【作者】 王一浩；

【导师】 邵宗鸿； 付蓉；

【作者基本信息】 天津医科大学 ， 内科学， 2009， 博士

【摘要】 目的初步探讨骨髓单个核细胞库姆(BMMNC-Coombs)试验(+)血细胞减少(又称免疫相关性全血细胞减少，immunorelated pancytopenia，IRP)患者骨髓造血细胞自身抗体IgG破坏和／或抑制造血机制。方法选取IRP患者为研究对象，以重型再生障碍性贫血(SAA)及正常人分别为病例及正常对照，进行下述研究：第一部分IRP患者骨髓红系造血岛(EI)形成机制。(1)采用流式细胞术(FACS)检测IRP患者骨髓细胞(CD34+、CD15+及GlycoA+细胞)膜抗体(IgG、IgM)类型；(2)采用免疫荧光(IF)法标记48例IgG(+)的IRP患者骨髓EI，观察自身抗体IgG在EI分布；(3)探讨EI自身抗体分布与IRP患者病情相关性。第二部分骨髓巨噬细胞(M_Φ)数量、活化抗原表达及功能研究。(1)采用流式细胞术(FACS)检测IRP患者骨髓细胞(CD34+、CD15+及GlycoA+细胞)膜抗体(IgG、IgM)类型；(2)采用FACS检测61例IgG(+)IRP患者骨髓M_Φ数量[(CD68+／CD45+)％]、活化抗原(CD69)表达水平[(CD68+CD69+／CD68+)％]；(3)采用鸡红细胞(CRBC)吞噬试验评价骨髓M_Φ吞噬率和吞噬指数。第三部分骨髓造血细胞膜功能受体封闭研究。FACS检测骨髓GlycoA(+)细胞膜EPO受体(EPOR)和自身抗体IgG；在含不同浓度EPO的培养基中培养骨髓干祖细胞，7天检测红系集落(CFU-E)产率。结果第一部分(1)48例IRP患者中有14例(29．17％)骨髓“EI”的IF阳性，即EI中巨噬细胞和幼红细胞间沉积自身抗体IgG。(2)这14例患者骨髓GlycoA(+)细胞膜上均(100％)测及自身抗体IgG，贫血是首发症状，以重度贫血为主[重度贫血9例(64．29％)，中度贫血5例(35．71％)]；余34例未测及EI中有自身抗体IgG沉积者仅2例(5．89％)，其贫血程度轻。(3)14例EI有自身抗体IgG沉积患者的网织红细胞比例[(2．0±0．8)％]、胸骨红系比例[(44．1±13．9)％]及间接胆红素水平[(9．4±4．7)mmol／1]均明显高于EI中无IgG沉积者[(1．3±1．0)％，(29．8±8．2)％，(6．6±6．7)mmol／l](P＜0．05)，而前者的血红蛋白水平[(59．6±16．2)g／l]低于后者[(83．4±25．0)g／l](P＜0．05)。(4)EI有自身抗体IgG沉积患者3、6个月总有效率(57．14％、85．71％)优于EI无自身抗体IgG沉积患者(P＜0．05)(32．26％、61．29％)。第二部分61例IRP患者骨髓M_Φ数量、活化抗原表达、CRBC吞噬率及吞噬指数[(0．57±0．30)％，(40．30±18．49)％，(37．56±15．20)％和(0．75±0．34)]均高于10例SAA患者[(0．46±0．08)％，(32．44±19．37)％，(28．26±10．46)％和(0．59±0．39)]及13名正常人[(0．44±0．69)％，(29．71±11．67)％，(25．63±14．75)％和(0．55±0．16)](P＜0．05)；且M_Φ数量与活化抗原表达、吞噬率及吞噬指数均呈正相关(r=0．89，p＜0．01；r=0．43，p＜0．01；r=0．40，p＜0．01)。根据M_Φ数量将IRP患者分为A组(M_Φ≥0．5％)和B组(M_Φ＜0．5％)，A组34例患者中32例(94．12％)自身抗体为IgG，B组27例患者中仅2例(7．41％)自身抗体为IgG；A组患者M_Φ的活化抗原表达、CRBC吞噬率及吞噬指数[(49．19±16．63)％，(46．62±13．38)％，(0．91±0．36)]显著高于B组患者[(29．11±14．30)％，(28．67±12．59)％，(0．61±0．30)](P＜0．05)，而B组患者与SAA及正常人无显著性差异(P＞0．05)。将34例BMMNC-IgG(+)型患者分为M_Φ高(≥0．75％)、低水平(＜0．75％)2组，25例(73．53％)M_Φ低水平患者均仅能检测到一系骨髓细胞(CD34+／或CD15+／或GlycoA+)有IgG，9例(23．53％)M_Φ高水平患者有8例能检测到二系骨髓细胞有IgG，1例(2．94％)是三系骨髓细胞均有IgG；M_Φ高水平的IRP患者M_Φ的活化抗原表达、CRBC吞噬率及吞噬指数[(56．12±15．11)％，(60．22±12．51)％，(1．23±0．23)]显著高于M_Φ低水平患者[(44．58±18．16)％，(43．32±9．24)％，(0．84±0．24)](P＜0．05)。M_Φ高水平患者外周血红细胞计数、血红蛋白水平、血小板计数均显著低于M_Φ低水平患者(P＜0．05)；M_Φ高水平患者的网织红细胞比例、胆红素水平及胸骨红系比例均显著高于M_Φ低水平患者(P＜0．05)。第三部分(1)24例骨髓GlycoA(+)细胞膜IgG(+)的IRP患者骨髓GlycoA(+)细胞膜EPOR水平[(4．16±0．15)％]显著低于10名正常人[(6．04±0．48)％](P＜0．05)。(2)这些IRP患者骨髓细胞体外培养，CFU-E产率与EPO浓度的关系呈两种形式：EPOR低水平组1的拟合方程为：y=1．0028x-2．0056，(P＜0．05，g＜0．05)，回归检验有显著性，失拟检验回归方程拟合良好，呈线性关系，提示有增长趋势；EPOR低水平组2数据行拟和检验结果：F=0．25，P=0．6264，g=0．16，为非线性关系，无增长变化趋势；正常人拟合方程为：y=0．68915x-1．9783(P＜0．05，g＜0．05)，回归检验有显著性，失拟检验回归方程拟合良好，呈线性关系，提示有增长趋势。结论IRP患者自身抗体IgG抑制和／或破坏骨髓造血的机制可能是：(1)激活M_Φ吞噬和破坏带有自身抗体IgG的骨髓造血细胞导致骨髓造血功能衰竭；(2)部分自身抗体IgG通过与骨髓造血细胞膜功能抗原(如EPOR)结合，使骨髓造血细胞缺乏必需造血生长因子刺激从而影响其增殖和分化。更多还原

