巴细胞性白血病（ALL）患儿中进行胞浆糖皮质激素受体（GR）检测及其与治疗效果的相关性,探讨GR能否作为判断儿童ALL预后的因素。方法利用GR的单克隆抗体,通过流式细胞仪检测48例初治ALL患儿的骨髓液（起病时和疾病获缓解时）和15例随机长期持续缓解（CCR）状态下ALL患儿骨髓液及30例正常儿童外周血液单个核细胞胞浆GR表达水平,分析其与疾病疗效的关系。并对前期实验组进行临床追踪,分析前期实验数据与CCR或复发的关系。结果初发ALL患儿起病时GR与治疗后是否获得缓解（CR）无相关性（P=0.56）;起病时GR表达水平与CR时GR表达水平无统计学差异（P=0.10）;与疾病缓解时的骨髓微小残留病（MRD）指标无相关性（P=0.54）。患儿CR时其GR与正常对照组GR表达水平无统计学差异（P=0.21）。对前期实验组追踪结果显示,起病时GR表达水平与CCR或疾病复发不存在统计学差异（P=0.45）;15例CCR患儿GR表达水平,与正常对照组比较,二者无统计学差异（P=0.28）。结论ALL患儿起病时其基础水平胞浆GR表达与治疗后缓解率、缓解时MRD水平、疾病长期持续缓解或复发无相关性,不能作为独立判断疾病的预后因素。而ALL患儿缓解及长期持续缓解时其胞浆GR与正常儿童GR表达水平无差异,故动态监测胞浆GR表达水平意义不大。     

原发性肾病综合征患儿外周血GRα、GRβ的表达及临床意义

目的探讨糖皮质激素受体α(GRα)和β(GRβ)在原发性肾病综合征(PNS)中的作用及其介导耐药的可能机制。方法选择15例糖皮质激素(GC)敏感型PNS(SSNS)患儿和15例GC耐药型PNS(SRNS)患儿,以及10例健康对照儿童,应用逆转录-聚合酶链式反应(RT-PCR)检测各组外周血单个核细胞(PBMC)中GRα和GRβ mRNA的表达,并分析其与24 h尿蛋白定量(24 hUTP)及肾脏病理积分的关系。结果 PNS患儿PBMC中GRα和GRβ均有表达,并以GRα为主;三组间GRα mRNA的表达无差异(P>0.05);而SRNS组GRβ mRNA、GRα/GRβ水平均高于SSNS和对照组(P均<0.05)。GRβ mRNA的表达与24 hUTP和病理积分呈正相关(P<0.05),即GRβ mRNA的表达越高,病理积分越高,病理损伤越大。结论 GRβ mRNA可以作为监测PNS患儿病情以及预后的指标。针对SRNS患儿PBMC中GRβ升高,增加激素受体敏感性,有望为SRNS的治疗提供新的思路。     

特发性血小板减少性紫癜患儿糖皮质激素受体研究

目的探讨糖皮质激素受体(GR)在特发性血小板减少性紫癜(ITP)儿童外周血淋巴细胞的表达与临床意义。方法采用流式细胞术分别测定17例急性ITP、23例慢性ITP和20例正常儿童细胞内受体(iGR)与膜受体(mGR)水平。结果ITP组mGP和iGR水平均高于正常儿童组(P<0.01),急性ITP组和慢性ITP组间mGR差异无显著性(P>0.05),急性ITP组iGR水平高于慢性组(P<0.05)。结论mGR、iGR均可能在ITP的发病机制中起重要作用,iGR的表达可能更重要;iGR低者更易发展成慢性患者,iGR水平可作为预测ITP预后的一项参考指标。     

Reduced folate carrier and dihydrofolate reductase expression in acute lymphocytic leukemia may predict outcome: a Children's Cancer Group Study

PURPOSE: Methotrexate is a major component of current treatment regimens for children with acute lymphocytic leukemia (ALL). Potential mechanisms of methotrexate resistance include impaired drug uptake, decreased drug retention, and dihydrofolate reductase (DHFR) amplification. The purpose of this study was to assess whether reduced folate carrier (RFC) and DHFR expression in untreated leukemic blasts correlated with outcome. METHODS: Quantitative real-time RT-PCR was used to measure RFC and DHFR mRNA expression in leukemic blasts from 40 newly diagnosed patients with ALL obtained in a blinded fashion from Children's Cancer Group studies. RESULTS: Low RFC expression at diagnosis correlated significantly with an unfavorable event free survival. Surprisingly, low, not high, DHFR expression correlated significantly with an unfavorable event-free survival. Proliferative cell nuclear antigen (PCNA) expression demonstrated a weak inverse relationship between sample PCNA and DHFR or RFC expression, suggesting that DHFR and RFC expression may be markers for factors other than drug resistance. CONCLUSIONS: These results suggest that impaired transport may be an important mechanism of intrinsic methotrexate resistance in ALL, and DHFR expression also may be an important prognostic factor in ALL. Additional studies are necessary to clarify the mechanism for the correlation of low DHFR expression with poor outcome.     

