疏风凉血补肾法对CITP免疫调控的影响
|
摘要
目的：
免疫性血小板减少性紫癜(Immune thrombocytopenic purpura, ITP)是一种以血小板减少、皮肤粘膜和(或)内脏出血为特征的自身免疫性出血性疾病,属中医学“血证”、“肌衄”、“发斑”等范畴。ITP具体的发病机制尚未明确,随着研究的深入,发现除体液免疫外,细胞免疫功能失调亦可能是其发病机制之一,成为目前的研究热点。协同刺激分子表达的上调或下调维持淋巴细胞处于持续激活状态,又能防止淋巴细胞过度活化,其表达的失衡可导致多种免疫性疾病的发生。正性协同刺激分子过度表达可导致T淋巴细胞过度活化,细胞因子异常分泌,进而引起B淋巴细胞异常活化,分泌抗血小板相关抗体,导致血小板破坏增加。负性协同刺激分子则可抑制T淋巴细胞的活化增殖和细胞因子的产生。T淋巴细胞亚群Thl/Th2分别介导细胞免疫和体液免疫,其分泌的细胞因子能通过自身分泌形式刺激自身的增殖,并相互下调对方的分泌,在一定条件下还可相互转换,从而维持机体的正常免疫功能,其分泌的异常可导致多种免疫性疾病的发生
本研究旨在从临床和动物实验两方面来探讨正性协同刺激分子(CD80、CD86)和负性协同刺激分子(PD-1)及Thl/Th2细胞因子(IL-4、IFN-γ)在慢性免疫性血小板减少性紫癜(Chronic Immune Thrombocytopenic Purpura, CITP)发病中的可能作用机制,并运用疏风凉血补肾法(方)干预治疗,采用流式细胞术(Flow CytoMeter, FCM)及酶联免疫吸附法(enzyme linked immunosorbent assay, ELISA),通过检测小鼠CITP模型和患者外周血CD80、CD86、PD-1的表达及与免疫密切相关的Th1/Th2细胞因子IL-4、FN-γ的水平变化,评价疏风凉血补肾法(方)的疗效,试图从免疫网络调控角度出发阐明疏风凉血补肾法(方)治疗CITP的效应和机制,使有效治疗且避免目前药物副作用成为可能,为其免疫治疗开辟新的方法和思路,为中医治疗CITP提供具有新观点的理论依据,丰富和深化中医药治疗免疫性疾病的理论和实践。
方法：
1动物实验研究
先行预实验,将SPF级BALB/C小鼠20只随机分为对照组、模型组。模型组按100u L/20g体重剂量向小鼠腹腔内注入经稀释的1：4豚鼠抗小鼠血小板血清(APS),参考杨宇飞造模法改进,注射时间为第1,3,5,7,9,11,13天,共7天,于造模两周后,检测外周血血小板计数、骨髓巨核细胞数量及形态分类,以判定动物模型是否成功建立。
在预实验的基础上,小鼠CITP模型成功建立后,将小鼠CITP模型75只,随机分为模型组、强的松组、疏风凉血补肾法(方)高、中、低剂量组,每组各15只,并设对照组10只。于造模第6天起按5mL/Kg体重剂量每日1次、连续9天灌胃,灌胃药物分别为生理盐水(对照组)、生理盐水(模型组)、强的松溶液(0.5mg/mL,临床用量的10倍)、疏风凉血补肾法(方)高浓度水煎浓缩液(12.75g/Kg-1.d-1)、疏风凉血补肾法(方)中浓度水煎浓缩液(8.5g/Kg.d-1)、疏风凉血补肾法(方)低浓度水煎浓缩液(5.68g/Kg-1.d-1),于造模第15天取材。观察一般情况、不良反应、死亡情况、血小板计数变化、检测药物治疗后外周血PD-1、CD80、CD86的表达水平以及细胞因子IFN-γ、IL-4的变化情况。
2临床研究
将符合CITP诊断标准和纳入标准的39例患者随机分为疏风凉血补肾法(方)组(A组)、疏风凉血补肾法(方)加强的松组(B组)和强的松组(C组),每组13例。A组予疏风凉血补肾法(方)治疗,B组予疏风凉血补肾法(方)加强的松治疗(中药用法同A组,强的松用法同C组),C组予强的松治疗,强的松按lmg·kg·d-1。以上治疗均4周为一疗程,连续治疗3疗程后,观察不良反应,疗效,出血症状积分改善情况,血小板计数变化,强的松用量变化,药物治疗前后外周血协同刺激分子PD-1、CD80、 CD86的表达以及细胞因子IFN-γ、IL-4的水平变化情况。
结果：
1动物实验结果
1.1本研究分别在第1,3,5,7,9,11,13天于小鼠腹腔内注射经稀释的1：4豚鼠抗小鼠血小板血清(APS),每次按照100μ L/20g体重剂量的标准,可以成功复制小鼠CITP模型,豚鼠抗小鼠血小板血清停止注射后,模型维持时间约为72小时,与既往相关文献报道相符。
1.2在PLT计数方面,疏风凉血补肾法(方)高、中剂量组均能显著提升小鼠CITP模型外周血PLT计数(P<0.05)；疏风凉血补肾法(方)高、中剂量组之间无显著性差异(P>0.05),疏风凉血补肾法(方)低剂量组和强的松组与模型组比较无显著性差异(P>0.05)。
1.3在PD-1表达方面,疏风凉血补肾法(方)各剂量组和强的松组均能明显下调小鼠CITP模型外周血PD-1的表达(P0.05),与强的松组比较无显著性差异(P>0.05)。
1.4在CD80、CD86表达方面,疏风凉血补肾法(方)各剂量组和强的松组均能明显降低小鼠CITP模型外周血CD80、CD86的表达(F<0.05)；疏风凉血补肾法(方)各剂量组与强的松组比较无显著性差异(P>0.05)。
1.5在IL-4、IFN-γ水平方面,外周血IL-4水平升高,IFN-γ水平降低。与模型组比较,疏风凉血补肾法(方)各剂量组和强的松组均能明显降低小鼠CITP模型外周血IL-4的水平(P<0.05),提高IFN-γ的水平(F 2临床研究结果
2.1临床疗效方面,三组总有效率无显著性差异(P>0.05),但疏风凉血补肾法(方)组(A组)和疏风凉血补肾法(方)加强的松组(B组)具有疗效高于强的松组(C组)的趋势。在良效率方面,疏风凉血补肾法(方)加强的松组(B组)优于强的松组(C组)(P<0.05)。
2.2各组治疗后出血症状有显著性改善,疏风凉血补肾法(方)组(A组)和疏风凉血补肾法(方)加强的松组(B组)改善更为明显,与强的松组(C组)比较有显著性差异(P<0.05)。
2.3在强的松用量方面,祛风凉血补肾法(方)加强的松组和强的松组治疗三疗程后,强的松使用量都明显减少(P<0.01),疏风凉血补肾法(方)加强的松组比强的松组用量减少更为显著(P<0.01)。
2.4疏风凉血补肾法(方)组能明显提高CITP患者外周血PLT计数,治疗前后有极显著性差异(P<0.01),且其疗效明显优于强的松组(P 2.5疏风凉血补肾法(方)组可以明显下调CITP患者外周血CD80.CD86的表达,治疗后CD80.CD86均明显低于治疗前(P<0.01),三组间比较无显著性差异(P>0.05)。
2.6疏风凉血补肾法(方)组可以明显下调CITP患者外周血PD-1的表达,治疗后PD-1均明显低于治疗前(P>0.01),三组间比较无显著性差异(P>0.05)。
2.7治疗前,各治疗组IL-4水平均明显高于对照组(P<0.05),治疗后IL-4水平均明显低于治疗前(P 2.8各治疗组均可以提高CITP患者外周血INF-γ的水平。治疗前,三组INF-γ水平均明显低于对照组(P0.05),强的松组INF-γ升高不明显,与对照组和疏风凉血补肾法(方)加强的松组比较差异显著(P 结论：
CITP免疫功能存在紊乱现象,协同刺激分子(通路)存在异常以及T淋巴细胞亚群漂移,其漂移失衡表现为Thl细胞功能低下,Th2细胞功能占优势为主。协同刺激分子CD80、CD86和PD-1是参与CITP发病和免疫反应的重要协同刺激分子,其过度表达及Th1/Th2细胞因子IL-4、IFN-γ水平的异常与CITP发病及临床表现密切相关。疏风凉血补肾法(方)对CITP具有良好的治疗效果,其疗效机制之一就在于该法(方)能对协同刺激分子的异常表达进行调控,通过上调负性协同刺激分子的表达,以抑制正性协同刺激分子的过度表达,抑制T淋巴细胞的过度活化；另一机制可能是通过调节Th1/Th2细胞间的平衡,降低细胞因子IL-4和上调IFN-γ的水平,抑制过度的自身免疫反应,减少血小板的破坏。因此通过纠正协同刺激分子CD80、CD86和PD-1的异常表达及Th1/Th2细胞因子IL-4、IFN-γ的水平,调节其免疫失衡状态可能是CITP的治疗策略之一,具有重要的临床意义,值得进一步深入研究。
Objective
Immune Thrombocytopenic Purpura (ITP) is an autounmune disease characterized by a low platelet count and mucocutaneous bleeding, and is a common hemorrhagic disease of blood system in clinics, it be long to "blood syndrome'、"muscle apostaxis'、"macula syndrome" etc, in Traditional Chinese Medicine (TCM). Its definite pathogenic mechanism is unknown, And with the development to the depth, except for humoral immunity, cellular immunity may be one of the pathogenesises of ITP, it is one of the research hotspot about ITP in the world at present. Imbalances of positive and negative costimuLatory signals in immunocytes may leads to autoimmune diseases.High excepression of Positive costimulatory molecules may lead to overactivation of T lymphocytes and abnormal cytokine secretion. samely, overactivation of B lymphocytes may bring more antiplatelet antibodies, which mainly destroy platelet. While negative costimulatory molecules may regulate function of T lymphocytes such as the activation, proliferation and secreting cytokines. Thl and Th2lymphocytes mediated cellular and humoral immunity respectively, they can stimulate themself Proliferation by autocrine cytokines Down-Regulate cytokines Secretion each other and mutual conversion in some condition, so as to maintain the normal immune function. The abnormal secretion of cytokine can cause many immune disease.
