【作者】 赵春红；

【导师】 侯明；

【作者基本信息】 山东大学 ， 内科， 2009， 博士

【摘要】 特发性血小板减少性紫癜（idiopathic thrombocytopenic purpura,ITP）是临床最为常见的出血性、自身免疫性疾病,约占出血性疾病总数的30%。以外周血小板减少、骨髓巨核细胞正常或增多伴成熟障碍为主要表现。目前,治疗以长期使用肾上腺皮质激素、免疫抑制剂及脾切除等方法为主,容易引发感染、骨髓抑制等,并且约有1/3的患者上述治疗无效,迁延不愈,甚至危及生命。本病发病机制复杂,至今尚未完全阐明。长期以来由自身抗体介导的血小板破坏增多被看成是ITP的主要病因。即血小板膜表面糖蛋白（glycoprotein,GP）作为自身抗原,特别是GPⅡb/Ⅲa和GPⅠb/Ⅸ复合物,刺激机体免疫系统产生特异性自身抗体,通过其Fab段与自身抗原结合,致敏的血小板容易被机体的网状内皮系统所清除,特别是脾脏中的单核吞噬细胞吞噬破坏,从而导致血小板减少。但是ITP发病具有明显的异质性。随着研究的深入,体液免疫异常在ITP发病过程中起决定性作用的观点已日益受到挑战。由于血小板自身抗体只能在50%-70%ITP患者中检测到,并且阴性结果不能排除诊断。多数研究认为ITP患者呈Th1优势反应模式,而Th1优势反应除可促进抗体产生外,还能直接促进淋巴细胞的细胞毒作用。因此,近年来细胞免疫功能失调在ITP发病中的作用越来越受重视。研究发现ITP患者外周血CD₈⁺细胞增多,CD₄⁺/CD₈⁺下降。Olsson等应用基因芯片技术发现在ITP患者与细胞毒作用有关的基因以及与Th1细胞反应有关的基因表达增高,提示细胞介导的细胞毒作用可能在ITP发病中起重要作用。随后,Olsson等和张峰等的体外实验证实细胞毒性T淋巴细胞（cytotoxic T lymphocyte,CTL,CD8⁺T）能直接参与对ITP患者血小板的杀伤,导致血小板持续破坏。目前普遍认为,除血小板破坏增多外,血小板生成减少也参与ITP的发病。血小板动力学研究及骨髓巨核细胞形态学检查均证实ITP患者的血小板生成减少。既然巨核细胞表面亦表达GPⅡb/Ⅲa和GPⅠb/Ⅸ,血小板自身抗体同样可以作用于巨核细胞,影响巨核细胞生成或导致巨核细胞在骨髓内被活化的补体或吞噬细胞破坏。多项研究证实,纯化的血小板膜糖蛋白抗体能够抑制巨核细胞集落的增殖,进而影响血小板生成。Chang等和McMillan等发现ITP患者血浆和纯化的血小板自身抗体可抑制体外巨核细胞生长,导致巨核细胞数量明显降低。充分证明血小板特异性抗原的自身抗体不仅介导外周血小板破坏,还影响骨髓中巨核细胞的生长和发育成熟。但是多数ITP患者骨髓中巨核细胞数量正常或增多,其血小板生成减少不能用巨核细胞数量降低解释。血小板生成与成熟巨核细胞的凋亡密切相关,巨核细胞凋亡异常即会引起血小板生成障碍。对ITP患者骨髓巨核细胞超微结构的研究表明,患者的巨核细胞存在广泛的异常,这些异常的巨核细胞发生与凋亡相似但不同于凋亡的程序性细胞死亡,是导致血小板生成减少的重要原因。我们首先应用改良MAIPA技术检测ITP患者血浆中的特异性血小板膜糖蛋白（GPⅡb/Ⅲa、GPⅠb/Ⅸ）自身抗体水平,以双色流式细胞技术为基础的细胞毒实验技术检测ITP患者及正常对照CTL对自身血小板的杀伤效应,比较MAIPA阳性和阴性ITP患者CTL对自体血小板的杀伤及地塞米松的作用。我们还将ITP患者血浆及其组分与正常脐血CD34⁺细胞共同孵育,定向扩增巨核细胞,观察ITP血浆及其组分对巨核细胞和血小板形成的影响。第一部分细胞毒作用在ITP发病及地塞米松治疗中的意义研究目的:比较MAIPA阳性和阴性ITP患者细胞毒T淋巴细胞对自体血小板的杀伤及地塞米松的作用,分析自身细胞毒杀伤血小板与抗血小板自身抗体的关系。方法:*抽取48例ITP患者和17例正常人的外周血15ml,其中患者标本分别在大剂量地塞米松治疗前和治疗后两周采样。*改良MAIPA检测ITP患者血浆中抗GPⅡb/Ⅲa、GPⅠb/Ⅸ自身抗体。*从外周血中分离单个核细胞和血小板。*用免疫磁珠分离技术分离CD8⁺细胞。*以自身血小板为靶细胞,以CD8⁺细胞为效应细胞的细胞毒实验。*用CD41a和Annexin V标记血小板,流式细胞仪分析被Annexin V标记的血小板比例（血小板裂解率）,以间接反映效应细胞的杀伤能力。*在ITP患者MAIPA阳性组与阴性组间进行细胞毒作用的比较。结果:*经改良MAIPA技术检测,48例慢性ITP患者血浆中,22例（MAIPA阳性组）一种以上抗体阳性,26例（MAIPA阴性组）抗体阴性。*大剂量地塞米松治疗前,ITP患者两组的血小板裂解率均明显高于正常对照组（P＜0.05）。ITP患者MAIPA阴性组血小板裂解率明显高于MAIPA阳性组,两者差异有显著性（P＜0.05）。*大剂量地塞米松治疗前,在ITP患者MAIPA阴性组,血小板计数和血小板裂解率呈负性相关（r=-0.439,P＜0.05）;而在MAIPA阳性组两者无显著相关性（r=-0.322,NS）。*大剂量地塞米松治疗后,ITP患者两组的血小板裂解率均明显低于治疗前（P＜0.05）。结论:*证实了细胞介导的细胞毒作用参与了ITP患者血小板破坏,而且在检测不到抗GPⅡb/Ⅲa、Ⅰb/Ⅸ抗体的ITP患者发病中发挥更大作用。*地塞米松可能有抑制CTL介导的细胞毒杀伤血小板作用。保护自身血小板免受细胞介导的细胞毒作用是HD-DXM发挥治疗作用的机制之一。*干预CTL介导的血小板破坏可能成为ITP治疗的一个新靶点第二部分ITP患者血浆对体外巨核细胞和血小板形成的影响及机制探讨目的:研究ITP血浆及其组分对正常脐血CD34⁺细胞定向扩增巨核细胞和血小板形成的影响。方法:*留取49例ITP患者和22例正常人的外周血血浆。*用免疫磁珠分离技术分离脐血CD34⁺细胞。*亲和层析法提取ITP患者和正常对照血浆IgG。* ITP患者血浆的自身抗体吸附。* CD34⁺细胞与ITP患者或正常对照血浆、IgG、自身抗体吸附后血浆共同孵育,定向扩增巨核细胞。*流式细胞仪检测CD41a的表达。*检测培养体系中生成的血小板。* CD41a⁺细胞DNA倍体分析。* CD41a⁺细胞凋亡检测。* CD41a⁺细胞Bcl-xL检测。*流式细胞仪检测CD41a⁺细胞的周期蛋白B1或周期蛋白D3的表达。