摘要
第一部分
CD20表达比例和强度在慢性淋巴细胞白血病中的预后价值
目的：慢性淋巴细胞白血病(Chronic lymphocytic leukemia，CLL)患者CD20表达水平存在异质性，本项研究旨在探讨CD20表达水平在CLL患者的预后价值。
方法：2003年1月至2011年12月在我院住院的172例初诊CLL患者。患者外周血标本均进行流式细胞术(Flow cytometry，FCM)检测，分析CD19+细胞的CD20、CD38和ZAP-70表达水平。间期荧光原位杂交(Fluorescence in situhybridization，FISH)检测细胞遗传学异常。聚合酶链式反应(Polymerase chainreaction， PCR)结合DNA测序检测p53基因和免疫球蛋白重链可变区(Immunoglobulin variable heavy chain，IGHV)基因突变。
结果：172例CLL患者，CD20在CD19+细胞中位表达比例为97.82%(0~100%)，中位平均荧光强度(Mean fluorescence intensity，MFI)为731.45(0~9071.90)。IGHV突变组CD20平均表达比例高于IGHV无突变组，分别为92.10%和80.40%(P<0.001)，CD20MFI在各预后指标组间均无明显差异(如IGHV突变状态等)。采用受试者工作特征曲线(Receiver operating characteristic curve，ROC)确定CD20表达比例在预测CLL患者IGHV突变的最佳临界值，曲线下面积(Area under thecurve，AUC)为0.661(95%CI：0.569~0.753)。CD20表达比例在IGHV突变预测的最佳临界值为60.30%，敏感性和特异性分别为90.00%和38.46%。CD20表达比例高于60.30%组无治疗生成时间(Treatment-free survival，TFS)比CD20表达比例低于60.30%组长(HR=0.452，95%CI：0.232~0.884，P=0.020)，多变量Cox回归分析显示CD20表达比例和MFI均与TFS无相关性(P<0.05)。
结论：CD20表达水平为CLL的预后指标之一，且高表达水平患者预后优于低表达患者，但不是独立性的预后因素。
第二部分
CD20单核苷酸多态性与慢性淋巴细胞白血病细胞CD20低表达相关性研究
目的：利妥昔单抗(Rituximab，RTX)现已在临床中广泛应用，部分CLL患者存在对其原发或继发耐药，但具体机制仍未明确。本项研究以CD20低表达为研究重点，探讨CD20蛋白编码区是否存在突变进而影响CD20表达量。
方法：针对CD20基因编码区Exon3~8分别设计6对引物，通过PCR扩增技术检测92例初诊CLL患者及200例正常对照样本CD20基因，后行基因序列检测。同时提取CLL患者外周血标本总RNA行CD20mRNA相对定量分析。FCM检测CLL细胞CD20表达比例和MFI。
结果：3.26%(3/92)初诊CLL患者存在Exon-3c.246C>T (rs17155019)；8.69%(8/92)初诊CLL患者存在Exon-4c.632C>T (rs2070770)，其余外显子均未发现任何异常。CLL人群rs17155019和rs2070770两个单核苷酸多态性(Single nucleotidepolymorphism，SNP)位点C/C基因型频率明显高于正常对照人群，分别为96.74%和93%；91.31%和81.00%，且具有统计学差异(P<0.01)；提示两SNP的C/C基因型可能为CLL的易感基因型。两个SNP位点的不同基因型间CD20mRNA水平、CD20表达比例和MFI均无明显统计学差异(P<0.05)。
结论：CLL患者CD20低表达与CD20基因编码区SNP无相关性。
第三部分
microRNA与慢性淋巴细胞白血病细胞CD20低表达相关性研究
目的： microRNA(miRNA)在生物体发育、细胞凋亡、增殖及分化等生命活动中发挥重要作用。它主要通过与靶标基因3’UTR的完全或不完全配对，降解靶标基因mRNA或抑制其翻译。本实验目的为研究miRNA与CD20表达调控之间的关系。
方法：分别检索三个数据库预测可能调节CD20的miRNA，包括hsa-miR-1、hsa-miR-221、hsa-miR-499-3p、hsa-miR-576-5p、hsa-miR-632和hsa-miR-708等。体外化学合成6个miRNA mimics，应用lipofectamine2000体外转染13例初诊CLL患者原代细胞，培养24h，通过RT-PCR检测CD20mRNA表达水平；FCM检测CD20表达比例和CD20MFI的变化。
结果：通过RT-PCR方法分别检测hsa-miR-1、hsa-miR-221、hsa-miR-499-3p、hsa-miR-576-5p、hsa-miR-632和hsa-miR-708表达水平，提示转染效率可。对6组转染miRNAmimic和阴性对照(Negative control，NC)组分别检测CD20mRNA和蛋白水平，与对照组比较提示无明显统计学差异(P<0.05)。
结论：提示hsa-miR-1、hsa-miR-221、 hsa-miR-499-3p、hsa-miR-576-5p、hsa-miR-632和hsa-miR-708的靶基因不包括CD20，CLL细胞CD20的低表达量可能与其不相关。
第四部分
CD20基因启动子甲基化水平与慢性淋巴细胞白血病细胞CD20低表达相关性研究
目的：表观遗传学在肿瘤的病理过程中发挥重要的作用。CLL细胞CD20低表达可能与表观遗传学的调控相关。本研究拟探讨组蛋白乙酰化与启动子甲基化与CD20表达之间的关系。
方法：分别以1mM、2mM和4mM的组蛋白去乙酰化酶抑制剂(Histonedeacetylase inhibitors，HDACI)丙戊酸(Valproic acid，VPA)处理CLL原代细胞，培养24h和48h后通过RT-PCR和FCM检测CD20mRNA和蛋白水平变化；DNA甲基转移酶抑制剂5-氮-2’-脱氧胞苷(5-Aza-dC)(100μM)处理CLL原代细胞，分别培养24h、48h和96h后收集细胞，通过RT-PCR和FCM检测CD20mRNA和蛋白水平变化。生物信息学软件分析CD20启动子区；亚硫酸氢盐测序法(Bisulfite genomic sequencing，BSP)对CLL细胞CD20启动子区行甲基化分析，包括：用重亚硫酸盐处理基因组DNA；分别设计3对引物，对CD20启动子区6个CpG行PCR扩增；PCR产物割胶纯化后TA克隆，挑取10个克隆测序。