In this study we try to investigate the specific mechanism of Positive and negative costimulatory signals cause ITP through clinical and animal researches. In order to study the function of T cells changed by costimulatory signals (CD80、CD86、PD-1) and Thl and Th2lymphocytes cytokines (IL-4、IFN-γ) which cause ITP, treatment with ShuFeng LiangXue BuShen therapy. techniques of monoclonal antibody, Flow cytometry (FCM)and enzyme linked immunosorbent assay (ELISA) be used in the experiments.on this basis, we will research the effects of serum containing ShuFeng LiangXue BuShen Therapy on costimulatory molecules and Th1and Th2lymphocytes-secreting cytokines (IL-4、IFN-γ) from ITP patients and animal model. Try to evaluate the effect of ShuFeng LiangXue BuShen Therapy, Evaluate the effect of ShuFeng LiangXue BuShen Therapy,and explore the possible immune regulation mechanism of ShuFeng LiangXue BuShen Therapy on CITP. The study may provides the new theory basis and practice for treating CITP through chinese medical science.
Methods
1Animal Experiment Study
Twenty BALB/C mices were randolnly divided into normal group, model group according to the counts of peripheral platelet.ITP mouse model were made by injected intraperitoneally1:4guinea pig-antimouse platelet serum(GP-APS)at a dose of100μL/20g at the first, third, fifth, seventh, ninth, eleventh, thirteenth day, totally7days. Detect the peripheral platelet counts, bone marrow megakaryocyte counts and morphology classiflcation, observe the pathological changes in thymus and spleen to in order to judge whether the model was successful or not to be established after two weeks. In the animal experiment,85BALB/C mice with SPF, ordered were randomly divided into six groups (10mice for normalg roup;15mice per group respectively for Blank group、 prednisone group and high-dosage e/midst-dosage/low-dosage groups of ShuFeng LiangXue BuShen Therapy). The latter five groups were improved with yang yufei methods and were injected intraperitoneally APS (Dilution with1:4proportion)100μ L Per time at every other day in13days and the CITP mice models were made. From the beginning of sixth day, these groups were administrated intragastrieally with a single dose Per day of experiment substance (administration quantity0.2mL/10g,10times for clinical dosage) in the next nine days, which were gavaged respectively with saline prednisone (0.5mg/mL, decuple of clinical dose)、high-dosage (12.75g/Kg-1. d-1)、 midst-dosage(8.5g/Kg-1. d-1)、low-dosage (5.68g/Kg-1. d-1) of the ShuFeng LiangXue BuShen Therapy. At the same time, the general state of health, the adverse reaction and the death amount were observed. Afterwards all mice were sacrificed and the needed specimens were taken in order to observe the change of Platelet counts、CD80、CD86、PD-1、IFN-γ and IL-4.
2Clinical Study
39CITP (chronic immune thrombocytopenic purpura, CITP) Patients were randomly divided to three groups (13Patients administrated with the ShuFeng LiangXue BuShen Therapy as A group;13Patients with the same decoction And Prednisone as B group;13Patients with Prednisone as C group. To more explain, B group administration way referred to A and C group.C group was constituted of Prednisone. The Prednisone administration quantity was given lmg·kg·d-1Per day. The abovet reatment was4weeks as one time of therapy. After3times of therapy, a series of observation were made as before.
Results
1Animal Experiment Study results
1.1The experiment successfully made the CITP mouse model according to injecting intraperitoneally the APS(dilution proportion1:4)with100μL Per time resectively at the first, third, fifth, seventh, ninth, eleventh, thirteenth day.The duration time of the CITP model was72hours and the result was the same as that of correlative data.
1.2With regard to Platelet counts of the CITP mice, the high-dosage and midst-dosage could all improve Platelet counts and was considered significant compared with the control group (P<0.05).However, there was no significant difference among the high-dosage and midst-dosage decoction. there was no significant difference the high-dosage、Prednisone groups and model group (P>0.05)
1.3With regard to the expression of PD-1of the CITP mice, the amount of the high-dosage、midst-dosage and low-dosage decoction groups and Prednisone groups could all be lowered and was considered significant compared with the control group (P<0.05), There was no significant difference among the four groups (P>0.05)
1.4With regard to the expression of CD80、CD86of the CITP mice, the amount of the high-dosage、midst-dosage and low-dosage decoction groups and Prednisone groups could all be lowered (P<0.05), There was no significant difference among the four groups (P>0.05)
1.5With regard to the expression of IL-4of the CITP mice, the amount of the high-dosage、midst-dosage and low-dosage decoction groups and Prednisone groups could all be lowered and IFN-γ be improved (P<0.05). IL-4was lowered more significantly in the high-dosage group and the Prednisone group(P<0.01).
2Clinical Experiment Study results
2.1With regard to the therapeutic effect, there was no significant difference among the three groups (P>0.05), But the ShuFeng LiangXue BuShen Therapy group and the ShuFeng LiangXue BuShen Therapy combined with Prednisone group have a effective trend than Prednisone group. With regard to the markedly effective, the ShuFeng LiangXue BuShen Therapy combined with Prednisone group is superior to the Prednisone group.
2.2With regard to the integration of hemorrhage symptoms, Three groups were improved significantly after therapy(P<0.05), the ShuFeng LiangXue BuShen Therapy group and the ShuFeng LiangXue BuShen Therapy combined with Prednisone group are better than the Prednisone group(P<0.05).
2.3With regard to the dosage of prednisone, after three period, the ShuFeng LiangXue BuShen Therapy combined with Prednisone group and the Prednisone group significantly reduced (P<0.01), the ShuFeng LiangXue BuShen Therapy combined with Prednisone group more significantly reduced than the Prednisone group (p<0.01)
2.4With regard to Platelet counts, that of the ShuFeng LiangXue BuShen Therapy group was raised obviously after therapy and there was significant difference compared with that before therapy(P<0.05). But the result of the ShuFeng LiangXue BuShen Therapy group was similar to that of the ShuFeng LiangXue BuShen Therapy combined with Prednisone group.
2.5Aspects of the expression of CD80、CD86of the CITP patients, three groups could all be lowered and was considered significant compared with the control group(P<0.05). There was no significant difference among the three groups (P>0.05)
2.6The ShuFeng LiangXue BuShen Therapy group could lowered the expression of PD-1(P<0.05), There was no significant difference among the three groups.
2.7Post-therapy, the Prednisone group and The ShuFeng LiangXue BuShen Therapy group significantly higher than those in control group(P<0.05), the ShuFeng LiangXue BuShen Therapy combined with the Prednisone group was significantly lower than the Prednisone group and the ShuFeng LiangXue BuShen Therapy group(P<0.05).
2.8The ShuFeng LiangXue BuShen Therapy group could improved the expression of INF-γ (P<0.01), There was no significant difference among the three groups. Pre-therapy, the three groups significantly lower than those in control group. Post-therapy, the Prednisone group higher than those in control group (P<0.05).
Conclusion
The result showed that the disturbance of costimulatory molecules (pathway) and T lymphocytes subsets imbalance exist in CITP, the function of Thl cell is weak, but the function of Th2cell prevail in immune dysfunction of CITP. All the analysis shows that over-expression of costimulating molecules CD80、 CD86and PD-1, and abnormal expression of related cytokines IL-4、IFN-γ be one of the pathogenesises of CITP. The ShuFeng LiangXue BuShen Therapy has good clinical effect on CITP. one of its mechanism is that it can adjust the imbalance of costimulatory molecules (pathway), upregulate the expression of negative costimulatory molecules (pathway) and inhibit the over-expression of positive costimulatory molecules, down regulate immune responses mediated by T lymphocytes; The other mechanism is regulating the imbalance of Thl/Th2cells, decrease the expression level of IL-4and increase the level of IFN-γ, inhibit over-autoimmune reaction, Reduce the destruction of platelets, a good clinical effect was achieved. Therefore, by correcting the abnormal expression of CD80、 CD86、PD-1and Th1/Th2to regulate immune balance is one of the treatment strategy, which has important clinical significance, It worth further research.
