慢性淋巴细胞白血病患者糖皮质激素受体表达及其与预后相关性的研究

【作者】 王一；

【导师】 方美云；

【作者基本信息】 大连医科大学 ， 内科学， 2014， 博士

【摘要】 目的：探讨慢性淋巴细胞白血病(Chronic lymphocytic leukemia，CLL)患者糖皮质激素受体（Glucocorticoid，GR）亚型α、β、γ和p的表达情况，分析GR各亚型表达的相关性。研究CLL患者GR与预后的关系。方法：连续取我院2009年5月～2013年2月期间住院的慢性淋巴细胞白血病患者33例（女性10例，男性23例，中位年龄62岁），及健康对照者32例（女性18例，男性14例，中位年龄34岁）为研究对象。1采集晨空腹肘静脉血5ml，采用Ficoll密度梯度离心法分离外周血单个核细胞。应用实时荧光定量PCR SYBR Green染料法检测患者和正常人GRα、GRβ、GRγ和GRp mRNA的表达。2采用Spearman相关分析CLL患者GRα、GRβ、GRγ、GRP各亚型表达的相关性。3统计患者无进展生存时间（progression free survival，PFS），中位随访时间为28（1～57）个月。以本研究检测到的GRα mRNA均值为界（9.045），将患者分为高GRα mRNA组及低GRα mRNA组。分别统计两组患者的PFS。同样方法分组高GRp mRNA组和低GRp mRNA组，采用Kaplan-Meier生存曲线法分析各亚组患者的PFS差异。结果：1CLL患者组中GRα mRNA的表达量为1210.25，GRβ mRNA的表达量为39.42，GRγmRNA的表达量为164.04，GRp mRNA的表达量为761.51。2患者组中GRα、GRβ、GRγ、GRP占总GR的百分比分别为是55.65%、1.81%、7.54%、35.00%；而正常对照组中分别为：64.82%、0.46%、18.73%和15.99%。3患者组GRα mRNA和GRP mRNA的表达均高于正常对照组，（P<0.05）。而GRβ和GRγ mRNA表达量在患者组与对照组无统计学差异。4CLL患者GRpmRNA与GRα mRNA呈正相关（P<0.05）。而其它受体亚型之间无明显相关性。5高GRα mRNA组较低GRα mRNA组患者的PFS明显延长，高GRp mRNA组较低GRp mRNA组的PFS也明显延长，各组之间有显著性差异，（P<0.05）。结论：1CLL患者GRα亚型表达占绝对优势，其次为GRp亚型，而GRβ及GRγ亚型的表达量较少，提示GRα是GC发挥疗效的最主要亚型。2与正常对照组相比，CLL患者GRα mRNA及GRp mRNA的表达明显增高，而GRβ mRNA及GRγmRNA的表达较对照组无明显差异。提示GRα和GRp在CLL的发病发挥一定的作用。3CLL患者中高GRαmRNA及高GRp mRNA表达者PFS显著延长，提示GRα及GRp可作为判断CLL患者预后的独立因素，高GRα及GRp是CLL患者预后良好的标志。4CLL患者GRα mRNA及GRp mRNA的表达呈正相关，而GRα mRNA的表达量在CLL患者中明显增高，提示GRp可能是通过增强GRα的表达来影响患者的预后。目的：检测慢性淋巴细胞白血病(Chronic lymphocytic leukemia，CLL)患者血清巨噬细胞移动抑制因子(macrophage migration inhibitory factor，MIF)的水平，并分析其与CLL其他预后因素的关系，以及MIF对患者预后的影响。方法：1患者及对照者选择见论文12采用酶联免疫吸附试验（enzyme-linked immunosorbent assay，ELISA）检测CLL患者及对照组血清MIF水平。3制备患者染色体标本，采用R显带技术进行核型分析。4应用荧光原位杂交法（FISH）法检测患者p53缺失。5检测患者临床指标包括：血清乳酸脱氢酶（LDH）、外周血淋巴细胞计数、血清β2微蛋白等。6统计患者无进展生存时间（progression free survival，PFS)，采用Kaplan-Meier生存曲线法分析GR表达对患者PFS的影响。结果：1CLL患者血清MIF水平（1199.79±691.01pg/ml）较对照组(176.74±105.82pg/ml)明显增高（P<0.01）。同时，临床分期较晚的患者血清MIF水平（Binet B、C，1292.26±529.96pg/ml）与早期患者（Binet A，1067.71±877.31pg/ml）相比，无统计学差异。2高LDH组（>250μ/L）患者血清MIF水平明显高于低LDH组（<250μ/L），P<0.05；高β2微球蛋白组（>3mg／L）的患者血清MIF水平明显高于低β2微球蛋白组（<3mg／L），P<0.05。而不同性别、年龄、淋巴细胞计数、脾大和染色体异常核型组别中，MIF水平无明显差别。3FISH法检测12例CLL患者p53缺失均为阴性。4依据MIF表达水平将33例CLL患者分为高MIF组（高于中位值）及低MIF组（低于中位值），采用Kaplan–Meier生存曲线分析两组的预后，结果显两组PFS无明显差异，P=0.875，结论：1CLL患者血清MIF水平明显增高，MIF可作为帮助诊断CLL的参数，但不能作为判断疾病进展的指标。2在高LDH组及高β2微球蛋白组患者血清MIF水平明显增高，提示MIF与患者的预后有关。3所有患者均未检测到p53缺失，提示MIF并不能引起p53基因的缺失，其对P53的调节可能是在翻译或转录水平实现的。4MIF不能对CLL患者的PFS产生影响，不能作为判断预后的独立因素。目的：检测miR-142-3p在慢性淋巴细胞白血病(Chronic lymphocytic leukemia，CLL)患者中的表达及对预后的影响；探讨miR-142-3p与CLL患者糖皮质激素受体各亚型的关系。方法：1患者及对照者选择见论文12采集晨空腹肘静脉血5ml，采用Ficoll密度梯度离心法分离外周血单个核细胞。应用实时荧光定量PCR SYBR Green染料法检测患者和正常人miR-142-3pmRNA的表达。3采用Pearson法分析miR-142-3p与GR各亚型的相关性。4依据实验中miR-142-3p mRNA表达水平将33例CLL患者分为高miR-142-3p表达组（高于中位值）及低miR-142-3p表达组（低于中位值），统计各组的无进展生存时间（PFS），采用Kaplan–Meier生存曲线分析两组的预后。结果：1CLL患者组miR-142-3p mRNA中位值0.86（0.559-2.75）明显高于对照组0.14（0.082-1.65），P＜0.05。2CLL患者miR-142-3p mRNA的表达与GRα及GRp呈负相关，P＜0.05。而与GRβ和GRγ无明显相关性，3高miR-142-3p表达组CLL患者较低表达组的PFS明显缩短。二者有统计学差异，P＜0.05，结论：1CLL患者miR-142-3p mRNA表达明显增高，提示miR-142-3p可以作为协助诊断CLL的参数。2CLL患者miR-142-3p与GRα及GRp mRNA呈负相关，提示miRNA142-3p抑制GRα及GRp的表达。3高miR-142-3p的表达水平是预后的不利因素，可作为CLL患者判断预后的独立指标。更多还原