结果： VPA1mM培养24h CD20mRNA水平增加明显，但CD20蛋白水平未见明显增加；5-Aza-dC处理24h后可明显提高CD20mRNA水平、处理48h后可明显提高蛋白水平。BSP结果显示6个CpG均存在部分甲基化，分别为28%(14/50)、24%(12/50)、8%(4/50)、20%(10/50)、6%(3/50)和16%(8/50)。CD20表达量明显增高的患者其CD20启动子总的甲基化比例仅为11.67%(7/60)。CD20表达比例和强度与CD20启动子甲基化比例无明显相关性。
结论：表观遗传学机制可能与CLL的CD20低表达相关；去甲基化药物可提高CLL细胞CD20表达。
Part Ⅰ
The prognostic value of CD20percent and fluorescence intensity inchronic lymphocytic leukemia
Objectives: Heterogeneity of CD20expression was existed in chronic lymphocyticleukemia (CLL), so we explore the prognostic significance of CD20expression inChinese patients with CLL.
Methods: One hundred and seventy-two consecutive Chinese patients with CLLwere enrolled from January2003to December2011. Blood samples collected atdiagnosis were available for CD20, ZAP-70and CD38analyses.Multi-parameter flowcytometry was used to detect the expression of CD20on CD19+cells. Molecularcytogenetic aberrations were detected by florescence in situ hybridization (FISH).p53mutation and immunoglobulin variable heavy chain (IGHV) mutation status wereanalyzed by polymerase chain reaction (PCR) and direct sequencing.
Results: In172CLL patients, the median expression percent of CD20was97.82%(range,0–100), and the median mean fluorescence intensity (MFI) of CD20on CLLcells was731.45(rang,0–9071.90). The percentage of CD20+cells in cases of IGHVmutated groups was higher than unmutated groups (mean,92.1%vs.80.4%,P<0.001). There were no difference in the MFI of CD20+cells in cases of eachprognostic factor group. The data of percentage of CD20using a receiver operatingcharacteristic plot (ROC) reflects separation between the two IGHV groups, with anarea under the curve (AUC) of0.661(95%CI:0.569to0.753). At the cutoff value ofpredicting IGHV mutation is60.30%of CD20percentage, the sensitivity and thespecificity were90.00%and38.46%. Patients whose percentage of CD20antigen above60.30%had a better treatment free survival (TFS)[Hazard Ratio (HR),0.452,95%CI:0.232to0.884, P=0.020]. Percentage and MFI of CD20were the variablesnot associated with TFS by multivariate Cox regression analysis (P<0.05).
Conclusions: Expression levels of CD20is one of the prognostic markers of CLL,high level of CD20expression in CLL appears to be associated with a good prognosis,but not independent prognostic factor.
PartⅡ
Study on the relation between single nucleotide polymorphisms ofCD20and low expression of CD20in chronic lymphocytic leukemiacells
Objectives: Rituximab (RTX) is now widely used in clinical practice. Some CLLpatients with primary or secondary resistance to the drug, the mechanism was not yetclear. This research focused on the relationship between CD20coding regionmutations with CD20expression.
