【作者】 赵丹丹；

【导师】 于力； 高春记； 张翼鷟；

【作者基本信息】 中国人民解放军军医进修学院 ， 内科学， 2010， 博士

【摘要】 骨髓增生异常综合征(Myelodysplastic syndrome, MDS)是血液系统的一组异质性克隆性疾病,主要表现为骨髓无效造血、外周血细胞一系或多系细胞减少,临床易转化为急性白血病。由于发病机制迄今为止尚未完全探明,故该病的诊断、分型及治疗等方面仍存在诸多悬疑。骨髓造血基质是造血系统的重要组成部分,通过产生和分泌细胞因子及细胞外基质对造血细胞的生成和分化发挥着极其重要的作用。间充质干细胞(Mesenchymal stem cell,MSC),也被称为骨髓基质细胞(Marrow stromal cell, MSC),是造血基质的重要组成部分。基质细胞衍生因子-1(SDF-1)是骨髓基质细胞表达的高效趋化因子,与其受体即表达于CD34+造血干／祖细胞(HSC/HPC)上的CXCR4特异性结合后,在HSC/HPC的归巢、骨髓定居、正常造血的维持及由骨髓动员至外周血的过程中发挥起着重要的作用。为研究SDF-1/CXCR4在MDS发病中的作用,我们将临床骨髓标本用CD34+细胞阳性分选柱进行CD34+造血祖细胞的分选后,在60ng的SDF-1α因子作用下进行Transwell迁移实验,结果显示,低危组、对照组和高危组的迁移细胞百分比分别为(16.7±3.5)%、(18.9±2.4)%和(31.7±3.3)%,低危组与对照组相比没有明显差异,而与高危组相比则有显著差异(P<0.05)。随后对42例临床骨髓血浆标本用酶联免疫反应法测定SDF-1α因子的浓度,结果显示,在MDS低危组、高危组和正常对照组中的表达量为2301.3±413.1pg/mL、1173.3±501.1pg/mL、689.3±189.7g/mL,其组间比较P值均小于0.05,无论MDS低危组还是高危组其SDF-1α的表达量均明显高于正常对照,而MDS低危组SDF-1α的表达量明显高于高危组。同时,用流式细胞术检测分选的CD34+细胞表面CXCR4的表达,结果显示,CD34+细胞CXCR4的表达率在MDS高危组、低危组和正常对照组分别为(59.0±9.5)%、(21.4±5.0)%及(18.8±3.4)%,高危组与正常组和MDS低危组之间相比有显著差异(P<0.01),而MDS低危组与正常对照组之间没有显著差异(P>0.05)。最后,用流式细胞术测定CD34+细胞的凋亡率,结果显示,CD34+细胞凋亡率在MDS低危组、MDS高危组和正常对照组中分别为(56.8±10.2)%、(24.34±7.9)%及(18.5±8.7)%,MDS低危组与高危组或正常对照组的CD34+细胞凋亡率比较有明显差异,P值均小于0.05,而高危组与正常对照组没有明显差异,P值大于0.05。所以我们的结论是,相对于正常对照组,MDS低危组患者CD34+细胞表面表达的CXCR4的水平是正常的,但骨髓基质细胞产生的SDF-1α因子的浓度显著高于正常骨髓,导致MDS患者骨髓增生极度活跃但细胞不能动员至外周血中,并且由于凋亡率显著高于正常对照组,从而导致MDS低危组患者骨髓增生活跃但外周血细胞一系至三系细胞减少的典型临床特征；而MDS高危组,相对于低危组患者,其CD34+细胞表达CXCR4水平明显增高,骨髓基质中SDF-1α水平下降,使骨髓中的细胞容易动员到外周血中,并且其凋亡率明显下降,导致MDS高危患者原始细胞明显增高。所以SDF-1/CXCR4轴在MDS低危组中的骨髓无效造血及MDS由低危转为高危的发病机制中起重要作用。JAK/STAT途径是一种多种细胞外信号导致靶细胞上基因表达快速改变的经典途径,该途径异常信号的传递将导致造血紊乱,与白血病等的发生有密切联系。SHP-1基因是近年来发现的抑癌基因,参与JAK/STAT途径的负性调控。启动子甲基化是SHP-1基因沉默的主要机制。为研究SHP-1在MDS发病中的作用,我们对共33例骨髓标本及1个由MDS转为AML的细胞系SKK-1进行甲基化特异PCR扩增(methylation specific PCR, MSP),结果发现在MDS低危组中SHP-1甲基化比例为18.2%,而高危组中为71.4%,SKK-1细胞系中则出现部分甲基化,正常对照组没有出现甲基化,测序分析SHP-1证实甲基化产物的正确性。我们推断SHP-1基因启动子区的甲基化可使SHP-1基因表达沉默,从而激活JAK/STAT途径参与MDS的发生发展过程。总之,骨髓基质细胞通过SDF-1/CXCR4轴,以及造血细胞中SHP-1基因启动子甲基化激活JAK/STAT途径,参与MDS疾病的发生发展过程,在疾病演化中起重要的作用,我们可以以此作为探索MDS治疗新手段的切入点。更多还原