免疫性血小板减少性紫癜(Immune thrombocytopenic purpura, ITP)是一种以血小板减少、皮肤粘膜和(或)内脏出血为特征的自身免疫性出血性疾病,属中医学“血证”、“肌衄”、“发斑”等范畴。ITP具体的发病机制尚未明确,随着研究的深入,发现除体液免疫外,细胞免疫功能失调亦可能是其发病机制之一,成为目前的研究热点。协同刺激分子表达的上调或下调维持淋巴细胞处于持续激活状态,又能防止淋巴细胞过度活化,其表达的失衡可导致多种免疫性疾病的发生。正性协同刺激分子过度表达可导致T淋巴细胞过度活化,细胞因子异常分泌,进而引起B淋巴细胞异常活化,分泌抗血小板相关抗体,导致血小板破坏增加。负性协同刺激分子则可抑制T淋巴细胞的活化增殖和细胞因子的产生。T淋巴细胞亚群Thl/Th2分别介导细胞免疫和体液免疫,其分泌的细胞因子能通过自身分泌形式刺激自身的增殖,并相互下调对方的分泌,在一定条件下还可相互转换,从而维持机体的正常免疫功能,其分泌的异常可导致多种免疫性疾病的发生
本研究旨在从临床和动物实验两方面来探讨正性协同刺激分子(CD80、CD86)和负性协同刺激分子(PD-1)及Thl/Th2细胞因子(IL-4、IFN-γ)在慢性免疫性血小板减少性紫癜(Chronic Immune Thrombocytopenic Purpura, CITP)发病中的可能作用机制,并运用疏风凉血补肾法(方)干预治疗,采用流式细胞术(Flow CytoMeter, FCM)及酶联免疫吸附法(enzyme linked immunosorbent assay, ELISA),通过检测小鼠CITP模型和患者外周血CD80、CD86、PD-1的表达及与免疫密切相关的Th1/Th2细胞因子IL-4、FN-γ的水平变化,评价疏风凉血补肾法(方)的疗效,试图从免疫网络调控角度出发阐明疏风凉血补肾法(方)治疗CITP的效应和机制,使有效治疗且避免目前药物副作用成为可能,为其免疫治疗开辟新的方法和思路,为中医治疗CITP提供具有新观点的理论依据,丰富和深化中医药治疗免疫性疾病的理论和实践。
方法：
1动物实验研究
先行预实验,将SPF级BALB/C小鼠20只随机分为对照组、模型组。模型组按100u L/20g体重剂量向小鼠腹腔内注入经稀释的1：4豚鼠抗小鼠血小板血清(APS),参考杨宇飞造模法改进,注射时间为第1,3,5,7,9,11,13天,共7天,于造模两周后,检测外周血血小板计数、骨髓巨核细胞数量及形态分类,以判定动物模型是否成功建立。
在预实验的基础上,小鼠CITP模型成功建立后,将小鼠CITP模型75只,随机分为模型组、强的松组、疏风凉血补肾法(方)高、中、低剂量组,每组各15只,并设对照组10只。于造模第6天起按5mL/Kg体重剂量每日1次、连续9天灌胃,灌胃药物分别为生理盐水(对照组)、生理盐水(模型组)、强的松溶液(0.5mg/mL,临床用量的10倍)、疏风凉血补肾法(方)高浓度水煎浓缩液(12.75g/Kg-1.d-1)、疏风凉血补肾法(方)中浓度水煎浓缩液(8.5g/Kg.d-1)、疏风凉血补肾法(方)低浓度水煎浓缩液(5.68g/Kg-1.d-1),于造模第15天取材。观察一般情况、不良反应、死亡情况、血小板计数变化、检测药物治疗后外周血PD-1、CD80、CD86的表达水平以及细胞因子IFN-γ、IL-4的变化情况。
2临床研究
将符合CITP诊断标准和纳入标准的39例患者随机分为疏风凉血补肾法(方)组(A组)、疏风凉血补肾法(方)加强的松组(B组)和强的松组(C组),每组13例。A组予疏风凉血补肾法(方)治疗,B组予疏风凉血补肾法(方)加强的松治疗(中药用法同A组,强的松用法同C组),C组予强的松治疗,强的松按lmg·kg·d-1。以上治疗均4周为一疗程,连续治疗3疗程后,观察不良反应,疗效,出血症状积分改善情况,血小板计数变化,强的松用量变化,药物治疗前后外周血协同刺激分子PD-1、CD80、 CD86的表达以及细胞因子IFN-γ、IL-4的水平变化情况。
结果：
1动物实验结果
1.1本研究分别在第1,3,5,7,9,11,13天于小鼠腹腔内注射经稀释的1：4豚鼠抗小鼠血小板血清(APS),每次按照100μ L/20g体重剂量的标准,可以成功复制小鼠CITP模型,豚鼠抗小鼠血小板血清停止注射后,模型维持时间约为72小时,与既往相关文献报道相符。
1.2在PLT计数方面,疏风凉血补肾法(方)高、中剂量组均能显著提升小鼠CITP模型外周血PLT计数(P<0.05)；疏风凉血补肾法(方)高、中剂量组之间无显著性差异(P>0.05),疏风凉血补肾法(方)低剂量组和强的松组与模型组比较无显著性差异(P>0.05)。
1.3在PD-1表达方面,疏风凉血补肾法(方)各剂量组和强的松组均能明显下调小鼠CITP模型外周血PD-1的表达(P
1.4在CD80、CD86表达方面,疏风凉血补肾法(方)各剂量组和强的松组均能明显降低小鼠CITP模型外周血CD80、CD86的表达(F<0.05)；疏风凉血补肾法(方)各剂量组与强的松组比较无显著性差异(P>0.05)。
1.5在IL-4、IFN-γ水平方面,外周血IL-4水平升高,IFN-γ水平降低。与模型组比较,疏风凉血补肾法(方)各剂量组和强的松组均能明显降低小鼠CITP模型外周血IL-4的水平(P<0.05),提高IFN-γ的水平(F
2.1临床疗效方面,三组总有效率无显著性差异(P>0.05),但疏风凉血补肾法(方)组(A组)和疏风凉血补肾法(方)加强的松组(B组)具有疗效高于强的松组(C组)的趋势。在良效率方面,疏风凉血补肾法(方)加强的松组(B组)优于强的松组(C组)(P<0.05)。
2.2各组治疗后出血症状有显著性改善,疏风凉血补肾法(方)组(A组)和疏风凉血补肾法(方)加强的松组(B组)改善更为明显,与强的松组(C组)比较有显著性差异(P<0.05)。
2.3在强的松用量方面,祛风凉血补肾法(方)加强的松组和强的松组治疗三疗程后,强的松使用量都明显减少(P<0.01),疏风凉血补肾法(方)加强的松组比强的松组用量减少更为显著(P<0.01)。
2.4疏风凉血补肾法(方)组能明显提高CITP患者外周血PLT计数,治疗前后有极显著性差异(P<0.01),且其疗效明显优于强的松组(P
2.6疏风凉血补肾法(方)组可以明显下调CITP患者外周血PD-1的表达,治疗后PD-1均明显低于治疗前(P>0.01),三组间比较无显著性差异(P>0.05)。
2.7治疗前,各治疗组IL-4水平均明显高于对照组(P<0.05),治疗后IL-4水平均明显低于治疗前(P
CITP免疫功能存在紊乱现象,协同刺激分子(通路)存在异常以及T淋巴细胞亚群漂移,其漂移失衡表现为Thl细胞功能低下,Th2细胞功能占优势为主。协同刺激分子CD80、CD86和PD-1是参与CITP发病和免疫反应的重要协同刺激分子,其过度表达及Th1/Th2细胞因子IL-4、IFN-γ水平的异常与CITP发病及临床表现密切相关。疏风凉血补肾法(方)对CITP具有良好的治疗效果,其疗效机制之一就在于该法(方)能对协同刺激分子的异常表达进行调控,通过上调负性协同刺激分子的表达,以抑制正性协同刺激分子的过度表达,抑制T淋巴细胞的过度活化；另一机制可能是通过调节Th1/Th2细胞间的平衡,降低细胞因子IL-4和上调IFN-γ的水平,抑制过度的自身免疫反应,减少血小板的破坏。因此通过纠正协同刺激分子CD80、CD86和PD-1的异常表达及Th1/Th2细胞因子IL-4、IFN-γ的水平,调节其免疫失衡状态可能是CITP的治疗策略之一,具有重要的临床意义,值得进一步深入研究。
Objective
Immune Thrombocytopenic Purpura (ITP) is an autounmune disease characterized by a low platelet count and mucocutaneous bleeding, and is a common hemorrhagic disease of blood system in clinics, it be long to "blood syndrome'、"muscle apostaxis'、"macula syndrome" etc, in Traditional Chinese Medicine (TCM). Its definite pathogenic mechanism is unknown, And with the development to the depth, except for humoral immunity, cellular immunity may be one of the pathogenesises of ITP, it is one of the research hotspot about ITP in the world at present. Imbalances of positive and negative costimuLatory signals in immunocytes may leads to autoimmune diseases.High excepression of Positive costimulatory molecules may lead to overactivation of T lymphocytes and abnormal cytokine secretion. samely, overactivation of B lymphocytes may bring more antiplatelet antibodies, which mainly destroy platelet. While negative costimulatory molecules may regulate function of T lymphocytes such as the activation, proliferation and secreting cytokines. Thl and Th2lymphocytes mediated cellular and humoral immunity respectively, they can stimulate themself Proliferation by autocrine cytokines Down-Regulate cytokines Secretion each other and mutual conversion in some condition, so as to maintain the normal immune function. The abnormal secretion of cytokine can cause many immune disease.