结果:*ITP患者血浆、IgG、自身抗体吸附后血浆对巨核细胞生成的影响。平行检测49例ITP患者血浆、IgG、非IgG对巨核细胞生成的影响。培养第15天时,26例ITP患者血浆孵育下的CD41a⁺细胞数＞正常血浆孵育组的（?）+1s[（5.10+0.90）×10⁵],定义为A组;14例ITP患者血浆孵育下的CD41a⁺细胞数＜正常血浆孵育组的（?）-1s[5.10—0.90)×10⁵],定义为B组;余9例介于正常血浆孵育组（?）-1s与x+1s之间,定义为C组。IgG作用下的A组[（3.15±0.93）×10⁵]和B组[（3.02±1.01）×10⁵]的巨核细胞生成,显著低于C组[（4.57±0.78）×10⁵]和正常对照组[（4.90±0.48）×10⁵]（P＜0.05）。ITP患者血浆自身抗体吸附后,A组[（7.85±1.30）×10⁵]的巨核细胞数量升至甚至超过正常对照水平[（5.36±0.58）×10⁵]（P＜0.05）。*多数患者ITP血浆促使巨核细胞生成增加,但成熟障碍和血小板生成减少,且抑制凋亡。A组[16.35%±4.90%,（7.51±2.41）×10³]和B组[16.110%±5.66%,（7.21±2.45）×10³]的多倍体和血小板计数,均显著低于正常对照[24.57%±2.83%,（11.21±1.82）×10³]和C组[24.66%±2.49%,（10.12±1.91）×10³]（P＜0.05）。并且A组[21.88%±3.53%]的巨核细胞凋亡比B组[27.36%±4.31%]、C组[28.21%±4.02%]和正常对照[29.43%±3.80%]显著受抑（P＜0.05）。同时,A组血浆作用下的巨核细胞Bcl-xl高表达。*多数ITP患者的IgG抑制巨核细胞生成,且成熟障碍和血小板生成减少,但不影响凋亡。A组[14.12%±6.09%,（5.95±2.27）×10³]和B组[15.68%±5.98%,（6.15±2.37）×10³]的倍体分布和血小板释放,显著低于C组[23.14%±2.27%,（9.85±1.61）×10³]和正常对照组[23.98%±2.23%,（10.97±1.92）×10³]（P＜0.05）。组A[28.63±4.32]、B[29.42±4.72]和C[29.30±5.55]的巨核细胞凋亡,与正常对照[30.93±3.86]无显著差别。* ITP患者的IgG作用下的巨核细胞周期蛋白B1/D3表达异常。周期蛋白D3在正常对照组培养第9天出现低水平表达（3.5%）,而ITP患者IgG组在培养第12天仍无表达。周期蛋白B1在正常对照组培养第9天出现表达降低的现象（第5天44.3%,第9天21.6%）,而ITP患者IgG组表达量较正常对照低并且未出现表达降低的现象（第5天21.4%,第9天20.5%）。*多数ITP患者的自身抗体吸附后血浆促使巨核细胞生成,无成熟障碍和血小板生成减少,但抑制凋亡。A组[22.30%±2.86%,（9.77±2.28）×10³]的倍体分布和血小板释放升至甚至超过正常对照水平[23.42%±2.17%,（10.28±2.76）×10³]（P＜0.05）。而与B组[30.24%±3.71%]、C组[30.08%±3.83%]和正常对照[30.73%±3.99%]比较,A组[22.44%±3.56%]的巨核细胞凋亡显著受抑（P＜0.05）。结论:*部分ITP患者的IgG可能在体外抑制巨核细胞生成并使周期蛋白B1/D3异常表达而使巨核细胞成熟障碍。*巨核细胞凋亡减少可使巨核细胞形成受抑及血小板生成减少,提示巨核细胞凋亡异常参与ITP发病。更多还原

【Abstract】 Idiopathic thrombocytopenic purpura（ITP） is one of the most common forms of autoimmune disease characterized by a low platelet count and normal or increased number of megakaryocytes in bone marrow.It is usually a persisting disease,which relapses frequently and requires a long-term treatment.In some cases,it progresses rapidly and even threatens the patients’ survival.The severe side effects of routine treatment lead to a poor prognosis.The pathogenic mechanism of ITP is not completely clear yet.The earliest studies have suggested that in the majority of patients,thrombocytopenia is mediated by antiplatelet autoantibodies,in which anti-GPⅡb/Ⅲa and GPⅠb/Ⅸantibodies are generally considered to be the most common ones.Binding of autoantibodies to these target antigens eventually results in platelet destruction by the reticuloendothelial system.Since platelet autoantibodies can be detected in only 50-70%of ITP patients and remission can occur despite the presence of platelet autoantibodies,there must be other mechanisms.Olsson et al reported several cytotoxic genes,such as Apo-1/Fas, granzyme B and perforin,together with genes involve in