TIM-3在免疫性血小板减少症发病机制中的初步研究

【作者】 杜伟廷；

【导师】 杨仁池；

【作者基本信息】 中国协和医科大学 ， 内科学， 2009， 博士

【摘要】 课题一TIM-3在免疫性血小板减少症发病机制中的作用第一部分TIM-3在ITP患者免疫细胞中的表达变化研究背景及目的:免疫性血小板减少症(Immune Thrombocytopenia,ITP)是一种获得性器官特异性自身免疫性疾病。患者体内产生抗血小板自身抗体,导致网状内皮系统破坏吞噬血小板,从而引起血小板减少。尽管目前认为ITP的发病机制是由自身抗体介导的,但T细胞对于自身抗体的产生起着非常重要的作用,因此T细胞调节功能的紊乱可能在ITP发病中发挥重要致病作用。对于T细胞的异常目前已有大量报道,包括血小板特异的自身反应性T细胞的活化,增强的针对血小板的细胞毒作用,调节性T细胞的缺失等。T细胞免疫球蛋白粘蛋白分子-3(T-cell immunoglobulin mucin-3,TIM-3)是一种Ⅰ型膜表面蛋白分子,属于新近发现的T细胞免疫球蛋白粘蛋白分子家族的一员。TIM-3分子只选择性表达在分化的Th1细胞而不是Th2细胞上,可以作为新的区分Th1和Th2细胞的表面标志。随着研究的深入,人们发现TIM-3也表达在固有免疫细胞中,提示TIM-3可能在自身免疫反应中发挥重要作用。大多数研究认为ITP是Th1介导的自身免疫性疾病,而TIM-3分子选择性表达在分化的Th1细胞上,本研究旨在明确TIM-3是否在ITP患者中存在异常。研究方法:通过荧光定量PCR法和流式细胞术检测ITP患者外周血单个核细胞中TIM-3表达的变化,并探讨TIM-3表达水平与各种临床参数间是否存在联系。同时,我们分析了ITP患者外周血CD4+T细胞,单核细胞和树突状细胞中TIM-3的表达变化。抗CD3/CD28抗体刺激CD4+T细胞后检测T细胞凋亡的变化。结果:荧光定量PCR法和流式细胞术结果均显示:活动期ITP患者组外周血单个核细胞中TIM-3表达显著低于正常对照组,而缓解期患者TIM-3表达明显升高,并且TIM-3表达水平与患者血小板数量呈显著正相关。活动期患者CD4+T细胞和单核细胞中TIM-3表达下调,而树突状细胞中TIM-3表达升高。缓解组患者CD4+T细胞和单核细胞中TIM-3表达升高,而树突状细胞中TIM-3表达下调。抗CD3/CD28抗体刺激后,活动期患者CD4+T细胞发生凋亡比例减少。结论:活动期ITP患者组CD4+T细胞和单核细胞中TIM-3表达下调,而树突状细胞中TIM-3表达升高。同时,在刺激后CD4+T细胞发生凋亡比例减少,提示这可能与CD4+T细胞中TIM-3表达下调有关。因此,TIM-3在ITP的发病机制中可能发挥重要作用。第二部分TIM-3基因多态性在免疫性血小板减少症中的作用研究背景与目的:T细胞免疫球蛋白粘蛋白分子(TIMs)家族在免疫调节中发挥重要的作用。TIM-3是一种优先表达在终末分化的Th1细胞上的跨膜蛋白,并在Th1介导的自身免疫性疾病中起重要作用。免疫性血小板减少症(ITP)是一种Th1极化的获得性器官特异性自身免疫性疾病。本研究的目的是探索TIM-3基因-1516G＞T,-574T＞G,4259G＞T三个位点单核苷酸多态性与ITP遗传易感性的关系。