In this study we try to investigate the specific mechanism of Positive and negative costimulatory signals cause ITP through clinical and animal researches. In order to study the function of T cells changed by costimulatory signals (CD80、CD86、PD-1) and Thl and Th2lymphocytes cytokines (IL-4、IFN-γ) which cause ITP, treatment with ShuFeng LiangXue BuShen therapy. techniques of monoclonal antibody, Flow cytometry (FCM)and enzyme linked immunosorbent assay (ELISA) be used in the experiments.on this basis, we will research the effects of serum containing ShuFeng LiangXue BuShen Therapy on costimulatory molecules and Th1and Th2lymphocytes-secreting cytokines (IL-4、IFN-γ) from ITP patients and animal model. Try to evaluate the effect of ShuFeng LiangXue BuShen Therapy, Evaluate the effect of ShuFeng LiangXue BuShen Therapy,and explore the possible immune regulation mechanism of ShuFeng LiangXue BuShen Therapy on CITP. The study may provides the new theory basis and practice for treating CITP through chinese medical science.
Methods
1Animal Experiment Study
Twenty BALB/C mices were randolnly divided into normal group, model group according to the counts of peripheral platelet.ITP mouse model were made by injected intraperitoneally1:4guinea pig-antimouse platelet serum(GP-APS)at a dose of100μL/20g at the first, third, fifth, seventh, ninth, eleventh, thirteenth day, totally7days. Detect the peripheral platelet counts, bone marrow megakaryocyte counts and morphology classiflcation, observe the pathological changes in thymus and spleen to in order to judge whether the model was successful or not to be established after two weeks. In the animal experiment,85BALB/C mice with SPF, ordered were randomly divided into six groups (10mice for normalg roup;15mice per group respectively for Blank group、 prednisone group and high-dosage e/midst-dosage/low-dosage groups of ShuFeng LiangXue BuShen Therapy). The latter five groups were improved with yang yufei methods and were injected intraperitoneally APS (Dilution with1:4proportion)100μ L Per time at every other day in13days and the CITP mice models were made. From the beginning of sixth day, these groups were administrated intragastrieally with a single dose Per day of experiment substance (administration quantity0.2mL/10g,10times for clinical dosage) in the next nine days, which were gavaged respectively with saline prednisone (0.5mg/mL, decuple of clinical dose)、high-dosage (12.75g/Kg-1. d-1)、 midst-dosage(8.5g/Kg-1. d-1)、low-dosage (5.68g/Kg-1. d-1) of the ShuFeng LiangXue BuShen Therapy. At the same time, the general state of health, the adverse reaction and the death amount were observed. Afterwards all mice were sacrificed and the needed specimens were taken in order to observe the change of Platelet counts、CD80、CD86、PD-1、IFN-γ and IL-4.
2Clinical Study
39CITP (chronic immune thrombocytopenic purpura, CITP) Patients were randomly divided to three groups (13Patients administrated with the ShuFeng LiangXue BuShen Therapy as A group;13Patients with the same decoction And Prednisone as B group;13Patients with Prednisone as C group. To more explain, B group administration way referred to A and C group.C group was constituted of Prednisone. The Prednisone administration quantity was given lmg·kg·d-1Per day. The abovet reatment was4weeks as one time of therapy. After3times of therapy, a series of observation were made as before.
Results
1Animal Experiment Study results
1.1The experiment successfully made the CITP mouse model according to injecting intraperitoneally the APS(dilution proportion1:4)with100μL Per time resectively at the first, third, fifth, seventh, ninth, eleventh, thirteenth day.The duration time of the CITP model was72hours and the result was the same as that of correlative data.
1.2With regard to Platelet counts of the CITP mice, the high-dosage and midst-dosage could all improve Platelet counts and was considered significant compared with the control group (P<0.05).However, there was no significant difference among the high-dosage and midst-dosage decoction. there was no significant difference the high-dosage、Prednisone groups and model group (P>0.05)
1.3With regard to the expression of PD-1of the CITP mice, the amount of the high-dosage、midst-dosage and low-dosage decoction groups and Prednisone groups could all be lowered and was considered significant compared with the control group (P<0.05), There was no significant difference among the four groups (P>0.05)
1.4With regard to the expression of CD80、CD86of the CITP mice, the amount of the high-dosage、midst-dosage and low-dosage decoction groups and Prednisone groups could all be lowered (P<0.05), There was no significant difference among the four groups (P>0.05)
1.5With regard to the expression of IL-4of the CITP mice, the amount of the high-dosage、midst-dosage and low-dosage decoction groups and Prednisone groups could all be lowered and IFN-γ be improved (P<0.05). IL-4was lowered more significantly in the high-dosage group and the Prednisone group(P<0.01).
2Clinical Experiment Study results
2.1With regard to the therapeutic effect, there was no significant difference among the three groups (P>0.05), But the ShuFeng LiangXue BuShen Therapy group and the ShuFeng LiangXue BuShen Therapy combined with Prednisone group have a effective trend than Prednisone group. With regard to the markedly effective, the ShuFeng LiangXue BuShen Therapy combined with Prednisone group is superior to the Prednisone group.
2.2With regard to the integration of hemorrhage symptoms, Three groups were improved significantly after therapy(P<0.05), the ShuFeng LiangXue BuShen Therapy group and the ShuFeng LiangXue BuShen Therapy combined with Prednisone group are better than the Prednisone group(P<0.05).
2.3With regard to the dosage of prednisone, after three period, the ShuFeng LiangXue BuShen Therapy combined with Prednisone group and the Prednisone group significantly reduced (P<0.01), the ShuFeng LiangXue BuShen Therapy combined with Prednisone group more significantly reduced than the Prednisone group (p<0.01)
2.4With regard to Platelet counts, that of the ShuFeng LiangXue BuShen Therapy group was raised obviously after therapy and there was significant difference compared with that before therapy(P<0.05). But the result of the ShuFeng LiangXue BuShen Therapy group was similar to that of the ShuFeng LiangXue BuShen Therapy combined with Prednisone group.
2.5Aspects of the expression of CD80、CD86of the CITP patients, three groups could all be lowered and was considered significant compared with the control group(P<0.05). There was no significant difference among the three groups (P>0.05)
2.6The ShuFeng LiangXue BuShen Therapy group could lowered the expression of PD-1(P<0.05), There was no significant difference among the three groups.
2.7Post-therapy, the Prednisone group and The ShuFeng LiangXue BuShen Therapy group significantly higher than those in control group(P<0.05), the ShuFeng LiangXue BuShen Therapy combined with the Prednisone group was significantly lower than the Prednisone group and the ShuFeng LiangXue BuShen Therapy group(P<0.05).
2.8The ShuFeng LiangXue BuShen Therapy group could improved the expression of INF-γ (P<0.01), There was no significant difference among the three groups. Pre-therapy, the three groups significantly lower than those in control group. Post-therapy, the Prednisone group higher than those in control group (P<0.05).
Conclusion
The result showed that the disturbance of costimulatory molecules (pathway) and T lymphocytes subsets imbalance exist in CITP, the function of Thl cell is weak, but the function of Th2cell prevail in immune dysfunction of CITP. All the analysis shows that over-expression of costimulating molecules CD80、 CD86and PD-1, and abnormal expression of related cytokines IL-4、IFN-γ be one of the pathogenesises of CITP. The ShuFeng LiangXue BuShen Therapy has good clinical effect on CITP. one of its mechanism is that it can adjust the imbalance of costimulatory molecules (pathway), upregulate the expression of negative costimulatory molecules (pathway) and inhibit the over-expression of positive costimulatory molecules, down regulate immune responses mediated by T lymphocytes; The other mechanism is regulating the imbalance of Thl/Th2cells, decrease the expression level of IL-4and increase the level of IFN-γ, inhibit over-autoimmune reaction, Reduce the destruction of platelets, a good clinical effect was achieved. Therefore, by correcting the abnormal expression of CD80、 CD86、PD-1and Th1/Th2to regulate immune balance is one of the treatment strategy, which has important clinical significance, It worth further research.
引文
[1]Malinio,CoopamahM, BernadetteGarvey. etal自身免疫性血小板减少性紫癜中的细胞免疫机制[J].国外医学输血及血液学分册,2003,26(4)：365-368.
[2]范颖,马骥.特发性血小板减少性紫癜免疫致病机制的研究[J].中医药学刊,2003,21(3)：401-403.
[3]张朝霞.特发性血小板减少性紫癜研究进展[J].中国小儿血液与肿瘤杂志,2006,11(6)：85.
[4]范磊,阮长耿.特发性血小板减少性紫癜发病机制[J].江苏医药,2006,32(4)：357-359.
[5]蓝海峰,杨仁池.特发性血小板减少性紫癜发病机制的认识[J].内科急危重症杂志,2006,12(6)：258-261.
[6]田翠时,宋广杰.特发性血小板减少性紫癜病因及发病机制的研究进展[J].北方学院学报(医学版),2006,23(4)：78-80.
[7]张欣,高清平,王琼玉.B淋巴细胞的细胞周期蛋白表达及周期分布在特发性血小板减少性紫癜发病机制中的意义[J].中华血液学杂志,2006,27(9)：630-631.
[8]魏金恺,刘惠哲.儿童特发性血小板减少性紫癜发病机制[J].人民军医,2007,50(9)：558-559.
[9]Bretscher P, Cohn M. A Theory of self-nonself discrimination [J].Scince 1970, 169:1042-1049.
[10]Hodge JW, Greiner JW, Tsang KY,et al. Costimulatory molecules as adjuvants for immunotherapy [J].Front Biosci.2006,11:788-803.
[11]Ishida Y, Agata Y, Shibahara K, et al.Induced expression of PD-1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death [J]. EMBO.1992, 11:3887-3895.