Glucocorticoid receptors in acute lymphoblastic leukemia in children]

Glucocorticoid receptor (GR) levels were quantitated in leukemic blasts from bone marrow aspirates of 53 children with acute lymphoblastic leukemia (ALL) and compared to the clinical and immunological features of the disease and its prognosis. GR levels did not correlate with the patients' sex, peripheral white blood cell count and cellular immunophenotype, but depended on the patients' age and histological type of leukemia. 43 patients were monitored during 3 or more years. The relapse-free and overall survival of ALL patients receiving standard courses of polychemotherapy including glucocorticoids was highly dependent on the GR level in bone marrow blasts. The best prognosis (67% relapse-free and 73% overall 3-years' survival) was observed in patients with GR levels over 6,000 sites/cell and patients with GR levels under 3,000 sites/cell had poor prognosis (31% relapse-free and 50% overall survival; median 1st remission duration 12 months). These correlations were most pronounced in ALL patients with "common" antigen.     

p38丝裂原活化蛋白激酶调节急性淋巴细胞性白血病CEM细胞株糖皮质激素受体功能的分子机制研究

目的探讨p38丝裂原活化蛋白激酶（p38MAPK）对地塞米松（DEX）诱导急性淋巴细胞性白血病CEM细胞株凋亡过程中糖皮质激素受体α（GRα）功能的影响。方法台盼蓝拒染法绘制增殖曲线;流式细胞仪解析细胞凋亡;Western blot检测糖皮质激素受体蛋白及磷酸化p38MAPK蛋白表达。结果（1）p38MAPK特异阻滞剂SB203580预处理后继续培养24-72h,细胞的增殖率分别由单用DEX时的62．3％、35．5％、11．6％升至82．8％、54．7％和48．1％（P〈0．01）;（2）5μmol／LDEX处理CEM细胞36h时,亚二倍体细胞为26．2％,显著高于对照组（P〈0．01）。SB203580预处理后,亚二倍体细胞由26．2％降至7．1％（P〈0．01）;（3）5μmol／L的DEX处理CEM细胞后,GRα总蛋白的表达水平从12h开始分别上调至117％（12h）、121％（24h）、122％（36h）、125％（48h）。未与DEX结合的GRα主要存在于细胞浆中,细胞核GRα与细胞浆GRα之比为0．27。从6-48h,GRα的胞核与胞浆之比分别为0．48、0．59、0．95、2．16、4．08;（4）5μmol／LDEX处理CEM细胞后,p-p38MAPK蛋白在15min时即隐约可见,1h开始表达增强并持续增强至6h,之后减弱至48h仍可见。SB203580预处理后,未检测到p-p38MAPK蛋白的表达;（5）SB203580预处理后,GRα的核浆比例由4．08降至0．43（P〈0．05）,GRα总蛋白无明显变化。结论DEX在诱导CEM细胞凋亡时上调GRα的蛋白表达水平并促进转位人核。p38MAPK信号转导通路的活化促进了GRα转位人核。     

Clinical significance of glucocorticoid receptors in acute leukaemia. Preliminary observations in Hungary and review of the literature

Glucocorticoid receptor (GR) levels were quantitated in leukaemic blast cells separated from peripheral blood of 15 children with acute lymphocytic leukaemia (ALL) and of 6 children with acute non-lymphocytic leukaemia (ANLL). Using a whole-cell assay, it was found that specific (3H)-dexamethasone binding exhibited a wide range in both types of acute leukaemia: GR levels scattered between 0-22,346/cell and 0-8772/cell in ALL and ANLL patients, respectively. In this paper we discuss our observations together with current knowledge on GR levels in leukaemic blast cells and their relationship to glucocorticoid sensitivity and disease outcome.     