【Abstract】 OBJECTIONSTo explore the pathogenetic mechanism of destruction and/or supp-ression of hematopoiesis by auto-antibodies(IgG)on bone marrow cellsin pancytopenia patients with positive BMMNC-Coombs Test(immunorelatedpancytopenia,IRP).METHODSIRP cases,severe aplastic anemia (SAA)and normal controls wereenrolled in this study.The categories of auto-antibodies(IgG、IgM)onBMMNC (CD34+/CD15+/GlycoA+ hematocytes)were assayed by fluorescenceactivated cell sorting (FACS).First Section The auto antibodies(IgG)in the“EI”on the BM smears of forty-eight cases with IRP、ten SAA andeleven normal controls were detected by immuno-histofluorescence (IF)staining.Clinical and laboratory data of all cases and controls werealso analyzed respectively.Second Section Sixty-one cases with IRP,tenSAA and thirteen normal controls were enrolled in this study.The quantity(the percent of CD68+/CD45+)and the expression of activated antigens (thepercent of CD69+CD68+/CD68+)of macrophages in the bone marrow of allcases and controls were measured by FACS and the function of macrophageswere evaluated according to the phagocytosis ratio and index of red bloodcells(CRBC)phagocytosed by macrophages which was cultured in vitro.Third Section EPO receptors(EPOR)on GlycoA(+)BMMNCs of IRP patientswith IgG(+)on GlycoA(+)BMMNCs were measured by FACS.The hemopoieticprecursors of BM were cultured in different levels of erythropoietin (EPO)in vitro and colony-forming units-erythoid (CFU-E)were observed at7-day-culture. RESULTSFirst Section (1)Mouse anti-human IgG could be detected on the BMsmear of 14 cases(29.17%),which deposited on the junction betweenmacrophagesanderythroblasts in the“EI”s,IgGon theGlycoA(+)cellsthese 14 cases were all founded.The initial clinical feature of the 14cases with auto antibodies(IgG)in the EI was anemia〔severe anemia (64.29%),middle degree anemia (35.71%)〕,concentration of hemoglobin〔(59.6±16.2)g/l〕in peripheral blood of cases with auto antibodies(IgG)inthe EI were lower than those〔(83.4±25.0)g/l〕of 34 cases without autoantibodies(IgG)in the EI (P＜0.05),but the percent ofreticulocyte(Ret)count〔(2.0±0.8)%〕、the ration of erythroid in the bone marrow fromsternum〔(44.1±13.9)%〕and the level of IBIL of the formers〔(9.4±4.7)mmol/l)higher (P＜0.05)(P＜0.05);Cases with auto antibodies (IgG)in the EI had good reponse to high doses of IVIG (HDIVIG)or /andGlucocorticoids therapies.Total curative rate at 3 and 6 month(indivi-dually 57.14%、85.71%)of cases with auto antibodies(IgG)in the EIwere better than those of the cases without auto antibodies(IgG)in theEI (P＜0.05)respectively.Second Section the quantity,expression ratioof activatedantigen,phagocytosisratioandindex〔(0.57±0.30)%,(40.30±18.49)%,(37.56±15.20)%和(0.75±0.34)〕of BM macrophages in IRPpatients were respectively significantly higher than those in all thecontrol cases〔(0.46±0.08)%,(32.44±19.37)%,(28.26±10.46)%和(0.59±0.39)〕(P＜0.05).And the quantity present high-positive correlationwith the expression ratio of activated antigen phagocytosis ratio andindex of BM macrophages (r=0.89,p＜0.01;r=0.43,p＜0.01;r=0.40,p＜0.01).Patients with IRP were classified into two subgroups according to the quantity of macrophages:Group A(M_Φ≥0.5%)(34casese)and Group B(M_Φ＜0.5%)(27cases),32 cases (94.12%)with auto antibodies(IgG)in