[12]Greenwald R J,Freeman G J, Sharpe A H. The B7 family revisited [J].Annu Rev Immunol 2005,23:515-548.
[13]Agata Y, Kawasaki A, Nishimura H, et al. Expression of the PD-1 antigen on the surf ice Of stimulated mouse T and B lymphocytes [J].Int Immunol,1996,8:765.
[14]Totsutka T, Kanai, T, Makaita S, et al. Regulation of murine chronic colitis by CD4+D25-Programmed death-l+T cells [J]. Eur J Immunol.2005:35(6):1773.
[15]Augello A, Tasso R, Negrini SM, et al. Bone marrow mesenchymal Progenitor cells inhibit lymphocyte Proliferation by activation of the Programmed death 1 Pathway [J].Eur J Immunol.2005:35(5):1482.
[16]Petrovas C, Casazza JP, Brenchley JM, et al. PD-1 is a regulator of virus-specific CD8+T cell survival in HIV infection [J].J. Exp.Med.2006,203:2281-2292.
[17]Latehman, Y, Wood CR, Chernova T, Chaudhary D, Borde M, Chernova I, I wai Yet al. PD-L2 is a Second ligand for PD-1 and inhibit T ceel activation [J].Nat Immunol,2001, 2(3):261-267.
[18]Dong HD, Zhu GF, Tamada K, et al. B7-H1,a third member of the B7 family, costimulates T-cell Proliferation and interleukin-10 secretion [J].Nat Med,1999:5:1365.
[19]Koga N.Blockade of the interaction between PD-1 and PD-L1 accelerates graft arterialdisease in cardiac allografts[J]. Arterioscler Thromb Vasc Bio,1 2004,24: 2057-2062.
[20]Arlene H S. The function of programmed cell death 1 and its ligands in regulating autoimmunity and infection [J]. Nature Immunology,2007(8):239-245.
[21]Riley JL. PD-1 signaling in primary T cells. [J] Immunol Rev.2009,229:114-125.
[22]Nurieva R, Thomas S, Nguyen T,et al.T-cell tolerance or function is determined by combinatorial costimulatory signals [J]. EMBO.2006,25:2623-2633.
[23]Okazaki T, Maeda A, Nishimura H, et al.PD-1 immunoreceptor inhibits B cell receptor-mediated signaling by recruiting src homology 2-domain-containing tyrosine phosphatase 2 to phosphotyrosine [J]. Proc. Natl. Acad. Sci. USA.2001,98:13866-13871.
[24]Latchman Y, Wood CR, Chernova T, et al. PD-L2 is a second ligand for PD-1 and inhibits T cell activation [J].Nat Immunol.2001,2:261-268.
[25]Coyle AJ, Gutierrez-Rmaos JC. The expanding B7 superfamily:increasing complexity in costimulatory signals reg μ Lating T cell function [J].Nat Immunol,2001,2 (3):203.
[26]Latchman Y. PD-L2 is a second ligand forPD-1 and inhibitsT cell activation[J]. Nat Immuno,2001(2):261-268.
[27]KuipersH. Contribution of the PD-1 ligands/PD-1 signaling pathway to dendritic cell-mediated CD4+ T cell activation [J].Eur J Immuno,1 2006,36:2472-2482.
[28]BlockiF A. Induction of a gene expression program in dendritic cells with a crosslinking IgM antibody to the costimulatory molecule B7-DC [J].FASEB J,2006,20:2408-2410.
[29]Van Keulen V P.Immunomodulation using the recombinantmonoclonalhuman B7-DC cross-linking antibody rHIgML2[J]. Clin Exp Immuno,1 2006,143:314-321.
[30]Dong H, Zhu G, Tamada K, et al. B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion [J].Nat Med,1999,5(12):1365-1369.
[31]Tamura H, Dong H,Zhu GF, et al.B7-H1 costim μ Lation preferentially enhances CD28-independent T-helper function.Blood.2001,97:1809-1816.
[32]Laura L, Carter, Lynette A. Fouser, Jason jussif,et al.PD-1:PD-L inhibitiory pathway affects both CD4+ and CD8+T cells and is overcomed by IL-2 [J]. Eur. J. Immunol,2002, 32:634.
[33]Freeman GJ, Long AJ, Iwai Y, et al. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation [J].J. Exp. Med.2000,192:1027-1034.
[34]Barber DL, Wherry EJ, Masopust D,et al. Restoring function in exhausted CD8+T cells during chronic viral infection[J]. Nature,2006,439(7077):682-687.
[35]Latchman Y, Wood CR, ChernovaT, et al. PD-L2 is a second ligand for PD-1 and inhibits T eell activation [J].Nat Immunol,2001,2:261.
[36]Freeman GJ, Gribben JG, Boussiotis VA, etal. Cloning of B7.2:CTLA-4 counter-receptor that costimulate human T cell proliferation [J]. Science,1993,262(5135):909-911.
[37]MocizukiK, HayashiN, KatayamaK, et al. B7/BB-lexpression and hepatitis activity in liver tissues ofpatients with chronic hepatitis C. Hepatol [J].1997,25(3):713-71.
[38]NDEJEMBI M P, TEIJARO J R, PATKE D S, et al. Control of memory CD4 T cell recall by the CD28/B7 costimulatory pathway[J].J Immunol,2006,177:7698-7706.
[39]吴松泉.细胞免疫中共刺激分子研究进展[J].国外医学免疫学分册.2000,23：16-18.
[40]Jaume JC, Parry SL, Madec AM, et al. Suppressive effect of glutamic acid decarboxylase 65-specific autoimmune B lymphocytes on processing of T cell determinants located within the antibody epitope [J].J Immunol.2002,169:665-672.
[41]SANCHEZ-LOCKHART M, MILLER J. Engagement of CD28 outside of the immunological synapse resultsin up-regulation of IL-2 mRNA stability but not IL-2 transcription[J].J Immunol,2006,176:4778-4784.
[42]MARINARI B, COSTANZO A, MARZANO V, et al.CD28 delivers a unique signal leading to the selective recruitment of RelA and p52 NF-kappa B subunits on IL-8 and Bcl-xL gene promoters[J].PNAS,2004,101:6098-6103.
[43]FIELDS P E, FINCH R J, GRAY G S, et al. B7.1 is aquantitatively stronger costimulus than B7.2 in the activation of naive CD8+TCR-transgenic T cells[J].J Immunol,1998,161:5268-5275.
[44]Kuchroo VK, Das MP, Brown JA,et al. B7-land B7-2costimulatory molecules activate differentiallytheThl/Th2 developmental pathways:application to autoimmune disease therapy [J].Cell,1995,80:707-718.
[45]Jacqueline MS, Jill EH, Barbara EB. CD80 and CD86 are not equivalent in their ability to induce the tyrosine phosphorylation of CD28 [J]. J. Biol. Chem.1999,274 (5):3116-3126.
[46]Azuma M, Yssel H, PhilliPs JH, et al. Functional expression of B7/BBI on activated T lymphocytes [J]. J EXP Med.1993 Marl,177(3):845-50.
[47]Jens L, BirgitK, Shuwei J, et al. The inhibitory function of B7 costimulators in T cell responses to foreign and self-antigens [J].Nature Immunology,2003,4(7):664-669.
[48]Helmut J, Edgar S, Michael S, et al. Identification and functional characterization of human CD4+CD25+T cells with regulatory properties isolated from peripheral blood [J].J Exp Med,2001,193(11):1285-1294.
[49]Luis G,Stephen P,Cobbold HW. Identification of regulatory T cells in tolerated allografts [J].J Exp Med,2002,195(12):1641-1646.
[50]Yong Z,Claire NM, Michael L, et al. CD80and CD86 differentially modulate the suppressive function of human regulatory T cells [J]. J Immunol,2004,172(5):2778-2784.
[51]McMillan R.Autoantibodies and autoantigens in chronic immune thrombocytopenic purpura [J].Semin Hemato,1 2000,37(3):239-248.
[52]MocizukiK,HayashiN, KatayamaKetal. B7/BB-1 expression and hepatitis activity in liver tissues of patientswith chronic hepatitis C [J]. Hepato.11997,25(3):713-710.
[53]李立元.Th1/Th2细胞因子与移植免疫耐受的研究进展[J].心血管病学进展.2010,31(1)：141.
[54]Roncarolo MG, Levings MK. The role of different subsets of T regulatory cells in controlling autoimmunity[J]. CurrOpin Immuno,1 2000,12(6):676-683.
[55]Lucey DR,Clerici M, Shearer G M. Type-1 and type-2 cytokine dysregulation in human infectious, neopastic, and in flammatory diseases [J].Clin Microbiol Rev,1996,9(4):532-562.
[56]Panitsas F P, The odoropoulou M, Kouraklis A, et al. Adult chronic idiopathic thrombocytopenic purpura (ITP) is the manifestation of a type-1 polarized immune response[J]. Blood,2004,103 (7):2645-2647.
[57]Zhou YM, Huang ZQ, Hu MH, et al. Clinical study on the effect Shengxueling on idiopathic thrombocytopenic purpura[J]. Chin J Integr Med,2005,11(1):60-64.
[58]Freeman GL, Long AJ, Iwai Y, et a.l Engagement of the PD-1 immnoinhibitory receptorby a novel B7 familymember leads to negative regulation of lymphocyte activation[J].J Exp Med,2000,192:1027-1034.
[59]Loke P, Allison J. PD-L1 and PD-L2 are differentially regulated by Thl and Th2 cells[J]. Proc Natl Acad Sci U S A,2003,100:5336-5341.
