【作者】 张薇；

【导师】 邵宗鸿；

【作者基本信息】 天津医科大学 ， 内科学， 2012， 博士

【摘要】 目的从免疫表型、分子生物学等方面寻找与骨髓增生异常综合征(Myelodysplastic Syndromes, MDS)异常造血克隆相关分子标志,并研究该分子标志在MDS中的作用及其临床意义。方法研究对象为天津医科大学总医院自2010年3月至2012年3月新诊断的MDS患者63例、急性髓系白血病(AML)13例及正常对照40名。第一部分采用流式细胞术(FCM)检测白介素-3受体α(CD123)和粘附因子CD96在MDS患者骨髓中的表达情况,从免疫表型方面寻找MDS异常造血克隆的分子标志,并检测CD96阳性细胞的异常特征。第二部分采用半定量RT-PCR的方法检测TET2(TET癌基因家族成员2)基因在MDS患者骨髓中的表达情况,并分析其临床意义,从分子生物学角度探寻恶性克隆的分子标志。采用荧光定量PCR的方法检测TET2和DLK1(delta like1)基因在MDS患者骨髓中CD3+和CD34+细胞中的表达情况,并分析其临床意义。第三部分体外培养正常对照骨髓CD34+细胞,采用RNA干扰(RNA interference, RNAi)技术敲低TET2的表达；应用流式细胞术检测评价细胞增殖和凋亡等生物学特征变化,以探讨其在MDS中的作用。第四部分体外培养MDS患者骨髓CD34+CD38-细胞,采用RNA干扰(RNAinterference, RNAi)技术敲低DLK1的表达；应用流式细胞术检测评价细胞增殖和凋亡等生物学特征变化,进一步明确该分子标志在MDS异常造血克隆中的作用。结果第一部分MDS患者CD34+CD38-细胞占CD34+的比例为(22.72±23.91)%,显著高于正常对照组(8.63±9.35)%(P<0.01),低于AML组(25.15±22.70)%；MDS患者骨髓CD34+CD38-细胞中CD96+及CD123+细胞比例(21.18±24.06,31.62±28.42)%显著高于正常对照组(11.23±17.91,9.29±12.63)%(P<0.01)。高危MDS组CD34+CD38-细胞中CD96+及CD123+细胞比例(26.25±27.64,37.56±31.01)%显著高于低危组(11.44±10.88,15.53±10.96)(P<0.05);CD34+CD38-CD96+细胞中CD114(7.23±10.25%),CD110(0.52±1.23%)及AnnexinV(2.96±3.84%)表达率均明显低于其在CD34+CD38-CD96-细胞中表达率(24.35±21.74%,12.03±10.91%,13.69±17.98%,P<0.05)。第二部分MDS患者BMMNC中TET2mRNA表达明显低于正常对照(P<0.05),MDS各亚组TET2mRNA表达无差异。MDS患者TET2mRNA表达≥0.9者原始细胞比例(1.04%±1.68%)明显低于<0.9者(6.13%±8.17%,P<0.05)。以骨髓异常染色体核型细胞数目占总分裂像细胞数目的比值作为MDS患者恶性克隆负荷,TET2基因的相对表达量随恶性克隆负荷增高而降低(r=-0.398,P<0.05)；并随IPSS指数的增高而降低(r=-0.480,P<0.05)。MDS患者骨髓CD3+T细胞中TET2mRNA表达是正常对照组的(0.16±0.15)倍(P<0.001)；TET2mRNA表达水平与血清补体C3呈显著正相关(r=0.404,P<0.05)；MDS患者骨髓CD3+T细胞中DLK1mRNA表达是正常对照组的(1.61±0.88)倍(P<0.05)；依据染色体分组,染色体异常组DLK1表达水平是染色体正常组的(1.45±0.44)倍(P<0.05)；DLK1mRNA表达水平与骨髓原始细胞比例呈显著正相关(r=0.343,P<0.05)。MDS患者骨髓CD34+细胞中TET2mRNA表达显著低于正常对照组[(0.58±0.26)vs(1.25±0.94)](P<0.005)；MDS患者骨髓CD34+细胞中DLK1mRNA表达显著高于正常对照组[(2.72±1.02)vs(1.31±1.00)](P<0.001)。第三部分正常人骨髓CD34+细胞转染siRNA靶向TET2后,TET2被敲低,其mRNA表达及蛋白表达水平下降；20例正常对照者CD34+细胞对照组和siRNA-scr转染组细胞G0/G1期分别(93.60±5.54)%和(92.65±7.06)%,S期分别为(5.95±5.53)%和(6.92±7.04)%；G2/M期分别为(0.53±1.07)%和(0.30±0.42)%；siRNA-TET2转染组细胞G0/G1期为(90.82±8.25)%,S期细胞增加为(8.50±8.31)%,G2/M期为(0.47±0.89)%,G0/G1期和S期比例与对照组差别具有统计学意义(P<0.005)。siRNA-TET2转染组细胞凋亡率较CD34+细胞对照组和siRNA-scr转染组细胞凋亡率显著降低[(23.28±9.73)%vs(26.20±9.78)%vs(26.17±9.88)%](P<0.05)。第四部分MDS患者骨髓CD34+CD38-细胞转染siRNA靶向DLK1后,DLK1被敲低,其mRNA表达及蛋白表达水平下降；23例MDS患者骨髓CD34+CD38-细胞对照组和siRNA-scr转染组细胞G0/G1期分别(91.22±10.82)%和(91.29±10.39)%,S期分别为(8.38±10.65)%和(8.41±10.33)%；G2/M期分别为(0.40±0.55)%和(0.34±0.49)%；siRNA-DLK1转染组细胞G0/G1期为(95.81±3.87)%,S期细胞降低为(3.90±3.61)%,G2/M期为(0.29±0.46)%,G0/G1期和S期比例与对照组差别具有统计学意义(P<0.05)。siRNA-DLK1转染组细胞凋亡率较CD34+CD38-细胞对照组和siRNA-scr转染组细胞凋亡率显著增加[(38.97±14.32)%vs(32.94±12.64)%vs(33.48±12.44)%](P<0.001)。结论(1)MDS患者骨髓中CD34+CD38-CD96+细胞与CD34+CD38-CD123+细胞增高,这些细胞存在分化异常、凋亡逃逸等恶性特征,且与骨髓原始细胞比例呈正相关。(2)TET2基因在MDS患者骨髓单个核细胞、CD3+T细胞及CD34+细胞中表达减低,提示TET2基因可能是MDS的“保护基因”,其表达水平可作为评价MDS患者病情的辅助指标；MDS患者骨髓CD3+T细胞及CD34+细胞中DLK1mRNA表达增高,提示DLK1基因在MDS发生发展过程中起到一定作用。同时MDS患者骨髓CD3+T细胞TET2及DLK1基因的表达异常,提示CD3+T细胞有“质”的异常,这可能是导致机体免疫监视功能下降的机制之一。(3)敲低TET2后,正常人骨髓CD34+细胞凋亡率降低,细胞周期S期增加,具有恶性变的倾向,由此推测TET2表达不足是导致髓系恶性肿瘤形成的机制之一。(4)敲低DLK1后,MDS患者骨髓CD34+CD38-细胞恶性程度减低,即凋亡增加,S期减少,G0/G1期阻滞,由此推测DLK1表达增加可能是导致MDS发生发展的机制之一。更多还原