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目的探讨选用不同中效糖皮质激素(GC)诱导缓解治疗儿童原发性肾病综合征(PNS)的疗效。方法 2008年11月至2010年2月于天津市儿童医院住院治疗的初治PNS患儿54例,随机分为泼尼松组、曲安西龙组、甲泼尼龙组,3组分别给予相应足量激素治疗。动态监测各组24h尿蛋白定量(Upro)、血浆白蛋白(Alb)、血浆胆固醇(Tcho)的变化,同时记录服用GC后尿蛋白转阴的时间。采用半定量逆转录-聚合酶链反应(RT-PCR)方法检测患儿外周血单个核细胞(PBMCs)中糖皮质激素受体(GR)GRα和GRβ的mRNA表达水平。结果甲泼尼龙对24h Upro的降低和血浆Alb的升高作用优于曲安西龙和泼尼松,尿蛋白转阴时间甲泼尼龙组及曲安西龙组较泼尼松组短,甲泼尼龙上调GRα的能力优于曲安西龙及泼尼松,而甲泼尼龙及曲安西龙抑制GRβ表达亢进的能力优于泼尼松。结论服用GC后,尿蛋白的转阴时间、Upro与血Alb的动态变化、GR的水平均可作为PNS诱导缓解期综合评价GC疗效的有价值的客观指标。初治儿童PNSGC诱导缓解的治疗阶段选用甲泼尼龙或曲安西龙的疗效优于泼尼松。     

Decreased sensitivity of cytostatic drugs in glucocorticoid receptor-free acute myeloid leukaemia cells. Clinical and experimental observations.

Preliminary clinical observations have suggested that low cellular glucocorticoid receptor (GR) levels might have been connected with multidrug resistance in children with acute myeloblastic leukaemia (AML). To test this possibility, we have developed glucocorticoid resistant subclones of two recently established human myeloid leukaemic cell lines. The cause of glucocorticoid resistance was GR negativity in these subclones. GR positive parent cell lines or GR negative subclones were incubated for 1 h in the presence of Adriamycin, Cytosine-arabinosid, Etoposide or Vincristine, respectively. After short-term (1 h) incubation in suspension cultures cells were washed and plated in clonogenic agar cultures. Each anticancer drug was more potent against both GR positive parent cell lines than against the GR negative subclones. The results of this study suggest that the absence of GRs is a useful marker of multidrug resistance in childhood AML.     

Transgenic glucocorticoid receptor expression driven by the SP-C promoter reduces neonatal lung cellularity and midkine expression in GRhypo mice

BACKGROUND: Congenital truncation of the glucocorticoid receptor (GR) is known to lead to lethal lung immaturity in newborn mice associated with increased lung cellularity (ratio of tissue to airspace) and, as we previously showed, prolonged expression of the retinoid-responsive growth factor midkine. OBJECTIVES: We sought to determine if these changes would be reversed by transgenic expression of GR exclusively in the distal airway epithelium. METHODS: Mice were generated with expression of transgenic rat (r) GR driven by the human (h) SP-C promoter, on a background of congenital GR truncation. RESULTS: Transgenic epithelial GR expression reduced lung cellularity and midkine expression to levels comparable to wild-type littermates. Nevertheless, the newborn transgenic mice still displayed respiratory failure. Moreover, epithelial expression of the GR transgene did not alter expression of a number of important markers of lung maturation. CONCLUSIONS: Our data demonstrating normalization of the lung tissue to airspace ratio in neonatal mice expressing transgenic GR in the distal airway epithelium is consistent with the concept that normal mesenchymal cell loss is due to GR-responsive stimulation from epithelial cells. However, we could find no evidence of altered apoptotic activity between the groups of mice. We speculate that correction of the severe neonatal lung phenotype of GR-deficient mice will require expression of normal GR in non-epithelial as well as epithelial tissues.     

特应性体质对毛细支气管炎患儿血清糖皮质激素受体水平的影响

目的探讨特应性体质(简称特应质)对毛细支气管炎(简称毛支)患儿糖皮质激素受体(GR)表达的影响。方法采用ELISA法测定和比较毛支组(77例,包括34例特应质患儿)和肺炎组(68例)患儿血清中GRα、GRβ水平的变化。38例同期住院的非感染性小儿外科术前患儿,且无特应性及过敏性疾病家族史的患儿作为对照组。结果与对照组比较,毛支组和肺炎组中血清GRα、GRβ水平均明显升高(P0.01);毛支组血清GRα、GRβ水平高于肺炎组(P0.01)。毛支组GRα/GRβ比值较对照组和肺炎组明显升高(P0.01)。毛支组特应质和非特应质患儿血清GRα、GRβ水平与对照组比较均明显升高(P0.01);毛支组非特应质患儿GRβ水平明显高于特应质患儿(P0.01);毛支组特应质患儿GRα/GRβ比值较对照组和毛支组非特应质患儿明显升高(P0.01)。结论毛支患儿血清GRα、GRβ表达水平增强;特应质毛支患儿表现为GRα/GRβ比值增加,提示特应质毛支患儿对糖皮质激素有较高的敏感性。     