[60]Skapenko A, Lipsky PE, Kraetsch HG, et al Antigen-independent Th2 cell differentiation by stim μ Lation of CD28:regulation via IL-4 gene expression andmitogen-activated protein kinase activation[J]. J Immunol 2001,166:4283-4292.
[61]何广胜,周玲,吴德沛.Thl和Th2细胞的分化调节机制[J].中华血液学杂志,2005：26(2)：125.
[62]Webber NP,Mascarenhas JO, Crow MK, etal. Functional properties of Lymphocytes in idiopathic thrombocytopenic purpura[J]. Hum Immunol,2001,62 (12):1346-1355.
[63]范颖,马骥,陶淑春.细胞因子对ITP发病机理与治疗的研究[J].中医药学刊.2002,20(1)：31-33
[64]Garcia-Suarez J, Prieto A, Reyes E, et al. Abnormal y IFN and a IFN secretion in Purified CD2+cells from autoimmune thrombocytopenic Purpura(ATP) Patients:Their implication in the clinicalCourse of the disease[J]. AM J Mematol,1995,49(4):271-276.
[65]Olsson B, Andersson PO, Jemas M, et al. T-cell-mediated cytotoxicity toward Platelets in Chronic idopathtic thrombocytopenic purpura [J].Nat Med,2003,9(9):1123-1124.
[66]Zhou Z,Yang L, Chen Z, et al. Health-related quality of life measured by the short form 36 in immune thrombocytopenic purpura:a cross-sectional survey in China [J]. Eur J Haematol,2007,78(6):518.
[67]杨仁池.特发性血小板减少性紫癜治疗中应该注意的几个问题[J].国际输血及血液学杂志,2007,30(6)：484.
[68]杨仁池.特发性血小板减少性紫癜的规范化治疗[J].中国实用内科杂志,2007,27(14)：1095.
[69]Portielje JA, Westendorp RGJ,Kluin-Nelemans HC,et al. Morbidity and mortality in Adults with with idiopathic thrombocytopenic purpura [J]. Blood,2001,97(9):2549.
[70]Zhao H, Li H,Zhang L, et al. Retrospective analysis of 472 Chineses Children with chronic idiopathic thrombocytopenic purpura:a single center experience[J]. Haematologica, 2005,90 (6):860.
[71]Li H, Zhang L, Zhao H, et al. Chronic idiopathic thrombocytopenic purpura in Adult Chinese patients:a retrospective single-centered analysis of 1791 cases [J].Chin Med J,2005,118(1):34.
[72]Cheng Y, Wong RSM, Soo YOY, et al. Initial treatment of immune thrombocytopenic purpura with high-dose dexamethasone [J]. N Engl J Med,2003,349(9):831.
[73]Zhou Z, Yang R. Rituximab treatment for chronic refractory idiopathic thrombocytopenic purpura [J]. Crit Rev Oncol Hematol,2008,65(1):21.
[74]杨菊妃,李娟,莫义玲,等.美罗华治疗难治性特发性血小板减少性紫癜的护理[J].护理学杂志,2010,25(15)：89-90.
[75]Zaja F, Baccarani M, Mazza P, et al. Dexamethasone plus rituximab yields higher sustained response rates than dexamethasone monotherapy in Adults with primary immune thrombocytopenia [J]. Blood,2010,115(14):2755.
[76]隋涛,张磊,周泽平,等.两种小剂量利妥昔单抗治疗方案原发免疫性血小板减少症疗效比较[J].中华血液学杂志,2011,32(9)：583.
[77]隋涛,薛峰,赵海丰,等.小剂量利妥昔单抗治疗特发性血小板减少性紫癜疗效分析[J] 中华血液学杂志,2010,31(3)：161.
[78]Stasi R, Pagano A, Stipa E, et al. Rituximab chimeric anti-CD20 monoclonal antibody treatment for Adults with idiopathic thrombocyt openic purpura [J]. Blood,2001, 98(4):952-957.
[79]Nishimura S, Ishida T, Imai K, et al. Monoclonal antibody therapy for disorders of hemostasis and coag μLation [J].Nippon Rinsho,2002,60(3):525-530.
[80]Bussel J B. Fc receptor blockage and immune thrombocytopenic purpura [J]. Semin Hematol,2000,37(3):261-266.
[81]Wang T,Wang Z,Yang R. Thrombopoietic growth factorsin the treatment of immune thrombocytopenic purpura [J].Crit Rev Oncol Hematol,2011,77(3):172.
[82]王书杰,杨仁池,邹萍,等.重组人血小板生成素治疗特发性血小板减少性紫癜的多中心随机对照临床试验[J].血栓与止血学,2010,15(4)：149.
[83]Goto H,Kikuta T, Ota A, et al. Successful teratment of refractory idiopathic thrombocytopenic purpura by eradiciation helicobacter pylori [J]. Rinsho Ketsueki,2001, 42(12):1192-1194.
[84]Oyama Y, Papadopoulos EB,Miranda M, et al. Allogeneic stem cell transplantation for Evans syndrome [J].Bone Marrow Transplant,2001,28(9):903-905.
[85]Perotta A, Sunneberg JA, Scott V, et al. Rituxan in the treatment of chronic idiopathic thrombocytopenic purpur(ITP)[J]. Blood,1999,94(1):14.
[86]Tyndall A, Passweg J, Gratwohl A, et al. Haemopoietic stem cell transplantation in the treatment of severe autoimmune disease [J].Ann Rheum Dis,2001,60(7):702-707.
[87]Tao J, Huang Y, Li H, et al. Long-term outcomes of combination chemotherapy in chronic refractory idiopathic thrombocytopenic purpura in China [J].Chin Med J,2007, 120(18):1643.
[88]梁冰,血液病[M].北京：人民卫生出版社,2002,233.
[89]吴晓勇.傅汝林治疗特发性血小板减少性紫癜经验拾萃[J].辽宁中医杂志,2008,35(11)：1635.
[90]周永明,程军.原发性血小板减少性紫癜的病机特点和治疗对策[J].辽宁中医杂志,2008,12：714-715.
[91]李杨,曲道炜.特发性血小板减少性紫癜的中医病因病机探要[J].辽宁中医杂志,2007,12(5)：34.
[92]侯丕华.原发性血小板减少性紫癜的中医论治体会[J].中日友好医院学报,2002,16(1)：59.
[93]许毅,黄振翘.黄振翘教授调补泻火法治疗难治性特发性血小板减少性紫癜经验介绍[J].新中医,2005,37(9)：12-13.
[94]杨琳,蒋文明.蒋文明教授治疗特发性血小板减少性紫癜经验[J].湖南中医杂志,2007,23(2)：36-37.
[95]王延丰.生血消斑饮治疗特发性血小板减少性紫癜临床观察[J].天津中医,2001,18(4)：3.
[96]李扬,曲道炜,马骥.特发性血小板减少性紫癜的中医病因病机探要[J].辽宁中医杂志,2007,34(5)：574-575.
[97]田胜利.姚乃中运用温肾填精、凉血活血法治疗特发性血小极减少性紫癜经验[J].上海中医药杂志,2007,41(6)：12-13.
[98]裴明创,王兴丽,阮文让等.戴锡孟教授治疗原发性血小板减少性紫癜经验[J].天津中医 药,2007,24(1)：9-80.
[99]何文清.加减归脾汤治疗特发性血小板减少性紫癜疗效观察[J].辽宁中医杂志,2006,33(9)：1146.
[100]周永明,程军.原发性血小板减少性紫癜的病机特点和治疗对策[J].辽宁中医杂志,2001,28(12)：714.
[101]沈伟,徐旻,姚乃中.补肾方治疗难治性血小板减少性紫癜40例的临床疗效观察[J].黑龙江中医药,2007,6(4)：5-6.
[102]李海燕.紫癜冲剂治疗免疫性血小板减少性紫癜的临床研究[J].辽宁中医杂志,2002,29(1)：33.
[103]韩宁林.中西医结合治疗特发性血小板减少性紫癜疗效观察[J].安徽中医学院学报,2005,24(5)：11-12.
[104]徐瑞荣,李芮,焦中华,等.凉血消斑汤为主治疗特发性血小板减少性紫癜30例[J].四川中医,2002,6(1)：27.
[105]那丽,雍延礼,刘丽梅.孙伟正教授治疗原发性血小板减少性紫癜经验[J].中医药信息,2008,25(4)：-48.
[106]王忠武.从痰、从瘀、从虚论治难治性特发性血小板减少性紫癜[J].新中医,2004,36(5)：69-70.
[107]郭茜.难治性特发性血小板减少性紫癜辨治体会[J].中国中医急症,2008,17(6)：55-856.
[108]何煜舟,徐璎.徐志瑛辨治难治性特发性血小板减少性紫癜经验[J].浙江中医杂志,2009,44(11)：783.
[109]王蔚然,杨文华.从脾胃论治成人难治性特发性血小板减少性紫癜初探[J].辽宁中医杂志,2005,32(11)：1139-1140.
[110]赵伟,张飞宇,武君.特发性血小板减少性紫癜的中医药治疗概况[J].辽宁中医药大学学报,2008,10(4)：56.
[111]钱会南.张景治十三法[J].浙江中医学院学报1992,16(6)：28.
[112]王伟涛.吴澄治血证八法评析[J].中医药学报,2010,38(4)：11.
[113]陈永辉.特发性血小板减少性紫癜分期论治[J].中医药研究,2001,2(17)：29.