【Abstract】 ObjectiveTo explore the molecular markers of the abnormal hematopoietic clone in myelodysplastic syndromes and its related mechanisms involving immunophenotyping and cytogenetics.MethodsSixty-three cases of MDS, thirteen cases of acute myeloid leukemias as well as forty normal controls were enrolled in this study. First Section The expressions of CD96and CD123on CD34+CD38-bone marrow cells(BMC) of these patients and controls were measured by FACS.CD114(GCSFR), EPOR and CD110(TPOR) expression on their CD34+CD38-CD96+and CD34+CD38"CD96" BMC and these cells’ apoptosis(Annexin V) were also detected by FACS. Second Section The expression of TET2mRNA in the bone marrow cells of cases with MDS and normal controls were measured by RT-PCR, aiming to search for the cytogenetic marker of malignant clone in MDS. CD3+T cells and CD34+cells were sorted by magnetic activated cell-sorting system. The mRNA expressions of TET2and DLK1in bone marrow CD3+T cells and CD34+cells were detected by qPCR. Third Section Normal CD34+cells were sorted by magnetic activated cell-sorting system. The silencing effect in vitro of RNA interference (RNAi) on TET2expression in normal CD34+cells was identified by qPCR and Western blot analysis. The cell cycle kinetics and apoptosis of these cells were detected by flowcytometry. Fourth Section CD34+CD38-cells in MDS were sorted by magnetic activated cell-sorting system. The silencing effect in vitro of RNA interference (RNAi) on DLK1expression in CD34+CD38-cells of MDS patients was identified by qPCR and Western blot analysis. The cell cycle kinetics and apoptosis of these cells were detected by flowcytometry.ResultsFirst Section MDS patients had significant higher proportion of CD34+CD38-/CD34+BMC((22.72±23.91)%) than normal controls ((8.63±9.35)%)(P<0.01). The expressions of CD34+CD38-CD96+/CD34+CD38-BMC and CD34+CD38-CD123+/CD34+CD38-BMC of MDS patients ((21.18±24.06,31.62±28.42)%) were significantly higher than those of normal controls ((11.23±17.91,9.29±12.63)%)(P<0.01). The expressions of CD34+CD38-CD96+/CD34+CD38-BMC and CD34+CD38-CD123+/CD34+CD38BMC in high-risk MDS group((26.25±27.64,37.56±31.01%) was significantly higher than those in low-risk group((11.44±10.88,15.53±10.96%)(P<0.05). The expression levels of CD123and CD96were significantly correlated with the proportion of bone marrow blasts(r=0.384, P<0.05; r=0.625, P<0.05).The expressions of CD114, CD110and Annexin V in CD34+CD38-CD96+BMC ((7.23±10.25,0.52±1.23,2.96±3.84)%) were significantly lower than those in CD34+CD38-CD96-BMC ((24.35±21.74,12.03±10.91,13.69±17.98)%) of MDS patients (P<0.05).Second Section The expression of TET2mRNA was down-regulated in MDS compared with controls(P<0.05). There was no significant difference in expression of TET2among RA, RCMD and RAEB cases. Patients with higher expression of TET2(≥0.9) presented significantly lower proportion of bone marrow blasts (1.04±1.68)%than those with lower expression (<0.9) of TET2(6.13±8.17)%(P<0.05). The expression of TET2mRNA in BMMNC of MDS patients was significantly positively correlated with their malignant clone burden (r=-0.398, P<0.05) and IPSS (r=-0.480, P<0.05). The expression of TET2mRNA in CD3+T cells was down-regulated in MDS (0.16±0.15) fold compared with that in normal controls (P<0.05). The expression of TET2mRNA in CD3+T cells of MDS patients was positively correlated with serum complement C3(r=0.404, P<0.05). The expression of DLK1mRNA in CD3+T cells was