Methods: Primers were designed for the CD20gene coding region.CD20wasamplified by PCR in92cases of newly diagnosed CLL patients and200healthydonors. Relative quantitative analysis of CD20mRNA was conducted in peripheralblood specimens of CLL patients. Proportions of CD20expression and fluorescenceintensity were detected by flow cytometry.
Results: Exon-3c.246C>T (rs17155019) was present in3.26%(3/92) of newlydiagnosed CLL patients. Exon-4c.632C>T (rs2070770) was present in8.69%(8/92) of newly diagnosed CLL patients. There were not found of any abnormal in theremaining exons. The frequency of C/C genotype of rs17155019and rs2070770wassignificantly higher than the normal control population,96.74%and93%;91.31%and81.00%, respectively (P<0.01). C/C genotype of two SNP may be susceptiblegenotype of CLL. No statistically significant difference in the CD20mRNA,proportion and intensity of CD20expression was found in the different genotypes oftwo polymorphic loci (P<0.05).
Conclusions: There was no correlation between low expression of the CD20andmutation of CD20gene coding region.
Part Ⅲ
Study on the relationship between microRNA and low expression ofCD20in chronic lymphocytic leukemia cells
Objectives: microRNA has been shown to play an important role in the development,apoptosis, proliferation and differentiation of organisms. It is mainly through thecomplete or incomplete matching with3'UTR of the target gene, which leaded to thedegradation of the target gene mRNA or inhibiting its translation. The purpose of thisexperiment was to study the relationship between miRNA and CD20expression.
Methods: To found potential miRNA which might regulate CD20expression, threedatabases, hsa-miR-1, hsa-miR-221, hsa-miR-499-3p, hsa-miR-576-5p, hsa-miR-632and hsa-miR-708were chosen. In vitro chemical synthesis of six miRNA mimicswere transfected to primary cells of13patients with newly diagnosed CLL bylipofectamine2000and cultured24h. Relative quantitative analysis of CD20mRNA was conducted in peripheral blood specimens of CLL patients. Proportions of CD20expression and fluorescence intensity were analysed by flow cytometry.
Results: The expression levels of hsa-miR-1, hsa-miR-221, hsa-miR-499-3p,hsa-miR-576-5p, hsa-miR-632and hsa-miR-708were detected by RT-PCR,suggesting transfection efficiency was acceptable. CD20transcriptome and proteinlevels were detected in transfected with miRNA mimic and negative control (NC)group, respectively. No statistically significant difference was found between miRNAmimic groups and control group (P<0.05).
Conclusions: The target of hsa-miR-1, hsa-miR-221, hsa-miR-499-3p,hsa-miR-576-5p, hsa-miR-632and hsa-miR-708does not include CD20. Lowexpression levels of CD20in CLL cells may not be regulated by microRNA.
Part Ⅳ
Study on the relations between methylation levels of CD20promoterand low expression of CD20in chronic lymphocytic leukemia cells
Objectives: Epigenetics has a pivotal role in many pathological processes of cancer.Low expression of the CD20antigen in CLL may be associated with epigeneticregulation. This study focused on the relationship between histone acetylation,promoter methylation and expression of CD20.
Methods: Primary CLL cells were treated with1mM,2mM, and4mM of VPA(Valproic acid), which was histone deacetylase inhibitors. After cultured24h and48h, relative quantitative analysis of CD20mRNA was conducted by PCR; Proportion of CD20expression and fluorescence intensity were detected by flow cytometry. ForCLL primary cells treated with5-Aza-dC (100μM) for24h,48h and96h, relativequantitative analysis of CD20mRNA was conducted by PCR; Proportion of CD20expression and fluorescence intensity were detected by flow cytometry. CD20promoter region was analysed by bioinformatics software. Methylation analysis ofCD20promoter region in CLL cells by Bisulfite genomic sequencing (BSP),including: genomic DNA with treated by bisulfite; Three pairs of primers weredesigned,6CpG in CD20promoter region were amplified by PCR.; Tapping andpurification of PCR products, TA cloning, picking10clones for sequencing.
Results: CLL cells were treated with1mM VPA for24h, CD20mRNA levelsincreased significantly, but no significant increase in the level of CD20protein. CD20mRNA level can obviously increase after24h of5-Aza-dC treatment; The proteinlevel can obviously increase after48h. There were partially methylated in six CpGby BSP,28%(14/50),24%(12/50),8%(4/50),20%(10/50),6%(3/50) and16%(8/50), respectively. In one patient, the CD20expression was significantly increased by5-Aza-dC, the CD20promoter methylation ratio was11.67%(7/60). No significantcorrelation between the proportion and intensity of CD20expression and promotermethylation was detected.
Conclusions: Epigenetic mechanisms may be associated with low CD20expressionin CLL; Demethylation drugs can increase the expression of CD20in CLL cells.
引文
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