儿童急性淋巴细胞白血病抗原错译表达的临床意义

目的 研究儿童急性淋巴细胞白血病(ALL)抗原错译表达的临床意义。方法 对54例ALL患儿的骨髓标本分别进行细胞形态学及细胞化学染色，确定其FAB类型，运用一组相关的单克隆抗体，采用流式细胞仪及直接免疫荧光标记技术进行免疫分型，采用吉姆萨G显带技术进行核型分析。结果 儿童ALL54例髓系抗原阳性表达率为29.63％，其中CD13为25．93％，CD33为20．37％，CD14为11．11％。T—ALL和B—ALL髓系抗原阳性表达差异无统计学意义(30．77％vs529．27％ P＝0.918)。CD34表达阳性ALL髓系抗原阳性表达率明显高于CD34表达阴性ALL髓系抗原阳性表达率(40.63％vs13.64％P＝0．039)；ALL髓系抗原阳性表达与髓系抗原阴性表达的完全缓解(CR)率差异无统计学意义(81．25％vs94．74％ P＝0．148)；但生存率曲线比较分析，ALL髓系抗原阳性表达的生存时间短(P＝0.031)。结论 儿童ALL的抗原错译表达率为29．63％；CD34阳性的ALL抗原错译表达率明显高于CD34表达阴性的ALL抗原错译表达率；ALL髓系抗原阳性表达的生存时间短；ALL髓系相关抗原阳性表达可能是影响ALL患儿预后的不利因素。     

糖皮质激素受体在Perthes病股骨头软骨及髋关节滑膜组织中的表达及意义

目的 探讨糖皮质激素及糖皮质激素受体在Perthes病发病机制中的作用.方法 用免疫组织化学方法检测20例Perthes病和10例非Perthes病(3例外伤致髋关节离断及7例尸体标本)的小儿股骨头软骨及髋关节滑膜组织中糖皮质激素受体的表达,并做图像分析.结果 糖皮质激素受体在20例Perthes病股骨头软骨中均有表达,对照组10例股骨头软骨中,仅有1例有糖皮质激素受体表达,两者表达率比较,差异有统计学意义(χ~2=21.607,P<0.01);糖皮质激素受体在20例Perthes病髋关节滑膜中均有表达,对照组10例髋关节滑膜中,仅有1例有糖皮质激素受体表达,两者表达率比较,差异有统计学意义(χ~2=17.919,P<0.01).Perthes病组和对照组股骨头软骨的糖皮质激素受体阳性颗粒积分光密度值比较,差异有统计学意义(t=5.893,P<0.01);Perthes病组和对照组髋关节滑膜的糖皮质激素受体阳性颗粒积分光密度值比较,差异有统计学意义(t=11.720,P<0.01).糖皮质激素受体在Perthes病股骨头软骨与髋关节滑膜中表达呈正相关关系(r=0.787,P<0.01).结论 糖皮质激素受体可能与Perthes病的发生、发展有关.     

BIM基因与儿童急性淋巴细胞白血病对糖皮质激素耐药的相关性

急性淋巴细胞白血病(ALL)是儿童时期最常见的恶性血液系统疾病。糖皮质激素作为ALL化疗方案中的主要用药,可通过多种途径诱导白血病细胞的凋亡,但仍有约10%的儿童ALL对糖皮质激素不敏感。研究发现糖皮质激素通过上调BIM基因表达介导ALL细胞的凋亡,BIM基因与儿童ALL对糖皮质激素耐药有关。本综述概括了近年关于儿童急性淋巴细胞白血病对糖皮质激素耐药相关研究,主要包括BIM基因及其表达产物在该过程中的作用。     

无镁对体外原代培养胎鼠皮层神经元糖皮质激素受体表达的影响

目的：研究无镁诱导惊厥后发育中皮层神经元表达糖皮质激素受体（glucocorticoid receptor, GR）的影响。方法：以原代培养的胎鼠大脑皮层神经元无镁诱导的反复惊厥样放电为模型,根据对培养6 d皮层神经元的不同处理分为正常细胞外液组和无镁细胞外液组。神经元在上述2种液体中孵育3 h,然后恢复正常DMEM培养液继续培养,在无镁损害后1 d、7 d及12 d时应用免疫细胞化学技术及实时定量PCR测定发育中大鼠皮层神经元无镁细胞外液处理后GR表达的变化。结果：应用免疫细胞化学方法发现无镁损伤后12 d惊厥组GR蛋白表达较对照组显著下降（P<0.01）;实时定量PCR发现无镁损伤后1 d、7 d惊厥组GR mRNA表达较对照组显著增高（P<0.05）。结论：无镁细胞外液处理可以诱导发育中大鼠皮层神经元GR表达的改变。［中国当代儿科杂志,2010,12（3）：211-214］