【Abstract】 So far, the pathogenesis of MDS (Myelodysplastic syndrome, MDS) has not yet fully addressed, and the diagnosis, classification and treatment of this disease remain largely uncertain. Bone marrow stroma plays a very important role in the generation and differentiation of hematopoietic cells. Bone marrow stromal cell is an important component of hematopoietic stroma. Stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 play a crucial role on HSC/HPC homing, normal hematopoiesis and mobilization from the bone marrow to peripheral blood.To study the role of SDF-1/CXCR4 in MDS, CD34 positive cells from bone marrows of patients were cultured in 60ng SDF-1 to perform transwell invasion assay. The results show that the percentage of invasive cells for low risk group, normal control group and high risk group were (16.7±3.5)%, (18.9±2.4) % and (31.7±3.3)% respectively. There are no significant differences between the control group and the low risk group, but the high-risk group is significantly high (P<0.05). And the concentration of SDF-1αin bone marrow plasma was determined with enzyme-linked immunoassay. The results show the concentration of SDF-1 a in the low risk group, the high-risk group and the control group is 2301.3±413.1pg/ml,1173.3±501.1pg/ml, and 689.3±189.7g/ml respectively and it is statistically significant between either group (P<0.05). The expression of CXCR4 in CD34-positive cells was detected by flow cytometry. CXCR4 positivity in the high risk group, the low risk group and the control group is (59.0±9.5)%, (21.4±5.0)%, and (18.8±3.4)% respectively. Compared to the low risk group or the control group, the high risk group is significantly different (P <0.01), while there is no significant difference between the low risk group and the control group (P> 0.05). The apoptosis in CD34-positive cells was also determined by flow cytometry. In the low risk group, the high risk group and the normal control group, the percentage of apoptotic cells is (56.8±10.2)%, (24.34±7.9)% and (18.5±8.7)% respectively. Compared to the high risk group or the normal control group, the apoptosis rate in the low risk group is significantly different (P<0.01). Therefore, we conclude that, compared with the normal control, the CXCR4 expression is normal in the CD34-positive cells from the MDS patients with low risk, but the bone marrow stromal cells produce excess SDF-1a factor, resulting in extremely high bone marrow apoptosis, and leading to the typical clinical phenotype. However, in the high risk MDS patients, compared with low risk patients, the number of CD34 positive cells expressing CXCR4 is significantly increased, while the bone marrow SDF-la levels decrease and the apoptosis is significantly decreased. So the SDF-1/CXCR4 axis plays a very important role in the invalid hematopoiesis in bone marrow of low risk MDS patients and in the progression from low risk to high risk.SHP-1 gene was recently identified as a tumor suppressor gene which negatively regulates JAK/STAT pathway and promoter methylation is the main mechanism for silence of its expression. To figure out if SHP-1 plays a role in MDS, methylation specific PCR was used to detect methylation in promoter region of SHP-1 gene. In 18.2% of samples in the normal control and low risk group, SHP-1 gene was found to be methylated, while in high risk group, the percentage is increased to 71.4%, and in SKK-1 cell line SHP-1 is partially methylated. In normal control group no methylation was detected. The sequencing results of methylation PCR products further confirm the specificity of MSP. Our data suggest that promoter methylation of SHP-1 gene may activate the JAK/ STAT pathway and play a role in MDS development.In summary, bone marrow stromal cells through SKD-1/CXCR4 axis, and promoter methylation of SHP-1 gene, may be involved in MDS development and could be targeted for MDS therapy.更多还原