【作者】 李丽娟；

【导师】 邵宗鸿；

【作者基本信息】 天津医科大学 ， 内科学， 2011， 博士

【摘要】 目的对骨髓增生异常综合征(Myelodysplastic Syndrome, MDS)患者骨髓造血细胞的分化异常进行检测,并对其分化异常的部分相关机制进行研究；同时进一步寻找MDS恶性克隆细胞,并探讨其过度增殖、分化异常及凋亡逃逸等恶性特征。方法研究对象为天津医科大学总医院自2008年7月至2010年10月新诊断的MDS患者62例、MDS转化的急性髓系白血病(MDS-AML)8例、急性髓系白血病(AML)7例、病例对照组16例及正常对照32名。第一部分检测MDS患者骨髓造血细胞分化异常情况,采用流式细胞术(FCM)检测MDS患者骨髓粒系、单核系及红系分化抗原；干细胞分群；单个核细胞细胞周期分布,并分析分化异常情况与染色体恶性克隆负荷的相关性。第二部分研究导致MDS患者骨髓造血细胞分化异常的相关机制,采用FCM检测MDS患者干/祖细胞表面干细胞因子受体(SCF-R)、红细胞生成素受体(EpoR)、粒细胞集落刺激因子受体(G-CSFR)及血小板生成素受体(TpoR)的表达情况,并分析其临床意义；干/祖细胞转录因子GATA-1、GATA-2的表达情况；RT-PCR的方法检测GATA-1基因在MDS患者骨髓中的表达情况。第三部分寻找MDS恶性克隆细胞,检测MDS患者骨髓干细胞中白细胞介素-3受体α(IL-3Ra)表达情况,并探讨IL-3Ra阳性细胞的异常增殖及分化特征。结果第一部分粒系细胞分化抗原表达的模式和比例：选择CD13/CD11b CD13/CD16及CD11b/CD16组合来分析粒细胞分化抗原表达模式,对照组和病例对照组骨髓粒系细胞组合模式分别为“对钩”、“镰刀,或“反7”状,MDS患者骨髓粒系细胞发育分化中的抗原表达失去正常序贯表达模式,呈现抗原的连续性表达中断等不同程度的改变。高危组CDllb/CD11b+比值(0.32±0.29)明显高于低危组(0.16±0.23)、病例对照组(0.05±0.03)及正常对照组(0.07±0.05,P<0.01)；高危组CD16/CD16+比值(1.32±0.77)明显高于病例对照组(0.33±0.32)和正常对照组(0.39±0.31,P<0.05)；高危组骨髓粒细胞CD13的平均荧光强度(MFI)明显高于正常对照组(P<0.01),侧向角散射光信号(SSC)的MFI明显低于病例对照组和正常对照组(P<0.01)。高危组CD11b-HLA-DR+(4.08%±2.97%)、CD11b-HLA-DR’(14.90%±15.44%)、CD16-HLA-DR- (38.98%±21.59%)、CD11b+CD16-(32.57%±16.52%)及CD13+CD16-(43.97%±20.31%)细胞占粒细胞比例明显高于低危组、病例对照组及对照组(P<0.05),其他组间比较差异无统计学意义。单核系细胞分化抗原表达的模式和比例：选择CD13/CD16和CD11b/HLA-DR组合来分析单核细胞分化抗原表达模式,MDS患者骨髓单核系细胞发育分化中的抗原表达模式相对于对照组的序贯组合模式出现不同程度的改变。高危组CD11b-HLA-DR+(12.38%±8.97%), CD11lb+HLA-DR-(38.33%±18.43%)细胞占单核系细胞比例明显高于病例对照组和正常对照组(P<0.05)。高危组单核细胞CD14(56.74%±17.73%)表达率明显低于正常对照组,CD64的表达率各组之间比较,差异无统计学意义,CD56(32.45%±20.07%)、CD34(13.54%±13.73%)及CD7(15.31%±12.76%)表达率明显高于病例对照组及正常对照组。红系细胞分化抗原表达的模式和比例：应用CD71和血型糖蛋白A(glycophorin A,GlyA)的组合来分析红系细胞的分化,对照组和病例对照组两种抗原的组合模式均为双阳性表达,部分MDS患者可见CD71和GlyA表达不同步现象。低危组和高危组CD71+、GlyA+细胞占CD45-细胞的比例均显著低于病例对照组和正常对照组(P<0.05),CD71+GlyA+双阳性细胞分别占CD45-、CD71+细胞的比例均显著低于病例对照组和正常对照组(P<0.05), CD71+GlyA+双阳性细胞占GlyA+细胞的比显著低于正常对照组(P<0.05)。MDS患者粒系、单核系及红系抗原表达的比例和模式的异常数目与IPSS(国际预后积分系统)积分(r=0.662, P=0.000)、WPSS (WHO分型预后积分系统)积分(r=0.602,P=0.000)及恶性克隆负荷(r=0.477,P=0.001)均呈正相关。高危和MDS-AML组骨髓CD34+细胞比例(2.29%±2.17%、18.69±17.47%)明显高于对照组(0.36%±0.49%,P<0.05)。低危、高危及MDS-AML组CD34+CD38+细胞相对比例(86.09%±7.79%、81.68%±11.82%、82.88%±2.60%)显著低于对照组(92.21%±3.85%,P<0.05),而CD34+CD38-细胞比例(13.91%±7.79%、18.32%±11.82%、17.13%±2.60%)显著高于对照组(7.79%±3.85%,P<0.05)。MDS组CD34+CD38-细胞比例与IPSS (r=0.493, P=0.001)、WPSS积分(r=0.586,P=0.000)均呈正相关。CD34+CD38-细胞比例≤12.0%的MDS患者治疗有效率高于CD34+CD38-细胞比例>12.0%的患者,差异无统计学意义。低危、高危及MDS-AML组单个核细胞(BMMNC)中Go/G1期细胞比例(94.52%±4.32%,96.07%±3.88%,94.65%±4.55%)明显高于对照组(88.94%±7.30%,P<0.01),而3组患者S期(4.63%±3.34%,3.45%±3.80%,5.12%±4.55%)和G2/M期(0.84%±1.52%,0.48%±0.74%,0.22%±0.34%)细胞比例明显低于对照组(9.06%±6.50%,2.00%±2.93%,P<0.05),3组S+G2/M期细胞比例明显高于对照组(P<0.01)。第二部分MDS患者骨髓造血细胞造血因子受体及部分转录因子的检测,对照组骨髓CD34+CD38+细胞亚群EpoR表达率(18.68%±18.34%)显著低于CD34+CD38细胞亚群(63.61%±19.98%,P<0.01),两亚群之间SCF-R.G-CSFR及TpoR表达率差异无统计学意义。