【作者】 丁凯；

【导师】 邵宗鸿；

【作者基本信息】 天津医科大学 ， 内科学（专业学位）， 2013， 博士

【摘要】 目的：1.检测免疫相关性全血细胞减少症(immuno-related pancytopenia, IRP)患者自身抗体IgG在骨髓造血细胞膜上的作用靶点,探索IRP患者造血细胞膜上可能的靶抗原成分,为进一步纯化和克隆IRP患者自身抗原提供依据。2.检测已发现的红系膜抗原促红细胞生成素受体(erythropoietin receptor, EPOR)在树突状细胞(dendritic cell, DC)上的表达,以期初步验证IRP中抗原呈递的过程。方法：1.研究对象为32例初治IRP患者作为实验组,15例再生障碍性贫血(AA)患者作为病例对照组及15名正常健康者作为对照。FCM检测骨髓造血细胞膜抗体,提取细胞膜蛋白,同时使用辛酸法+盐析法提纯骨髓上清液中的IgG,然后使用免疫印迹法检测IRP患者骨髓上清液中自身抗体IgG的阳性率,进而应用聚丙烯酰胺凝胶电泳结合免疫印迹法分离和鉴定IRP患者自身抗体IgG在细胞膜上的靶抗原,并与正常对照及病例对照组进行对比；选取目标蛋白条带进行高压液相串联质谱(LC-MS/MS)分析以确定靶抗原成分。并对所鉴定出的部分蛋白成分检测其在造血细胞膜上的异常表达。2.研究对象为39例骨髓有核红细胞抗体阳性(GlycoA+)患者、30例GlycoA-患者、及17名正常健康者作为对照。使用FCM检测骨髓DC细胞胞膜及胞浆中EPOR的表达,并进行统计学分析,进一步使用RT-PCR的方法检测不同组病例骨髓除红系后造血细胞中EPOR的mRNA表达的差异。结果：1. IRP患者组骨髓上清液中IgG的阳性率为50%(16/32),明显高于病例对照组(0%)和正常对照组(13%，2/15)(均P<0.01)。在32例初治IRP患者中,其骨髓造血细胞膜上共有两种蛋白成分可被自身抗体IgG识别,其相对分子量分别为72-95kDa及55kDa,阳性率分别为56.25%(9/16)和31.25%(5/16)。切割蛋白条带进行高压液相串联质谱分析,发现乳铁蛋白和WD重复序列蛋白两种膜蛋白成分,其中乳铁蛋白第121位精氨酸存在替代点突变,被丝氨酸或天门冬氨酸所取代。正常对照组10-25kDa蛋白条带未鉴定成功。进一步检测乳铁蛋白在有核红细胞膜上的表达。GlycoA+组患者骨髓有核红细胞乳铁蛋白的平均阳性率为13.44±3.53%,明显高于GlycoA组患者的3.22±2.32%(P<0.001)及正常对照组的0.54±0.48%(P<0.001),后两者之间亦存在明显差异(P<0.05)。2. EPOR在骨髓DC细胞中的表达。a) GlycoA+组与GlycoA进行比较。i.DCl细胞膜上EPOR的表达,GlycoA+组表达率1.45±5.45%与GlycoA-组表达率9.89±4.03%及正常对照组表达率1.06±2.06%无明显差异,而后两者之间存在统计学差异(P=0.016)。DC1胞浆中EPOR的表达,GlycoA+组表达率24.67±31.15%,明显高于GlycoA组表达率8.45±3.04%(P<0.05)及正常对照组表达率12.45±28.46%(P=0.001),后两者之间无统计学差异。ii.DC2细胞膜上EPOR的表达,GlycoA+组表达率10.47±5.26%,明显高于正常对照组表达率0.94±2.67%(P<0.05),与GlycoA-组表达率13.23±7.27%无明显差异。DC2胞浆中EPOR的表达,GlycoA+组表达率30.85±11.20%,明显高于GlycoA组表达率15.44±6.53%(P<0.05)及正常对照组表达率10.34±4.88%(P=0.001),后两者之间亦有明显差异差异(P<0.05)。b)使用上述数据对两实验组进行组内DC1与DC2的比较、ⅰ. GlycoA+组,DC1与DC2胞膜EOPR表达率均明显低于胞内(P均<0.05)。DC2细胞EPOR表达率无论在细胞膜上还是在胞浆中均明显高于DC1细胞的表达(P<0.05)。ⅱ. GlycoA组DC1与DC2的EPOR胞膜胞浆上的表达均无明显差异。c)骨髓去除红系后细胞,三组EOPR的mRNA均未见表达。结论：1.IRP患者骨髓上清液中存在自身抗体IgG,可作用于骨髓细胞膜上多个靶抗原成分,乳铁蛋白及WD重复序列蛋白就是其中两种可能的抗原,且部分乳铁蛋白存在第121为精氨酸序列的替代点突变,被丝氨酸或天门冬氨酸所替代。另外IRP患者的有核红细胞膜上的确存在本不定位于其细胞膜的乳铁蛋白的异常表达。2. GlycoA+的IRP患者其DC2细胞胞浆中存在EPOR的异常表达,与其细胞膜表达并不匹配,可能是早期呈递EPOR作为IRP患者造血细胞靶抗原的关键细胞。更多还原