研究方法:采用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)方法分析了187例ITP患者和123例健康对照TIM-3基因-1516G＞T,-574T＞G,4259G＞T三个位点单核苷酸多态性。结果:ITP患者和正常对照TIM-3 -1516G＞T,-574T＞G,4259G＞T三个位点基因型频率和等位基因分布无显著差异。对三个位点进行连锁不平衡分析,发现-574T＞G和4259G＞T之间存在强连锁不平衡性(r~2=0.633)。而-1516G＞T与-574T＞G(r~2=0.007)和4259G＞T(r~2=0.002)之间均不存在连锁不平衡。结论:TIM-3 -1516G＞T,-574T＞G,4259G＞T三个位点基因多态性在ITP发病的遗传性危险因素中可能没有发挥主要作用。课题二RNA干扰逆转白血病多药耐药性研究研究背景与目的:化疗是治疗恶性血液病最有效的手段。大剂量联合化疗和早期定期强化治疗使现代白血病治疗取得了令人瞩目的成绩,但仍有许多患者因化疗失败而死亡。其主要原因是白血病细胞在治疗中产生多药耐药(multidrugresistance,MDR)。肿瘤的多药耐药性(multidrug resistance,MDR)是指肿瘤细胞对一种化疗药物产生耐药后,同时对未接触过的,结构和作用机制完全不同的化疗药物具有交叉耐药性。研究表明,由MDR1基因编码的p-糖蛋白(p-glycoprotein,P-gp或p170)在肿瘤细胞表面过度表达是导致肿瘤细胞多药耐药的主要机制。因此要提高恶性肿瘤化疗的有效性,就必须深入研究MDR的机制以及寻求有效逆转MDR的对策。RNA干扰(RNA interferance,RNAi)是一种进化保守的转录后基因沉默机制,是指双链RNA分子(dsRNA)被切割成21-23个核苷酸的小干扰RNA(small/short interference RNAs,siRNA),最终使其同源的mRNA特异性降解。目前,RNAi已成为最有应用价值的反义核酸技术之一,近两年来已被大量应用于肿瘤、抗病毒治疗及各种基因功能的研究中。本研究旨在应用shRNA干扰载体转染人白血病多药耐药细胞株K562/A02,通过体外和体内实验观察shRNA干扰载体对MDR1基因和P糖蛋白(P-gp)的表达及功能的影响,从而寻找最佳shRNA干扰载体。研究方法:构建针对MDR1序列特异性短发夹状RNA干扰载体,通过脂质体介导方式将含mdr1-shRNA质粒转入K562/A02细胞,经G418筛选得到稳定表达细胞株,用实时定量RT-PCR和Western blot分别检测干扰后MDR1 mRNA水平及蛋白水平的表达变化,通过CCK8测定干扰后细胞对化疗药的敏感性变化,Rhodamine123外排实验检测P-gp功能的变化,从而筛选得到最有效的干扰载体。随后通过转铁蛋白-脂质体介导方式将MDR1-shRNA质粒导入K562/A02裸鼠移植瘤并联合化疗,观察肿瘤生长及耐药表型变化。结果:与对照载体相比,构建的4个MDR1-shRNA质粒载体均能有效下调MDR1的表达,P-糖蛋白的功能也明显降低,其中508-526靶位点的shRNA作用效果最好。体内实验观察到该载体能够显著下调肿瘤表面P-糖蛋白的表达,并逆转耐药表型。结论:通过体外和体内实验筛选得到一个针对MDR1序列特异性短发夹状RNA干扰载体,这不仅为MDR1的RNA干扰研究提供了新靶点,还为以后的肿瘤基因治疗提供了实验支持。更多还原