[114]吴维海,刘清池,武大勇,等.凉血解毒法治疗慢性难治性血小板减少性紫癜38例[J].中医杂志,2005,46(7)：515.
[115]唐旭东,麻柔.特发性血小板减少性紫癜的中医病机和治疗[J].内科急危重症杂志,2006,12(6)：262-263.
[116]陈洪洲.中西医结合治疗原发性血小板减少性紫癜60例总结[J].湖南中医杂志,2008,24(3)：15-16.
[117]李克煦.清热凉血法治疗特发性血小板减少性紫癜[J].四川中医,2005,23(3)：61.
[118]周永明,黄振翘,胡明辉,等.生血灵治疗特发性血小板减少性紫癜的临床研究[J].中国中西医结合杂志,2005,11(1)：60-64.
[119]周永明,周韶红,黄振翘,等.泻火宁血健脾滋肾法治疗难治性原发性血小板减少性紫癜35例[J].上海中医药杂志,2002,36(1)：22-23.
[120]何文清.加减归脾汤治疗特发性血小板减少性紫癜疗效观察[J].辽宁中医杂志,2006,33(9)：1146.
[121]曾建平.扶正化癜汤治疗原发性血小板减少性紫癜[J].中国中医药信息杂志,2004,11(4)：336.
[122]李海燕,陶淑春.治疗原发性血小板减少性紫癜的经验[J].辽宁中医杂志,2003,30(1)：16-17.
[123]陆东亮.中医中药治疗特发性血小板减少性紫癜[J].医药产业资讯,2005(17)：96.
[124]熊德上.宁血饮干预治疗特发性血小板减少性紫癜临床观察[J].实用中医药杂志,2011,27(8)：530-531.
[125]杨淑莲,杜昊,周振环,等.柴莲生血颗粒治疗慢性难治性特发性血小板减少性紫癜50例[J].河北中医,2010,32(8)：1145.
[126]吴雪琴,白玉盛.益气活血方治疗难治性特发性血小板减少性紫癜的临床观察[J].新疆中医药,2010,28(2)：31-33.
[127]李树年,刘志,丁建华.“生精化血饮”治疗原发性血小板减少性紫癜43例临床研究[J].江苏中医药,2009,41(12)：23.
[128]陈志雄,戴嫩,于天启,等.祛风凉血补肾法治疗难治性特发性血小板减少性紫癜的临床观察[J].广州中医药大学学报,2008,25(6)：495-497.
[129]赵武能,蔡锐,李珊,等.门冬清肺饮加味治疗慢性难治性血小板减少性紫癜30例临床观察[J].湖南中医药大学学报,2008,28(3)：62-65.
[130]王志慧,窦晶晶.仙连升板汤治疗慢性特发性血小板减少性紫癜30例[J].河南中医,2007,27(3)：47-48.
[131]周全良,陈学达.陈学达运用柴平生血汤治疗特发性血小板减少性紫癜经验[J].中医药临床杂志,2006,18(3)：219-220.
[132]宋振邦等.自拟羊枣黄芪汤加味治疗特发性血小板减少性紫癜36例[J].辽宁中医杂志,2006,33(3)：316-317.
[133]潘迎英.益气升板汤治疗特发性血小板减少性紫癜65例[J].中华临床医学杂志,2005,18(22)：1606.
[134]赵海东.益气生血汤治疗特发性血小板减少性紫癜45例[J].山西中医,2005,21(4)：17-18.
[135]周清华,魏自敏.消斑饮口服液治疗慢性原发性血小板减少性紫癜100例[J].实用中医内科杂志,2004,18(8)：2.
[136]宋华.紫癜宁治疗特发性血小板减少性紫癜48例小结[J].甘肃中医,2004,17(3)：26.
[137]张晓琳,彭军,侯明.特发性血小板减少性紫癜中协同刺激信号的作用研究[J].中国实验血液学杂志,2006,14：1053-1055.
[138]王忠武,于天启,杨宏光.祛风凉血法治疗特发性血小板减少性紫癜的理论基础[J].广州中医药大学学报,2004,21(2)：91-93.
[139]王忠武,孙莺,杨宏光,等.祛风凉血法对慢性特发性血小板减少性紫癜患者T细胞亚群及NK细胞的影响[J].广州中医药大学学报,2005,22(5)：344-347.
[140]曾英坚.祛风凉血补肾法对CITP骨髓巨核细胞分化成熟及TGF_β1的调节.博士论文.广州中医药大学,45,2008.
[141]戴媺,陈志雄,于天启,等.祛风法对凉血补肾中药治疗慢性特发性血小板减少性紫癜疗效的影响[J].广州中医药大学学报.2009,26(2)：128-130.
[142]郑其进,于天启.祛风凉血补肾法治疗难治性ITP15例疗效观察[J].河南中医,2010,(30)6：64-66.
[143]刘俊忠,姚大莉,姚雯.益气补肾活血法治疗原发性血小板减少性紫癜25例[J].国医论坛,1999,14(5)：36.
[144]朱珊.血得安颗粒治疗特发性血小板减少性紫癜的临床研究[J].河南中医.2000,20(3)：333-34.
[145]徐旻,沈伟,姚乃中.补肾填髓活血方对慢性特发性血小板减少性紫癜患者T细胞亚群的影响[J].陕西中医,2010,31(6)：680-681.
[146]计磊,刘方军,王玉萍,等.蕾苏茵陈汤治疗难治性特发性血小板减少性紫癜[J].中国中医药信息杂志,2000,7(1)：56-57.
[147]俞亚琴,孙伟正,孙风.补肾化瘀解毒方药对ITP患者血浆β-血小板球蛋白和血小板第4因子影响的研究[J].云南中医学院学报,2003,26(4)：1.
[148]武君紫癜圣愈散对小鼠CITP模型外周血共刺激分子CD86影响的实验研究.硕士论文.黑龙江中医药大学硕士学位论文,42,2008.
[149]尹艳,刘耒,李喜伟.桑椹三仙胶对慢性特发性血小板减少性紫癜患者共刺激分子CD80和CD86表达的影响[J].中华中医药学刊,2010,28(11)：2403-2405.
[150]杨宇飞,周霭祥,麻柔.免疫性血小板减少性紫癜动物模型的建立[J].中华血液学杂志,1994,15(3)：160-161.
[151]富琦,范颖,王家辉,等.免疫性血小板减少性紫癜病证结合动物模型建立与评价[J]中国中医基础医学杂志,2004,10(2)：30-32.
[152]曲道炜,林庶茹,范颖.免疫法建立特发性血小板减少性紫癜动物模型过程的改进及其体会[J].中医药学刊,2005,23(5)：928.
[153]宋崇顺,周立孝.医用中药药理学[M].人民卫生出版社,1996,106.
[154]中华医学会编著.临床诊疗指南血液学分册[M].人民卫生出版社,2006,106.
[155]张之南.血液病诊断及疗效标准[M].北京：科学出版社,2007,3：172-174.
[156]武大勇,庞宇慧,崔永芳,等.再生11号胶囊为主治疗特发性血小板减少性紫癜96例临床观察[J].中西医结合学报,2004,2(5)：332.
[157]张爱军,侯明.免疫性血小板减少性紫癜动物模型的研究进展[J].中国实验血液学杂志,1991,12(2)：623-626.
[158]Harrington W J, M innichV, Hollingseorth JW, etal. Demonstration of a thrombocytopenic factor in the blood of patients with thrombocytopenic purpura [J]. J Lab Clin Med,1951, 38:1-10.
[159]杨宇飞,麻柔,周霭祥,等.免疫性血小板减少性紫癜实验动物模型研究进展[J].中国中医基础医学杂志,1995,1(3)：43.
[160]Stenberg. Susained thromboeytopenia in mice serial studies of megarkaryoetes and plate [J]. Experimantal Hematology,1990,18:124.
[161]杨宇飞,周霭祥,麻柔,等.免疫性血小板减少性紫癜动物模型建立[J].中华血液学杂志,1994,15(4)：160-162.
[162]富琦,范颖,王家辉,等.免疫性血小板减少性紫癜病证结合动物模型建立与评价[J].中国中医基础医学杂志,2004,10(2)：30-32.
[163]曲道炜,林庶茹,范颖.免疫法建立特发性血小板减少性紫癜动物模型过程的改进及其体会[J].中医药学刊,2005,23(5)：928.
[164]M izutaniH, Engelman RW, Kurata Y, et al. Development and characterization of monoclonal antiplatelet autoantibodies from autoimmune thrombocytopenic purpura-prone [J] (NZW x BXSB) F1 mice. Blood,1993,82:837-844.
[165]MusajiA, Vanhoorelbeke K, Deckmyn H, et al.New model of transient strain-dependent autoimmune thrombocytopenia in mice immunized with rat platelets [J]. ExpHemato,12004; 32:87-94.
[166]MeKenzie SE, Taylor SM, MalladiP, etal. The role of the human Fc receptor/Fc γ TR11A in the immune celearance of platelets a transgenic mouse model [J].J Immunol,1999, 162:4311-431.
[167]张爱军,侯明.免疫性血小板减少性紫癜动物模型的研究进展[J].中国实验血液学杂志, 1991,12(2)：623-626.
[168]Cines DB, McKenzie SE, Siegel DL. Mechanisms of action of therapeutics in idiopathic thrombocytopenic purpura [J]. J Pediatr Hematolonco,1 2003,25(Suppll):52-56.