the Th1 cell response,such as interferon-γand interleukin-2 receptor-β,showed increased expression in the ITP patients.Most recently,in vitro studies suggested that cytotoxic T-lymphocyte（CTL, CD8⁺） may be involved in the pathogenesis of chronic ITP through cell-mediated destruction of autologous platelets.On the other hand,platelet kinetic studies and morphologic alterations of ITP marrow megakaryocytes suggest that megakaryocytopoiesis and thrombopoiesis may be disrupted.Since the target antigens are present on both platelets and their precursors,megakaryocytes,it is possible that megakaryocytopoiesis and thrombopoiesis are also impaired during ITP,which could further aggravate the thrombocytopenia caused initially by increased peripheral destruction of platelets. Recent in vitro studies,showing reduced megakaryocyte production and maturation in the presence of autoantibodies against platelet glycoproteins in ITP plasma,provide evidence for autoantibody-induced suppression of megakaryocytopoiesis.In normal physiology platelet production and mature megakaryocyte apoptosis are closely related events.In disease,however,the decreased megakaryocyte apoptosis might disrupt platelet formation.Growing evidence suggests that ITP megakaryocytes demonstrate predominantly characteristics of apoptosis-like programmed cell death which contribute to thrombocytopenia.Whether the difference exits in CTL-mediated cytotoxicity toward autologous platelets between ITP patients with and without anti-platelet antibodies remains unknown.In the present work,we evaluated autoantibodies against GPⅡb/Ⅲa and GPⅠb/Ⅸin ITP patients and observed the cytotoxic effect of CTL toward autologous platelets.In addition,we prospectively measured the effect of high dose dexamethasone（HD-DXM） on CTL-mediated cytotoxicity in ITP patients.Besides,we investigated the effect of plasma from patients with ITP on in vitro megakaryocyte production.ⅠEffects of CTL-mediated cytotoxicity on the onset of ITP and mechanism of action of dexamethasone.Objective:To investigate the difference in cytotoxicity toward autologous platelets between ITP patients with and without autoantibodies against GPⅡb/Ⅲa and GPⅠb/Ⅸ,we measured platelet autoantibodies,and performed cytotoxic T cell assay in 48 patients. In addition,we prospectively measured CD8⁺ cytotoxic T-lymphocyte mediated cytotoxicity in patients during treatment with high dose dexamethasone.Methods:* 48 chronic ITP patients were enrolled in this study.Blood sampling was performed before and after treatment at the end of the second week with high dose dexamethasone.