up-regulated in MDS (1.61±0.88) fold compared with that in controls (P<0.05). Grouped by karyotype, patients with abnormal karyotype presented significantly (1.45±0.44) fold higher of DLK1mRNA expression than those with normal karyotype (P<0.05). The expression of DLK1mRNA in CD3+T cells of MDS patients was positively correlated with their proportion of bone marrow blasts (r=0.343, P<0.05). The expression of TET2mRNA in CD34+cells was down-regulated in MDS compared with that in normal controls [(0.58±0.26)vs(1.25±0.94)](P<0.005). The expression of DLK1mRNA in CD34+cells was up-regulated in MDS compared with that in normal controls[(2.72±1.02)vs(1.31±1.00)](P<0.001).Third Section The siRNA targeting TET2suppressed the expression of TET2in normal CD34+cells in vitro. Meanwhile, the proliferation activity was significantly enhanced [G0/G1phase:(90.82±8.25)%,(92.65±7.06)%,(93.60±5.54)%; S phase:(8.50±8.31)%,(6.92±7.04)%,(5.95±5.53)%; G2/M phase:(0.47±0.89)%,(0.30±0.42)%,(0.53±1.07)%. G0/G1phase and S phase was significantly different between control cells and cells which transfected with TET2siRNA (P<0.005)] And the apoptosis rate was declined in cells which transfected with TET2siRNA as compared to those of the cells transfected with scrambled siRNA and control cells [(23.28±9.73)%vs (26.20±9.78)%vs (26.17±9.88)%, the apoptosis rate was statistically significant between control cells and cells which transfected with TET2siRNA (P<0.05)].Fourth Section The siRNA targeting DLK1suppressed the expression of DLK1in MDS patients’CD34+CD38" cells. Meanwhile, the proliferation activity was significantly reduced [G0/G1phase:(95.81±3.87)%,(91.29±10.39)%,(91.22±10.82)%; S phase:(3.90±3.61)%,(8.41±10.33)%,(8.38±10.65)%; G2/M phase:(0.29±0.46)%,(0.34±0.49)%,(0.40±0.55)%. G0/G1phase and S phase was statistically significant between control cells and cells which transfected with DLK1siRNA (P<0.05)]. And the apoptosis rate was inclined in cells which transfected with DLK1siRNA as compared to those of the cells transfected with scrambled siRNA and control cells [(38.97±14.32)%vs(32.94±12.64)%vs (33.48±12.44)%], the apoptosis rate was statistically significant between control cells and cells which transfected with DLK1siRNA (P<0.05)].Conclusions(1) There were higher proportion of CD34+CD38-CD96+and CD34+CD38-CD123+cells in bone marrow of MDS patients. Those cells expressed lower cytokine receptors and displayed lower apoptosis. Leukemia stem cells(LSC) in MDS might be contained in those cells.(2) The mRNA expressions of TET2in BMMNC、CD3+T cells and CD34+cells of MDS patients decreased, and the mRNA expression of DLK1in CD3+T cells and CD34+ cells was increased, which might useful as an important indicator for the evaluation of MDS clone burden and an important indicator to explore the mechanism of immune tolerance.(3) Suppression of TET2expression renders the CD34+cells harboring more aggressive phenotype. This preliminary finding suggests that CD34+cells lowering expression of TET2may play an oncogenic role on myeloid tumor.(4) Suppression of DLK1expression renders the CD34+CD38" cells harboring lower aggressive phenotype. This preliminary finding suggests that CD34+CD38-cells with over-load expression of DLK1may play an oncogenic role in myeloid tumor.更多还原