在CD34+CD38+细胞亚群中,3组间SCF-R和TpoR表达率差异无统计学意义,而低危组和高危组EpoR的表达率(8.30%±6.55%、7.82%±7.98%)明显低于对照组(18.68%±18.34%,P<0.05),G-CSFR的表达率(29.78%±19.14%、28.66%±21.14%)明显低于对照组(44.37%±23.43%,P<0.05)；在CD34+CD38-细胞亚群中,3组间SCF-R和G-CSFR表达率差异无统计学意义,低危组和高危组EpoR的表达率(42.19%±21.87%、25.67%±15.64%)明显低于对照组(63.61%±19.98%,P<0.01),TpoR的表达率(5.42%±4.72%、4.05%±3.95%)明显低于对照组(10.13%±8.31%,P<0.05)。MDS患者骨髓CD34+CD38+和CD34+CD38-细胞亚群表面受体表达率低的患者其外周血血红蛋白水平、中性粒细胞及血小板计数减低的发生率明显高于受体表达率不低的患者(均P<0.05)。在CD34+CD38+细胞亚群中,低危组和高危组GATA-1的表达率高于对照组,差异无统计学意义,高危组GATA-2的表达率(12.47%±13.00%)明显高于对照组(5.64%±7.32%,P<0.05),余组间比较差异无统计学意义；在CD34+CD38-细胞亚群中,高危组GATA-1的表达率(28.16%±13.71%)明显高于对照组(12.76%±14.02%,P<0.05),低危组和高危组GATA-2的表达率(17.12%±10.61%、29.38%±7.71%)明显高于对照组(10.07%±9.26%,P<0.05)。GATA-1基因在高危组骨髓单个核细胞(BMMNC)中的表达量为(0.504±0.156),显著高于对照组(0.323±0.086,P<0.01),余组间比较差异无统计学意义。第三部分MDS骨髓干细胞中白细胞介素-3受体α(IL-3Rα)表达情况及IL-3Rα阳性细胞增殖及分化等特征的检测。低危组、高危组及AML组CD34+CD38细胞中CD123+细胞比例(42.48%±25.88%、51.62%±29.24%、56.19%±32.20%)明显高于对照组(9.06%±10.04%, P<0.01)。CD 123+CD34+CD38-/CD34+CD38表达率与恶性克隆负荷呈显著正相关(r=0.419,P=0.003),与MDS骨髓粒系、单核系及红系细胞分化模式和比例的异常数目呈显著正相关(r=0.462,P=0.001)。低危组、高危组及AML组CD34+CD38 CD 123+细胞中GATA.1.GATA.2及CD71表达率均明显高于其在CD34+CD38+CD123+细胞中的表达率(P<0.01)；3组CD34+CD38-CD123+细胞中EpoR表达率高于其在CD34+CD38-CD123细胞中的表达率,但差异无统计学意义；3组CD34+CD38-CD123+细胞中CDllb、CD114及Annexin V表达率均明显低于其在CD34+CD38-CD123-细胞中表达率(P<0.05)。结论(1)MDS患者骨髓粒系、单核系及红系细胞抗原不同步表达,表现为阶段特异性和系别特异性的异常,诸如成熟阶段细胞异常表达幼稚细胞抗原,SSC值降低即胞质低颗粒化\脱颗粒现象、连续性表达中断、抗原部分表达降低\缺失,髓系细胞异常表达淋巴系抗原等,并且由低危向高危进展的过程中存在分化异常的积累并与IPSS、WPSS积分及恶性克隆负荷成显著正相关。这提示分化抗原检测可能有助于MDS患者的早期诊断和预后判断。(2)MDS患者原代骨髓CD34+细胞亚群分化异常,CD34+CD38-/CD34+比例增高,并和IPSS、WPSS积分呈正相关性；骨髓单个核细胞存在G1期阻滞现象,提示MDS患者造血细胞增殖分化异常,CD34+细胞亚群和细胞周期测定有助于MDS患者的辅助诊断以及疗效和预后判断。(3)MDS患者原代骨髓CD34+细胞亚群膜表面部分造血细胞因子受体表达减低,CD34+细胞亚群转录因子GATA-1和GATA-2表达增高,这可能与MDS患者分化异常导致无效造血有关,且有望用于辅助诊断MDS。(4)MDS患者原代骨髓中CD34+CD38-CD123+细胞增高,并与恶性克隆负荷及骨髓造血.细胞分化异常项数目呈显著正相关,这些细胞表现为过度增殖、分化异常、部分造血因子受体的表达缺陷及凋亡逃逸等恶性特征,提示该团细胞可能是MDS恶性克隆细胞及最终转化为AML的恶性根源。更多还原

【Abstract】 ObjectiveThis study was to detect the differentiation abnormalities of bone marrow hematopoietic cells in myelodysplastic syndrome (MDS), its related mechanisms, MDS malignant clone cells and their malignant characteristics of excessive proliferation, abnormal differentiation and apoptosis escape.MethodsSixty-two cases of MDS patients,8 acute myeloid leukemias preceded by MDS (MDS-AML),7 acute myeloid leukemias and 16 case-controls, as well as 32 normal controls were enrolled in this study. PartⅠThe differentiation antigens on the membrane of bone marrow granulocytes, monocytes and erythroblasts, the subset of stem cell and bone marrow cell cycle were measured by flow cytometry. PartⅡThe expressions of stem cell factor receptor(SCF-R), erythropoietin receptor(EpoR), granulocyte colony-stimulating factor receptor (G-CSFR) and thrombopoietin receptor (TpoR) on hematopoietic stem/progenior cells and the expressions of GATA-1 and GATA-2 in nuclear of stem/progenitor cells were measured by flow cytometry. The