Group A,only 2 cases(7.41%)with auto antibodies(IgG)in Group B.There weresignificant differences in expression ratio of activated antigen,phagocytosis ratio and index of macrophages between Group A〔(49.19±16.63)%,(46.62±13.38)%,(0.91±0.36)〕andGroupB〔(29.11±14.30)%,(28.67±12.59)%,(0.61±0.30)〕(P＜0.05).Thirty-four cases of IRP wasdivided into two subgroups according to the quantity of M_Φ:high levelgroup(≥0.75%)(25cases)and Low level group (＜0.75%)(9cases),24 casesin M_Φhigh level group with auto antibodies(IgG)on one hemotopoietic celllineage,l on two lineages,while 8 cases in M_ΦLow level group with autoantibodies(IgG)on two cell lineages,and l on three cell lineages.Theexpression ratio of activated antigen,phagocytosis ration and index ofmacrophages were much higher in high level group〔(56.12±15.11)%,(60.22±12.51)%,(1.23±0.23)〕than those〔(44.58±18.16)%,(43.32±9.24)%,(0.84±0.24)〕in low level group(P＜0.05).The count of RBC、concentration of HbandBPCin peripheral blood of high level group wererespectively lower than those in low level group(P＜0.05).While thepercentage of Ret,the level of TBIL and the ratio of erythroid of sternalbone marrow in high level group were higher than those in low levelgroup(P＜0.05).Third Section The level of EPO receptors(EPOR)onGlycoA(+)BMMNCs of IRP patients with IgG(+)on GlycoA(+)BMMNCs〔(4.16±0.15)%〕was lower than that of normal controls〔(6.04±0.48)%〕(P＜0.05).There were two different relationships between CFU-E and EPO level.Therewere twenty patients in Group 1 and four cases in Group2.The equationtest of goodness of fit in Grouplwasy=l.OO28x-2.0056,(P＜0.05,g＜0.05). There was no linearity correlation between CFU-E and EPO level (F=0.25,P=0.6264,g=0.16)in Group2.The equation test of goodness of fit in normalcontrol was y=0.68915x-1.9783 (P＜0.05,g＜0.05).CONCLUSION(1)Macrophage connected with erythroblasts by auto antibodies(IgG)in the“EI”s of some of IRP patients.These“EI”s were not niches oferythroblastic developmentment and differenttiation but where macroph-ages devoured and destroyed erythroblasts in vivo.Macrophages didn’t nurse but devour erythroblasts.The pathogentic mechanism of IRP withauto antibodies(IgG)in the EI may be that macrophages devoured morehematopoietic cells.(2)The quantity、expression ratio of activatedangtigen and phagocytosis ratio and index of macrophages were enhancedin IRP patients with auto antibodies(IgG).When the numbers of hematopoi-eric cells with auto antibodies(IgG)of bone marrow phogocytosed bymacrophages which were activated were more than those of the hematopo-ietic cells which are produced by bone morrow,bone marrow failure canoccur.Macrophages might not involved in destruction of bone marrow inIRP with auto antibodies(IgM)or cold autoantibodies.(3)Maybe autoantibodies (IgG)which combinated with EPOR on hematopoietic cells blockEPO-EPOR signation and hematopoietic cells could not proliferate anddifferentiate,then the absence of EPO causes bone marrow failure.更多还原