【作者】 席微波；

【导师】 刘玉峰；

【作者基本信息】 郑州大学 ， 儿科学， 2013， 硕士

【摘要】 研究目的通过对骨髓增生异常综合征(myelodysplastic syndromes,MDS)患者Mitoferrin-1、Mitoferrin-2和ABCB10在基因水平和蛋白水平的表达研究,揭示MDS患者线粒体铁超载的原因,探讨线粒体铁代谢异常在MDS发病中的作用,为祛铁治疗MDS提供依据。研究对象和方法1.选择MDS初诊患者(成人23例和儿童8例)为实验组,免疫性血小板减少症(ITP)初诊患者(成人12例和儿童8例)为本实验的近似健康对照组。实验组入选标准参照WHO(2008)MDS诊断分型,且要求未行输注红细胞和祛铁治疗；实验组和对照组均排除其它血液系统疾病、严重感染、甲状腺功能亢进等铁代谢异常的疾病。2.用无菌注射器分别抽取实验组和对照组的骨髓液,迅速涂片数张,后再抽取骨髓液各2-4ml,并迅速打入EDTA抗凝管中。3.分别抽取实验组和对照组清晨空腹静脉血各3ml,肝素抗凝,送至我院生化室检测血清铁代谢指标。4.分别对实验组和对照组骨髓细胞涂片行铁染色,光学显微镜下观察骨髓细胞外铁、内铁的分布,记录并采集图像保存。5.应用密度梯度离心法分离骨髓单个核细胞,并分装2管分别用于总RNA和蛋白的提取,于-80℃保存。6.参照Trizol试剂说明书提取骨髓单个核细胞中总RNA,检测RNA的纯度及完整性；应用Fermentas第一链反转录试剂盒合成cDNA;采用Real-time PCR测定Mitoferrin-1mRNA. Mitoferrin-2mRNA和ABCB10mRNA的表达。7.使用RIPA裂解液提取骨髓单个核细胞中总蛋白,应用Western Blot分别测定Mitoferrin-1. Mitoferrin-2和ABCB10蛋白的表达。结果1.成人MDS患者Mitoferrin-1mRNA、Mitoferrin-2mRNA和ABCB10mRNA的表达与对照组比较均无明显统计学差异(P>0.05)。2.(1)成人低危组即MDS-RA、MDS-RARS患者Mitoferrin-1蛋白的表达高于对照组,差异有统计学意义(P<0.001)；(2)高危组RAEB患者Mitoferrin-1蛋白的表达与对照组比较,差异无统计学意义(P>0.05)；(3)RARS患者Mitoferrin-1蛋白的表达高于RA,差异有统计学意义(P<0.001)(4)MDS患者Mitoferrin-2蛋白的表达与对照组比较,差异无统计学意义(P>0.05)；(5)低危组ABCB10蛋白的表达高于对照组,差异有统计学意义(P<0.001)；(6)高危组ABCB10蛋白的表达与对照组比较,差异无统计学意义(P>0.05)；(7)RARS患者ABCB10蛋白的表达高于RA,差异有统计学意义(P<0.001)。3.MDS患儿Mitoferrin-1mRNA. Mitoferrin-2mRNA和ABCB10mRNA的表达与对照组比较均无明显统计学差异(P>0.05)。4.(1)2例RA患儿Mitoferrin-1蛋白的表达均高于对照组；(2)RAEB患儿Mitoferrin-1蛋白的表达与对照组相比,差异无统计学意义(P>0.05)；(3)MDS患儿Mitoferrin-2蛋白的表达与对照组相比,差异无统计学意义(P>0.05)；(4)2例RA患儿ABCB10蛋白的表达均高于对照组；(5)RAEB患儿ABCB10蛋白的表达与对照组相比,差异无统计学意义(P>0.05)。结论1.MDS患者Mitoferrin-1和ABCB10在基因水平上的表达较对照均无明显增高,但在蛋白水平上的表达呈现出异质性,低危组(RA、RARS)表达高于对照组,高危组(RAEB)与对照组相比无明显增高。2.MDS患者Mitoferrin-2在基因和蛋白水平上的表达较对照均无明显增高。3.MDS患者Mitoferrin-1的表达可能受转录后的调节,在ABCB10介导下不适当的稳定性的增加可能参与了部分MDS(尤其是RARS)患者线粒体铁超载的发生。4. Mitoferrin-2可能与MDS线粒体铁超载的发生无关。更多还原