【Abstract】 Objective: To study the expression of glucocorticoid receptor (GR) of mononuclearcells in peripheral blood of chronic lymphocytic leukemia (CLL) patients. To analyzethe correlation among the expression of GR subtypes including alpha, beta, gammaand P. To study on the relationship between GR expression and the prognosis of CLLpatients.Methods: Continuous samples of33chronic lymphocytic leukemia patients (10female and23male, median age62years old) hospitalized in our hospital from2009May to2013February were taken, as well as samples of32healthy controls (18females,14males, median age34years old).1Fasting elbow venous blood5ml in normal individuals and patients with CLLwas gathered. Peripheral blood mononuclear cells (PBMC) were collected usingFicoll density gradient centrifugation. Expression of GR α, GRβ, GRγ and GRpmRNA were detected by real-time fluorescence quantitative PCR and SYBR Green.2The correlation among the expression of GRα, GRβ, GRγ, GRP isoforms inCLL patients was analyzed by Spearman correlation.3Progression free survival (PFS) of the patients were analyzed. The medianfollow-up time was28(1~57) months. Kaplan-Meier survival curve was used toanalyze the effect of GR expression on patients PFS.Results: 1In the group of CLL patients, GR α mRNA expression amount was1210.25, theexpression of GRβ mRNA was39.42, the expression of GR γ mRNA was164.04,the expression of GRp mRNA was761.2The percentage of GR α, GR β, GR γ, GRP in patients group was55.65%,1.81%,7.54%, and35%respectively. While in the control group was64.82%,0.46%,18.73%, and15.9respectively.3The expression of GRαmRNA and GRP mRNA in patients group were higherthan that in control group,（P<0.05）. While the GRβ and GRγ mRNA expression inthe patient group and the control group showed no significant difference.4In CLL patients, GRp was positively correlated with GRαmRNA（P<0.05）. Butthere was no correlation between other receptor subtypes.5PFS in high GRαmRNA patients was significantly prolonged compared withthe lower GRαmRNA group, PFS in high GRp mRNA group were also significantlyprolonged compared with the lower GRp mRNA group.Conclusions:1The expression of GR alpha isoform in CLL patients occupies the absolutesuperiority, followed by subtype GRp, GRβ and GRγ, suggesting that GRαis themain effecting subtype.2The expression of GRαand GRp mRNA in patients with CLL were significantlyincreased compared with healthy controls, while the expression of GRβand GRγnosignificant difference between the patients and the control group. These datasuggested that GRα and GRp play a major role in the pathogenesis of CLL.3Patients with high GRαand high of GRp expression had a longer PFS,suggesting that GRα and GRp can be used in evaluating prognosis of CLL. HighGRαand GRp is a sign of good prognosis of CLL patients.4GRp was positively correlated with GRαmRNA in CLL patients, whileGRαmRNA were significantly increased, suggesting that GRp is likely to affect theprognosis of the patients by increasing the expression of GRα. Objective: To detect the serum macrophage migration inhibitory factor (MIF) level inchronic lymphocytic leukemia (CLL) patients, and analyze its relationship with otherprognostic factors of CLL. Investigate the effect of MIF on prognosis in CLL.Methods:1Continuous samples of33chronic lymphocytic leukemia patients (10femaleand23male, median age62years old) hospitalized in our hospital from2009May to2013February were taken, as well as samples of32healthy controls (18females,14males, median age34years old).2Serum MIF level were detected by ELISA in CLL patients and controls.3Analyze chromosome karyotypes of patients using R banding technique.4Mutations of P53in patients with CLL were detected by fluorescence in situhybridization (FISH) method.5Patients clinical indicators were detected, serum lactate dehydrogenase (LDH),peripheral blood lymphocyte count, serum β2micro protein level, etc.6Progression free survival (PFS) of the patients were analyzed, The