expression of GATA-1 mRNA in the bone marrow cells of cases with MDS and normal controls were measured by RT-PCR. PartⅢThe expression of interleukin-3 receptorα(CD123) on the bone marrow stem cells of cases with MDS, AML and normal controls were measured by flow cytometry. Further to investigate the characteristics of proliferation and differentiation of CD123+ cells.ResultsPartⅠThe granulocytic differentiation was analyzed with the combinations of CD13/CD11b, CD13/CD16 and CD11b/CD16. The "right hook", "sickle" and "retroflex 7" shape expressions were found in normal and case controls while there were various changes in MDS groups. The ratios of CD11b-/CD11b+(0.32±0.29) and CD16-/CD16+(1.32±0.77) were significantly higher in high-risk MDS group than those in case-control group(0.05±0.03 and 0.33±0.32 respectively) and control group (0.07±0.05 and 0.39±0.31 respectively) (P<0.05). The MFI (mean fluorescence index) of SSC (side scatter) in the granulocyte gate of high-risk MDS group was significantly lower while MFI of CD13 was significantly higher than the case-control and control groups. The mean percentages of CD11b-HLA-DR+(4.08%±2.97%), CD11b-HLA-DR-(14.90%±15.44%), CD16-HLA-DR-(38.98%±21.59%), CDllb+CD16-(32.57%±16.52%) and CD13+CD16-(43.97%±20.31%) granulocytes of high-risk MDS group were significantly higher than those of low-risk, case-control and control groups (P<0.05). The monocytic differentiation was analyzed with the combinations of CD13/CD16 and CDllb/HLA-DR. There were various changes in MDS group comparison with case-control and control groups. The mean percentages of CD11b"HLA-DR+(12.38%±8.97%), CD11b+HLA-DR-(38.33%±18.43%) monocytes of high-risk MDS group were significantly higher than those of case-control and control groups (P<0.05). The mean percentage of CD14+ (56.74%±17.73%) on monocytes of high-risk MDS group was significantly lower than those of control group(P<0.05). The mean percentages of CD56+(32.45%±20.07%), CD34+(13.54%±13.73%) and CD7+(15.31%±12.76%) of high-risk MDS group were significantly higher than those of case-control and control groups (P<0.05). The erythroid cell lineage differentiation was analyzed with CD71/glycophorin A combination. Double positive expression was found in all case controls and controls, but asynchronous expression of CD71/glycophorin A was found in some MDS cases. The mean percentages of CD71+and GlyA+cells in CD45" cell population were significantly lower in low-risk and high-risk MDS groups. The mean percentages of CD71+GlyA+double positive cells in CD45", CD71+and GlyA+cell population were significantly lower in low-risk and high-risk MDS groups. The abnormal numbers of the antigen expression of MDS cases per case correlated directly with their IPSS (international prognostic scoring system) (r=0.662, P=0.000), WPSS (WHO adapted prognostic scoring