【Abstract】 ObjectiveThe aim is to study the expressions of Mitoferrin-1,Mitoferrin-2and ABCB10at the gene and protein level respectively in patients with myelodysplastic syndromes(MDS),in order to reveal the reasons for mitochondrial iron overload in MDS patients,to explore the role of mitochondrial iron metabolism abnormalities in the pathogenesis of MDS,and to provide the basis for iron chelation therapy.Object and Methods1. To select newly diagnosed MDS patients (23cases of adults and8cases of children) for the experimental roup,immune thrombocytopenia (ITP) of newly diagnosed patients (12adults and8children) as the similar healthy control group of this experiment. The inclusion criteria of experimental group refered to the2008World Health Organization (WHO) reclassification of myelodysplastic syndromes (MDS). To be enrolled in this study,patients had to be previously untreated with red blood cell transfusions and iron chelating agents.The experimental and control group were excluded from other blood diseases,severe infection,thyroid hyperfunction and others with abnormal iron metabolism.2. To extract bone marrow fluid from the experimental group and the control group with sterile syringe,smear a few pieces quickly,extract each2-4ml of bone marrow fluid again and put into EDTA anticoagulant tube fastly.3. Each3ml of fasting venous blood was extracted from the experimental and control group in the early morning,anticoagulant with heparin and sent to the biochemistry department of our hospital to detect serum iron metabolism indicators.4. Bone marrow smears of the experimental and control group were done with iron staining respectively.To observe the distributions of extracellular iron and intracellular iron,record and capture images to save.5. To separate bone marrow mononuclear cells by density gradient centrifugation, distribute into2tubes for the extraction of total RNA and protein,and store them at-80℃.6. To extract the total RNA from bone marrow mononuclear cells with reference to the Trizol reagent manual,detect the purity and integrity of the extracted RNA.To synthesize the cDNA by applying Fermentas first strand reverse transcription kit. To detect the expressions of Mitoferrin-1,Mitoferrin-2and ABCB10mRNA by real-time PCR.7. To extract the total protein from bone marrow mononuclear cells using RIPA lysis buffer. To detect the expressions of Mitoferrin-1,Mitoferrin-2and ABCB10protein by Western Blot.Results1. The expressions of Mitoferrin-1mRNA,Mitoferrin-2mRNA and ABCB10mRNA in adult MDS patients compared with the control group showed no statistically significant difference (P>0.05).2.(1)The expression of Mitoferrin-1protein in adult low-risk MDS(RA and RARS) was higher than the control group,and the difference was statistically significant (P<0.001);(2) the expression of Mitoferrin-1protein in high-risk MDS(RAEB) compared with the control group showed no statistically significant difference (P>0.05);(3) the expression of Mitoferrin-1protein in RARS was higher than RA,and the difference was statistically significant (P<0.001);(4) the expression of Mitoferrin-2protein in MDS patients compared with the control group showed no statistically significant difference (P>0.05);(5) the expression of ABCB10protein in low-risk MDS was higher than the control group,and the difference was statistically significant (P<0.001);(6) the expression of ABCB10protein in high-risk MDS compared with the control group showed no statistically significant difference (P>0.05).(7) the expression of ABCB10protein in RARS was higher than RA,and the difference was statistically significant (P<0.001).3. The expressions of Mitoferrin-1mRNA,Mitoferrin-2mRNA and ABCB10mRNA in MDS children compared with the control group showed no statistically significant difference (P>0.05).4.(1)The expression of Mitoferrin-1protein in2cases of RA children was higher than the control group;(2) the expression of Mitoferrin-1protein in RAEB children compared with the control group showed no statistically significant difference (P>0.05);(3) the expression of Mitoferrin-2protein in MDS children compared with the control group showed no statistically significant difference (P>0.05);(4) the expression of ABCB10protein in2cases of RA children was higher than the control group;(5) the expression of ABCB10protein in RAEB children compared with the control group showed no statistically significant difference (P>0.05).Conclusion1. The expressions of Mitoferrin-1and ABCB10in MDS were not significantly higher than the control at the gene level.The expressions of Mitoferrin-1and ABCB10in MDS showed heterogeneity at the protein level,higher than the control in low-risk group (RA, RARS), no significantly higher than the control in high-risk group(RAEB).2. The expression of Mitoferrin-2in MDS was not significantly higher than the control at both gene and protein level.3. The expression of Mitoferrin-1in MDS may be regulated by post-transcriptional mechanism.The inappropriate increased stabilization of Mitoferrin-1mediated by ABCB10may be involved in the occurrence of mitochondrial iron overload in some patients of MDS(especially RARS).4. Mitoferrin-2may not be responsible for the occurrence of mitochondrial iron overload in MDS.更多还原