* Platelet autoantibodies against GPⅡb/Ⅲa and GPⅠb/Ⅸwere detected by modified monoclonal antibody specific immobilization of platelet antigens（MAIPA） assay.* Platelets were prepared from peripheral blood.* CD8⁺T lymphocytes were positively selected using CD₈⁺ magnetic microbeads, according to the manufacture’s recommendations（Miltenyi Biotec）.* The CD8⁺T lymphocytes used as effector cells and autologous platelets used as target cells were incubated for 4 h.To stimulate cytolytic effector T-cells, anti-CD₃-antibody was added.* Cells were labeled with PEcy5-CD41a mAb,PEcyS-IgG1 used as isotype control, incubated with FITC-Annexin V mAb,and then analyzed with a Becton Dickinson FACScan flow cytometer.Logarithmic amplifiers were used for fluorescence signals, and 10 000 events were collected.CD41a⁺ cells were gated and recognized as platelets,and apoptotic platelets were Annexin V⁺ cells within that population.Results were expressed in percent and analyzed using the CellQuest software.The specific lysis was calculated according to the following equation:induced lysis - spontaneous lysis,and expressed in%.Results:* In the plasma of 48 patients with ITP,antibodies against GPⅡb/Ⅲa and/or GPⅠb/Ⅸwere detected in 22 samples（groupⅠ）.Negative reactions to both glycoproteins were displayed in the remainders（groupⅡ）.* Before HD-DXM treatment,positive platelet lysis was seen in 11 of groupⅠand 21 of groupⅡ（groupⅠvs.groupⅡ,P＜0.05）,while 4 of groupⅠand 7 of groupⅡafter treatment（groupⅠvs.groupⅡ,no significant（NS）;pretreatment vs.posttreatment in groupⅠ,P＜0.05,and in groupⅡ,P＜0.01）.On the other hand,before treatment,both groupⅠand groupⅡhad increased platelet lysis compared with controls（groupⅠvs. controls,P＜0.05;groupⅡvs.controls,P＜0.01;groupⅠvs.groupⅡ,P＜0.01）,whereas platelet lysis was substantially decreased in both groups after treatment（pretreatment vs.posttreatment in groupⅠ,P＜0.01,and in groupⅡ,P＜0.01）.* Additionally,the platelet lysis was found to be negatively correlated with the platelet count in groupⅡ（r=-0.439,P＜0.05）,but not in groupⅠ（r=-0.322,NS）.Conclusions:We conclude that cytotoxicity toward autologous platelets is predominant in autoantibody-negative ITP patients and could be thwarted by HD-DXM,and protection against CTL-mediated destruction might be considered as a new therapeutic approach for ITP.ⅡEffects of plasma from patients with ITP on in vitro megakaryocytopoiesis and platelet production.Objective:To investigate the effects of plasma from patients with ITP on in vitro megakaryocyte production and platelet production.Methods:* Plasma samples were obtained from 49 patients with chronic ITP and 22 healthy blood donors.* IgG antibody was purified from plasmas by affinity chromatography.