system) (r=0.602, P=0.000) scores and malignant clone burden(r=0.477, P=0.001). The mean percentages of CD34+cells in bone marrow karyocyte of high risk(2.29%±2.17%) and MDS-AML groups(18.69%±17.47%) were significantly higher than that of control group (0.36%±0.49%, P<0.05). The mean percentages of CD34+CD38+cells were significantly lower in low risk, high risk and MDS-AML groups(86.09%±7.79%, 81.68%±11.82%and 82.88%±2.60% respectively) than that in control group (92.21%±3.85%, P<0.05), thus the percentages of CD34+CD38" cells were significantly higher in either MDS (low-risk and high-risk) or MDS-AML groups (13.91%±7.79%,18.32%±11.82%or 17.13%±2.60% respectively) than that in control group (7.79%±3.85%, P<0.05). The percentages of CD34+CD38’cells of MDS cases correlated directly with their IPSS (r=0.493, P=0.001) and WPSS (γ=0.586,P=0.000) scores. MDS patients with low percentages of CD34+CD38-(≤12.0%) cells presented higher therapeutic efficacy than those with high percentages of CD34+CD38’(> 12.0%) cells (P> 0.05), but not reveal significant differences. The percentages of bone marrow mononuclea cells(BMMNCs) in Go/Gi phase of in low-risk, high-risk and MDS-AML groups (94.52%±4.32%, 96.07%±3.88%and 94.65%±4.55%respectively) were significantly higher than that of control group(88.94%±7.30%, P<0.01), thus the percentages of BMMNCs in S and G2/M phase were significantly lower in either MDS (low-risk and high-risk) or MDS-AML groups than that in control group (P<0.05).Part II In control group, the mean percentage of antigen expression of EpoR was significantly lower in CD34+CD38+cells (18.68%±18.34%) than that in CD34+CD38- cells (63.61%±19.98%, P<0.01).The expressions of SCF-R,G-CSFR and TpoR were not significantly different between the two cell populations. The expression of EpoR on CD34+CD38+cells of low-risk and high-risk MDS groups(8.30%±6.55%,7.82%±7.98%) were significantly lower than that of control group(18.68%±18.34%, P<0.05). The expression of G-CSFR on CD34+CD38+cells of low-risk and high-risk MDS groups(29.78%±19.14%,28.66%±21.14%)were significantly lower than that of control group(44.37%±23.43%, P<0.05). The amount of EpoR on CD34+CD38- cells of low-risk and high-risk MDS groups(42.19%±21.87%,25.67%±15.64%) were significantly lower than that of control group(63.61%±19.98%, P<0.01), The expression of TpoR on CD34+CD38’ cells of low-risk and high-risk MDS groups(5.42%±4.72%,4.05%±3.95%) were significantly lower than that of control group(10.13%±8.31%, P<0.05). The incidence of cytopenia with low expression rates of hemopoietic cytokine receptors on CD34+ cells were higher than that of MDS with high expression rates of hemopoietic cytokine receptors. The expression of GATA-1 on CD34+CD38+cells of MDS groups were higher than that of control group, but not reveal significant differences.The expression of GATA-2 on CD34+CD38+cells of