【Abstract】 Objective:1.To investigate the auto-antigens targeted by auto-antibodies IgG on the membrane of bone marrow hematopoietic cells of the patients with immuno-related pancytopenia(IRP),for further purifying and cloning them.2. To investigate the expression of erythropoietin receptor (EPOR), which is a previously discovered auto-antigen, on dendritic cells (DCs) for elucidating the antigen presenting procedure in IRP patients.Method:1.32newly diagnosed IRP patients and15aplastic anemia patients were enrolled in the study, as well as15healthy donors as controls. The antibody IgG was examined by flow cytometry, and then the total membrane protein was extracted. At the same time, the IgG in the marrow supernatant was purified by organic acid and salting out method. The IgG in supernatant was also examined by western blot. And the bone marrow cell membrane auto-antigens targeted by IgG in IRP were identified from membrane protein extracts by SDS-PAGE, Western blot and liquid chromatography-mass spectrography/mass spectrography. Furthermore, the abnormal expression of a newly discovered auto-antigen on the membrane of bone marrow nucleared erythroid cells was investigated.2.39newly diagnosed GlycoA+IRP patients and30GlycoA-IRP patients were enrolled in the study, as well as17healthy donors as controls. The expression of EPOR on DCs (both on cytomembrane and in cytoplasm) was investigated, and then statistically analyzed. Furthermore, the mRNA expression of EPOR in bone marrow cells without erythroid cells was examined by PT-PCR.Result:1. Autoantibody IgG reacting with bone marrow cell membrane antigens could be found in bone marrow supernatant of IRP patients in a positive rate of50%(16/32), which was significantly higher than those of aplastic anemia(AA)(0%) or normal controls(13%,2/15)(P<0.01). Autoantibody IgG in IRP could react with several auto-antigens with approximate MWs of72-95kDa及55kDa. Positive rates of each auto-antigen were56.25%(9/16) 和31.25%(5/16) respectively. The positive bands on the gel were cut, and identified by liquid chromatography-mass spectrography/mass spectrography; the result suggested that the two proteins were lactoferrin (Lf) and WD repeat-containing protein. Furthermore, part of the discovered lactoferrin had the point replaced mutation in the amino acid of Arg121, which replaced by Ser or Asp. The10-25kDa proteins in normal controls were failed to be identified. And then, the expression of lactoferrin on the membrane of marrow nucleared erythroid cells was examined. The expression rate in GlycoA+group patients is13.44±3.53%, which is significantly higher than that in the GlycoA-group patients’3.22±2.32%(P<0.001) or controls’0.54±0.48%(P<0.001), and the later two also has significant difference.2. The expression of EPOR in DCs.a) Comparison between GlycoA+group and GlycoA-group,ⅰ. The expression rate of EPOR on the membrane of DC1was1.45±5.45%in the GlycoA+group,9.89±4.03%in the GlycoA-group, and1.06±2.06%in control group, except the later two had significant difference (P=0.016), no difference between other groups. And the expression rate of EPOR in the cytoplasm of DC1was24.67±31.15%in the GlycoA+group, which is significantly higher than that in GlycoA-group (8.45±3.04%, P<0.05) and control group (12.45±28.46%, P=0.001), there is no significant difference between the later two groups.ⅱ. The expression rate of EPOR on the membrane of DC2was10.47±5.26%in the GlycoA+group, which is significantly higher than that in control group (0.94±2.67%, P<0.05), but compared with the GlycoA+group’s13.23±7.27%, there is no significant difference. And the expression rate of EPOR in the cytoplasm of DC2was30.85±11.20%in the GlycoA+group, which is significantly higher than that in GlycoA-group (15.44±6.53%, P <0.05) and control group (10.34±4.88%, P=0.001), the later two also has significant difference (P<0.05) b) According to the date above, the expression comparisons were done in each group.i. In GlycoA+group, on the membrane of both DC1and DC2, the expression rate of EPOR is significantly lower than that in cytoplasm (P<0.05). The expression in DC2is significantly higher than that in DC1, both on the membrane and in cytoplasm,ii. In GlycoA-group, there is no difference between each group.c) The mRNA expression of EPOR in bone marrow cells without erythroid cells are all negative in the three groups.Conclusion:1. There are auto-antibodies in the bone marrow supernatant of IRP patients, which can target several antigens on hematopoietic cells’ membrane, and lactoferrin and WD repeat-containing protein just two probable candidates. But part of the lactoferrin had the point replaced mutation in the amino acid of Arg121. Furthermore, there is abnormal expression of lactoferrin on the bone marrow erythroid membrane of GlycoA+IRP patients.2. The expression of EPOR in the cytoplasm of DC2was abnormally higher in GlycoA+patients group than that in other groups. However, the EPOR expression on the membrane of DC2and in the cytoplasm of it is not match, suggesting that DC2may be a critical factor in the early stage of EPOR antigen presenting.更多还原