【Abstract】 Background:Immune thrombocytopenia(ITP) is an acquired autoimmune hematologic disorder in which platelets are prematurely destroyed in the reticuloendothelial system by platelet autoantibodies.Although the pathogenesis of the disease is thought to be antibody-mediated,the production of these IgG anti-platelet autoantibodies is regulated by T-cells and thus T-cells indirectly might play a major pathogenetic role in the development of ITP.A variety of T-cell mediated abnormalities have been described in patients with ITP,including the activation of platelet-specific autoreactive T cells,decreased expression of Killer-cell immunoglobulin-like receptor(KIR) on cytotoxic lymphocytes(CTL) resulting in lysis of autologous platelets in ITP,and defective Tregs.TIM-3,a member of the novel TIM(T cell immunoglobulin and mucin domain) family of molecules,is the first molecule identified to be specifically expressed on CD4+ Th1 and not on Th2 cells.Up to now,it has also been found that TIM-3 is expressed on multiple cells of innate immune system,therefore it may play an important role in autoimmunity. Accumulating datas have shown that a high Th1/Th2 ratio was reported in patients with chronic ITP,suggesting that ITP is one of Th1-mediated autoimmune diseases. Whereas differentiated Th1 cells selectively express TIM-3,in the present study,we have detected TIM-3 expression in active and remission patients with ITP,relative to control subjects.Methods:TIM-3 expression of Peripheral Blood Mononuclear Cells(PBMCs) in ITP patients was detected by real-time PCR and flow cytometry.The correlation between TIM-3 expression levels and clinical parameters were further analyzed.We also evaluated the expression of TIM-3 in CD4+T cells,monocytes and dendritic cells (DCs) in peripheral blood of ITP patients.And the apoptosis of CD3/CD28 antibody-activated CD4+T cells was determined.Results:The data of real-time PCR and flow cytometry both showed that the TIM-3 expression of PBMCs of patients in active phase was significantly lower than that in healthy controls.The expression level of TIM-3 was significantly increased in patients in remission and had a positive correlation with the number of platelet.The TIM-3 expression was downregulated in CD4+T cells and monocytes in patients of active phase,but it was upregulated in DCs.In contrary,its level was upregulated in CD4+T cells and monocytes and was downregulated in DCs of patients in remission. The ratio of apoptosis in CD3/CD28 antibody-activated CD4+T cells was decreased in patients of active phase.Conclusion:The TIM-3 expression was downregulated in CD4+T cells and monocytes in patients of active phase,but it was upregulated in DCs.The apoptosis in activated CD4+T cells was decreased,suggesting that it has related to the downregulation of TIM-3.Taken together,TIM-3 may play an important role in the pathogenesis of ITP. Objective T cell immunoglobulin-and mucin-domain-containing molecules(TIMs) family have an important role in immune regulation.TIM-3 is a transmembrane protein preferentially expressed on terminally differentiated Th1 cells,which plays a role in Th1-mediated autoimmune disease.Idiopathic thrombocytopenic purpura(ITP) is an acquired organ-specific autoimmune disease with a polarization of Th1.The purpose of this study was to investigate whether the -1516G＞T,-574T＞G,4259G＞T single nucleotide polymorphisms within TIM-3 gene contribute to the genetic susceptibility to ITP.Method Genotyping of TIM-3 -1516G＞T,-574T＞G and 4259G＞T were performed in 187 patients with ITP and 123 healthy individuals by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) assay.Result No significant differences were found in genotype and allele distributions between the patients with ITP and the controls in all 3 sites.There was strong linkage disequilibrium(LD)(r~2=0.633) between -574T＞G and 4259G＞T,whereas -1516G＞T was not in LD with -574T＞G(r~2 = 0.007) or with 4259G＞T(r~2=0.002).Conclusion The -1516G＞T,-574T＞G and 4259G＞T of TIM-3 gene polymorphisms might not play an important role as a genetic risk factor in the pathophysiology of ITP. Background:Chemotherapy is the best treatment option for malignant leukemia patients.At present,high-dose combination chemotherapy and early regular intensive therapy have got a series of remarkable achievements in treating leukemia,but many patients died of chemotherapy failure.Multidrug resistance(MDR) is the major cause of failure of drug treatment modalities in leukemia.After developing resistance to a single drug or a class of drugs,cancer cells show cross-resistance to other functionally and structurally unrelated drugs.This phenomenon is known as cancer multidrug resistance.Accumulating data have shown that the overproduction of the 170-kDa, membranespanning P-glycoprotein(MDR1/P-gp,P-170,ABCB1) is the main underlying mechanism conferring this MDR phenotype.It is believed that inhibition of MDR1/P-gp function or of its expression may reverse the "classical" MDR phenotype and improve the effectiveness of chemotherapy.RNA interference(RNAi) is a conserved biologic response to double-stranded RNA that resuRs in the sequence-specific silencing of target gene expression.Double-stranded RNAs (dsRNA) are cleaved into 21-23nt small/short interference RNAs(siRNA).And then it guides the specific degradation of its cognate RNA.In recent years,RNA interference(RNAi) technique has enormous potential for the functional study of various genes and the development of novel gene therapeutic treatment strategies against cancer and viral infection.In this study,we used shRNA-encoding plasmid to transfect leukemia multidrug resistant cell line K562/A02 and detected the expression and the function of MDR1 gene and P-glycoprotein(P-gp) in vitro and vivo to select the most efficient vector.Methods:In order to design and screen short hairpin RNA(shRNA)molecules targeting multidrug resistance gene(MDR1),MDR1-shRNA expression vector was transfected into K562/A02 cells by lipofectamine2000,and G418 was added to screen and establish the stable expression cell strain.The expression of MDR1 was detected by real-time RT-PCR and western blot.The proliferation of cells was tested by CCK8 assay.The function of p-glycoprotein was determined by Rhodamine123 efflux experiment.And we selected the most efficient vector to determine whether this vector combined with transferrin-liposome could increase the inhibition of doxorubicin to K562/A02 cells xenografts in vivo.Results:The results showed that all of four mdr1-shRNA expression vector can significantly knockdown the expression of p-glycoprotein as compared with control vector,moreover,the vector targeting 508-526 sites of MDR1 gene is the best one.In vivo experiments demonstrated that this vector was able to downregulate the expression of P-glycoprotein(P-gp) on tumor cells and reverse the resistant phenotype.Conclusion:Our study show that the mdr1-shRNA expression vector by screening can significantly knockdown the expression of MDR1 gene and reverse leukemia drug resistance,which provide a new target site for MDR1 and pave the way for the future cancer gene therapy.更多还原