* Patient plasma was mixed with the washed control platelets and incubated.* CD34⁺ cells were purified from healthy umbilical cord blood mononuclear cells （MNCs） obtained after Ficoll-Hypaque gradient centrifugation by using a magnetic cell separation method.* CD34⁺ cells were cultured in medium containing thrombopoietin,stem cell factor, interleukin-3,and 10%plasma or purified IgG or antibody-adsorbed plasma from patients or controls.* Megakaryocytes were recognized as CD41a⁺ events by fluorescence-activated cell sorter（FACS）.* Platelet count was analyzed in cultured cells.*Megakaryocyte ploidy was measured.CD41a⁺ cells were gated and ploidy distribution was assessed by the intensity of the PI fluorescence.* Apoptosis in megakaryocytes was measured using the Annexin V-FITC Apoptosis Detection Kit according to the manufacturer’s instructions.* Bcl-xl expression in megakaryocytes was performed by first incubating the cells with PEcy5-conjugated CD41a mAb.After staining,cells were fixed in 1% paraformaldehyde,permeabilized with 0.1%saponin,and incubated with FITC-conjugated Bcl-xl.CD41a⁺ cells were gated and Bcl-xl expression was shown as mean fluorescence intensity（MFI） within that population.*To analyze Cyclin B1/Cyclin D3 expression in megakaryocytes,PEcy5-CD41a mAb labeled cells were incubated with FITC-Cyclin B1/FITC-IgG1,or FITC-Cyclin D3/ FITC-IgG1κ.The labeled cells were resuspended after washing and analyzed within two hours using flow cytometer.Results:* Role of plasma or purified IgG or antibody-adsorbed plasma in megakaryocyte productionWith twenty-six ITP plasma samples（group A）,the number of megakaryocyte in the day- 15 culture was more than mean+1SD[（5.10+0.90）×10⁵]of control cultures. With fourteen ITP plasma samples（group B）,the yield of megakaryocyte was less than mean-1SD[（5.10-0.90）×10⁵]of control cultures.With other nine ITP plasma samples（group C）,the yield of megakaryocyte was between mean-1 SD and mean+1SD of control cultures.Megakaryocyte production was significantly suppressed in cultures containing IgG from both group A[（3.15±0.93）×10⁵]and B[（3.02±1.01）×10⁵]plasmas in comparison with cultures containing IgG from group C[（4.57±0.78）×10⁵]and control plasmas [（4.90±0.48）×10⁵]. After adsorption of autoantibody from patient plasmas,a significant increase in megakaryocyte number was seen in cultures with adsorbed group A plasmas [（7.85±1.30）×10⁵]than those cultured with control plasmas[（5.36±0.58）×10⁵].