medianfollow-up time was28(1~57) months. Kaplan-Meier survival curve was used toanalyze the effect of MIF on patients PFS.Results:1The serum MIF level in CLL patients (1199.79±691.01pg/ml) weresignificantly higher than the control group (176.74±105.82pg/ml)(P<0.01).Meanwhile, the serum MIF level had no significant difference between the patients inlate clinical stage (Binet B, C,1292.26±529.96pg/ml) and the patients in early stage(Binet A,1067.71±877.31pg/ml).2In the high LDH group (>250μ/L), the serum level of MIF patients was significantly higher than the low LDH group (<250μ/L), P<0.05; the serum level ofMIF patients in higher β2microglobulin group (>3mg/L) was significantly higherthan that of lower β2microglobulin group (<3mg/L), P<0.05. The MIF levels had nosignificant difference in the gender, age, white blood cell count, splenomegaly andabnormal karyotype of chromosome subgroups.3Detection of P53mutation by FISH in12CLL patients were negative.4According to the expression level of MIF,33patients with CLL were dividedinto high MIF group (a value higher than the medium) and low group (a value below),Analyzing prognosis of the two groups by Kaplan-Meier survival curves, resultsshow PFS had no significant difference in the two groups, P=0.875.Conclusion:1The serum MIF levels in CLL patients were significantly increased, MIF can beused as candidate markers for diagnosis of CLL, but should not be considered asindicators of disease progression.2In the high LDH group and high β2microglobulin group, the serum MIF levelswere significantly increased, suggesting that MIF were associated with the prognosisof CLL patients.3All patients had no detectable P53mutations, suggesting that MIF does notcause mutations in P53. The regulation of P53by MIF may occur in the translation ortranscription level.4MIF level has no effect on PFS of patients with CLL, so it is not considered asan independent prognostic factor. Objective: To detect the expression of miR-142-3P in chronic lymphocytic leukemia(CLL) patients, and its influence on prognosis; to explore the relationship betweenmiR-142-3p and glucocorticoid receptor isoforms in CLL patients.Methods:Continuous samples of33chronic lymphocytic leukemia patients (10femaleand23male, median age62years old) hospitalized in our hospital from2009May to2013February were taken, as well as samples of32healthy controls (18females,14males, median age34years old).1Fasting elbow venous blood5ml in normal individuals and patients with CLLwas gathered. Peripheral blood mononuclear cells (PBMC) were collected usingFicoll density gradient centrifugation. MiR-142-3p mRNA were detected by real-timefluorescence quantitative PCR and SYBR Green.2The correlation between miR-142-3p and GR isoforms was analyzed.3Based on experiment value of miR-142-3p mRNA, patients with CLL weredivided into high miR-142-3p expression group and low miR-142-3p expressiongroup, progression free survival (PFS) of each group was analyzes using Kaplan-Meier survival curves.Results:1The expression of miR-142-3p mRNA with a median value of0.86(0.559-2.75)in CLL patients was significantly higher than that in control group0.14(0.082-1.65),P <0.05.2The expression of miR-142-3p mRNA was negatively correlated with GR α and GRp in CLL patients, P <0.05. But there was no significant correlation betweenmiR-142-3p mRNA and GR β, GRγ.3PFS in high miR-142-3p expression group was shortened obviously comparedwith low miR-142-3p expression group. The PFS in two groups have significantdifference, P <0.05.Conclusion:1The expression of miRNA142-3p in CLL patients was significantly higher thanthat in control, suggesting that miRNA142-3p is closely related to the pathogenesis ofCLL.2The expression of miRNA142-3p in CLL patients were negatively correlated withGRα and GRp mRNA, suggesting that miRNA142-3p inhibits expression of GRα andGRp.3High level of miRNA142-3p expression is a negative prognostic factor, can beused as an independent indicator of prognosis of CLL patients.更多还原