high-risk MDS group (12.47%±13.00%) was significantly higher than that of control group(5.64%±7.32%, P<0.05). The expression of GATA-1 on CD34+CD38- cells of high-risk MDS group (28.16％±13.71%) was significantly higher than that of control group(12.76%±14.02%, P<0.05). The expression of GATA-2 on CD34+CD38" cells of low-risk and high-risk MDS groups(17.12%±10.61%,29.38%±7.71%) were significantly higher than that of control group(10.07%±9.26%, P<0.05). The expression of GATA-1 mRNA in BMMNC of MDS patients (0.504±0.156) was significantly higher than that of normal controls (0.323±0.086, P<0.01).PartⅢThe ratios of CD34+CD38"CD123+/CD34+CD38- in the bone marrow cells of low-risk, high-risk MDS and AML groups (42.48%±25.88%,51.62%±29.24%, 56.19%±32.20%) were significantly higher than that of normal control (9.06%±10.04%, P<0.01). The ratio of CD34+CD38"CD123+/CD34+CD38’in MDS groups was significantly positively correlated with the malignant clone burden(r=0.419,P=0.003) and the abnormal numbers of the differentiation antigen expression (r=0.462, P=0.001). The expressions of GATA-1, GATA-2 and CD71 in CD34+CD38-CD123+cells were significantly higher than those in CD34+CD38" CD123-cells of MDS and AML groups(P<0.01). The expression of EpoR on CD34+CD38-CD123+cells was higher than that on CD34+CD38-CD123- cells, but not reveal significant differences. The expressions of CD11b, CD114 and Annexin V on CD34+CD38-CD123+cells were significantly lower than those on CD34+CD38-CD123-cells of MDS and AML groups(P<0.05).Conclusions(1) There are abnormal expressions of differentiation antigens on bone marrow myeloied cells of MDS patients. And the severity is correlated with the IPSS, WPSS scores and malignant clone burden. The abnormal differentiation of myeloid cells is probably involved in the pathogenesis of MDS. So the examination of these antigenic expressions with flow cytometry might be helpful for diagnosis and prognosis of MDS.(2) There was abnormalitie of differentiation of CD34+bone marrow cells and high proportion of G0/G1 cells which indicated a G1 phase arrest in MDS. That indicated there were abnormal characteristics of differentiation and proliferation in hematopoietic cells of MDS. So the examination of CD34+bone marrow cells and cell cycle might be helpful for MDS diagnosis and assessment of prognosis and therapeutic effect. (3) There were abnormalities of some membrane hemopoietic cytokine receptors and transcription factors of CD34+bone marrow cells in MDS, which were associated with MDS cytopenia and might be useful for MDS diagnosis.(4) The percentage of CD34+CD38-CD123+cells in the bone marrow of MDS patients was increased. These cells manifest the malignant characteristics of excessive proliferation, abnormal differentiation and apoptosis escape. CD34+CD38" CD123+cells were probably the malignant clone cells in MDS.更多还原