* More ITP plasmas boosted megakaryocyte mass with impaired maturation, decreased platelet production,and inhibited apoptosis.The percentage of polyploidy（N≥4） and the platelet count in cultures with both group A[16.35%±4.90%,（7.51±2.41）×10³],and B[16.11%±5.66%,（7.21±2.45）×10³]ITP plasmas were significantly lower than those in control[24.57%±2.83%, （11.21±1.82）×10³]and group C[24.66%±2.49%,（10.12±1.91）×10³]cultures （P＜0.05）.On the other hand,reduced megakaryocyte apoptosis was observed in group A [21.88%±3.53%]cultures compared with that in group B[27.36%±4.31%],group C [28.21%±4.02%]and control[29.43%±3.80%]cultures（P＜0.05）.Meanwhile, megakaryocytes in different group cultures all showed a degraded expression of Bcl-xL during the culture process with a significantly higher level in group A cultures （P＜0.05）.* Patient IgG suppressed megakaryocyte yield,maturation and ability to produce platelet with no impact on apoptosis.A significant reduction in ploidy distribution and platelet release was seen in cultures containing IgG from both group A[14.12%±6.09%,（5.95±2.27）×10³]and B [15.68%±5.98%,（6.15±2.37）×10³]plasmas in comparison with cultures containing IgG from group C[23.14%±2.27%,（9.85±1.61）×10³]and control plasmas [23.98%±2.23%,（10.97±1.92）×10³].Besides,no significant difference in megakaryocyte apoptosis was seen between cultures containing patient IgG（from group A,B and C plasmas） and those containing control IgG.* More autoantibody-adsorbed plasmas increased megakaryocyte production not accompanied with more polyploidy cells and platelet produced,but inhibited apoptosis.After adsorption of autoantibody from patient plasmas,polyploidy percentage and platelet release rose to control level and even more[22.30%±2.86%,（9.77±2.28）×10³] than those cultured with control plasmas[23.42%±2.17%,（10.28±2.76）×10³].However,a significant reduced megakaryocyte apoptosis was observed in group A [22.44%±3.56%]cultures compared with that in group B[30.24%±3.71%],group C [30.08%±3.83%]and control[30.73%±3.99%]cultures（P＜0.05）.* Abnormal expression of cyclin B1/D3 in megakaryocytes cultured with patient IgGCultured with control IgG,megakaryocytes expressed cyclin B1 with a reduction from 44.3%to 21.6%at day 9 and then began to express cyclin D3（3.5%）.Whereas cultured with patient IgG（from group A and B）,megakaryocytes expressed cyclin B1 at a persistently low level（20.5%） and almost did not express cyclin D3 during the whole culture time including day 12.Conclusions:Our findings show that IgG in some ITP plasmas suppresses in vitro megakaryocytopoisis and abnormal expression of cyclin B1/D3 might be the mechanism by which patient IgG inhibits megakaryocyte maturation.On the other hand,decreased apoptosis of megakaryocyte also contributes to in vitro dysmegakaryocytopoiesis and reduced platelet production,suggesting that inhibited megakaryocyte apoptosis may be involved in the pathogenesis of ITP.更多还原