[169]张艳霞,周红艳,李建生.病证结合模型的研究进展[J].河南中医学院学报,2008,9(5)：97.
[170]刘丽梅,王瑞海,陈琳,等.病证结合方证相应在证候动物模型研究中的应用[J].中国中医基础医学杂志,2010,1(1)：90.
[171]Chambers C.The expanding world of co-stimulation:the two-signal model revisited [J].Trends immunol,2001,22:217-223.
[172]Hodge JW, Greiner JW, Tsang K Y, et al. Costimulatory molecules as adjuvants for immunotherapy [J].Front Biosci.2006,11:788-803.
[173]Freeman GJ, Long AJ, Iwai Y, et al. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation [J]. J. Exp. Med.2000,192:1027-1034.
[174]Latchman Y, Wood CR, Chernova T, et al. PD-L2 is a second ligand for PD-1 and inhibits T cell activation [J].Nat Immunol.2001,2:261-268.
[175]骆和生,罗鼎辉.免疫中药学：中药免疫药理与临床[M].北京大学医学出版社,1999,15.
[176]骆和生,罗鼎辉.免疫中药学：中药免疫药理与临床[M].北京大学医学出版社,1999,,25.
[177]王忠武,孙莺,杨宏光,等.祛风凉血法对慢性特发性血小板减少性紫癜患者T细胞亚群及NK细胞的影响[J].广州中医药大学学报,2005,22(5)：344-347.
[178]戴媺,陈志雄,于天启,等.祛风法对凉血补肾中药治疗慢性特发性血小板减少性紫癜疗效的影响[J].广州中医药大学学报.2009,26(2)：128-130.
[179]曾英坚.疏风凉血补肾法对CITP骨髓巨核细胞分化成熟及TGF_ β 1的调节[D].广州中医药大学博士学位论文,2008.
[180]陈桂玉,项东宇.防风的有效成分及药理学研究进展[J].黑龙江医药,2011,24(4)：601.
[181]邹升产.蝉蜕提取物诱导活动期SLE患者淋巴细胞凋亡的实验研究[D].成都中医药大学,2004.
[182]戴岳,毕培曦,陈耀邦.苍耳子对速发型过敏反应的抑制作用[J].中国野生植物资源,2002,21(6)：61.
[183]李发胜,杨光,徐恒瑰.补骨脂多糖的提取及免疫活性的研究[J].中国药师,2008,11(2)：140-142.
[184]陈小娟,李爱华,陈再智.巴戟多糖免疫药理研究[J].实用医学杂志,1995,11(5)：348-349.
[185]吕世静,黄槐莲.巴戟天对淋巴细胞增殖及产生细胞因子的调节作用[J].中医药研究,1997,13(5)：46-48.
[186]张庭廷,夏晓凯,陈传平,等.黄精多糖的生物活性研究[J].中国实验方剂学杂志,2006,12(7)：42-45.
[187]吴喜凤,韩淑英,朱丽莎,等.女贞子提取物对小鼠免疫功能的影响[J].华北煤炭医学院学报,2008,10(3)：303-304.
[188]金若敏,陈长勋,范广平,等.犀角与水牛角药理作用的研究[J].中成药,1997,19(7)：34.
[189]VasilikiP, ProkopiosM, Ioanna C, et a.1 Volatileswith antimicrobial activity fromthe roots ofGreek Paeonia taxa [J].J E thnopharm acol,2002,81(1):101.
[190]李逢春,周晓玲,磨红玲,等.丹皮酚注射增强免疫功能的实验研究[J].中国中西医结合杂志,1994,14(1)：37-38.
[191]朱作金,蔡福盛,杨志平,等.丹皮酚雾化吸入对大鼠免疫功能的影响[J].广西医科大学学报,1994,11(4)：386-387.
[192]王朴.生地黄的现代药理研究与临床应用[J].中国中医药现代远程教育,2008,6(8)：986.
[193]王丽雯,袁昉,魏伟.重组人白细胞介素对人外周血淋巴细胞功能的影响及芍药苷的作用[J].中国临床药理学杂志,2009,25(2)：129-133.
[194]葛志东.白芍总甙、芍药苷和白芍总甙去除芍药苷对佐剂性关节炎大鼠的免疫调节作用[J].中国药理学通报,1995,11(4)：303-305.
[195]Dong TX, Cui XM, Song ZH, et al. Chemical assessment of roots of anax notoginseng in China:regional and seasonal variations in its active constituents[J].J Agric Food Chem, 2003,51(16):4617.
[196]杨胜利,刘发.茜草双酯的免疫抑制作用[J].中国药学杂志,1996,31(7)：425-426.
[197]魏建宇,曹德来.黄芪的药理作用及黄芪多糖产品鉴别的方法[J].兽药,2012(4)：21.
[198]邓曼,窦晓兵,史亦谦,等.黄芪多糖定向诱生脐血来源树突状细胞及其对T细胞增殖作用的研究[J].中国免疫学杂志,2007,23(6)：539-550.
[199]程安玮,金征字,万发春.甘草多糖对小鼠腹腔巨噬细胞的激活作用[J].食品科学,2007,28(12)：431-434.
[200]刘霞,谢建新,李艳,等.甘草多糖免疫功能研究[J].中国公共卫生,2004,20(5)：572-573.
[201]高清平,曾艳,王琼玉,等.特发性血小板减少性紫癜患者B淋巴细胞的特征[J].中华微生物学和免疫学杂志,2005(25)：247.
[202]潘建玲,关秀茹,马学华,等.特发性血小板减少性紫癜患者外周血淋巴细胞共刺激分子的表达及白介素18的作用[J].中国实验血液学杂志,2007,15(3)：578-580.
[203]张春梅,赵春亭,滕清良,等.ITP患者外周血淋巴细胞共刺激分子[J].山东医药,2004,44(16)：7.
[204]马肖容,陈银霞,张王刚,等.特发性血小板减少性紫癜患者外周血共刺激分子的表达及与血小板抗体关系的临床研究[J].中国实验血液学杂志,2009,17(2)：483-486.
[205]Ling Y, CaoXS, Yu ZQ. Alteration and biological significance of peripheral dendritic cells in patients with chronic idiopathic thrombocytopenic purpura [J].Journal of experimental Hematology,2008,29:187-191.
[206]Chakir H,Wang H, Lefebvre DE, et al. T-bet/GATA3 ratio as a measure of the TH 1/TH2 cytokine profile in mixed cell populations predominant role of GATA3[J].J Immunol Methods,2003,278:157-169.
[207]Panitsas FP, Theodoropoulou M,Kouraklis A,etal Adult chronic idiopathic thrombocytopenic purpura(ITP) is the manifestation of a type-1 polarized immune response[J]. Blood,2004,103(7):2645-2647.
[208]Webber NP, Mascarenhas J 0, CrowMK, etal Functionalproperties of lymphocytes in idiopathic thrombocytopenic purpura[J].Hum Immunol,2001,62 (12):1346-1355.
[209]陈小芳,杨勤.原发性血小板减少性紫癜患儿部分白介素血清水平的研究[J].海南医学院学报,2006,12(4)：298-299.
[210]苏丽萍,郝国平.慢性特发性血小板减少性紫癜患者细胞免疫功能的研究[J].中华血液学杂志,2000,21(3)：144-145.
[211]Scott C S,Wheeler R,Ford P,etal T lymphocyte subpopulations in idiopathic thrombocytopenic purpura(ITP) [J].Scand J Haematol 1983,30(5):401-406.
[212]Webber NP, Mascarenhas JO, Crow MK, et al. FunCtional properties of lymphocytes inidiopathie thrombocytopenic purpura[J]. Hum immunol,2001,62(12):1346-1355.
[213]张丽萍,沈柏均,时庆,等.特发性血小板减少性紫癜患者血清IL-3、IL-4、IL-6水平的测定[J].中华血液学杂志,2000,21(3)：153.
[214]Krarauer T. Differential inhibitory effect of interleukin-10 and Interleukin-4 and dexamethason on staphylococcal exterotoxin-induced cytokine production and T cell activation[J]. J Leuka Biol,1995,57(3):450-454.
[215]蔡力生,汪明春,游伟文,等.成人慢性ITP患者Thl/Th2极化改变[J].临床医学,2005,4：391.
[2]范颖,马骥.特发性血小板减少性紫癜免疫致病机制的研究[J].中医药学刊,2003,21(3)：401-403.
[3]张朝霞.特发性血小板减少性紫癜研究进展[J].中国小儿血液与肿瘤杂志,2006,11(6)：85.
[4]范磊,阮长耿.特发性血小板减少性紫癜发病机制[J].江苏医药,2006,32(4)：357-359.
[5]蓝海峰,杨仁池.特发性血小板减少性紫癜发病机制的认识[J].内科急危重症杂志,2006,12(6)：258-261.
[6]田翠时,宋广杰.特发性血小板减少性紫癜病因及发病机制的研究进展[J].北方学院学报(医学版),2006,23(4)：78-80.
[7]张欣,高清平,王琼玉.B淋巴细胞的细胞周期蛋白表达及周期分布在特发性血小板减少性紫癜发病机制中的意义[J].中华血液学杂志,2006,27(9)：630-631.
[8]魏金恺,刘惠哲.儿童特发性血小板减少性紫癜发病机制[J].人民军医,2007,50(9)：558-559.
[9]Bretscher P, Cohn M. A Theory of self-nonself discrimination [J].Scince 1970, 169:1042-1049.
[10]Hodge JW, Greiner JW, Tsang KY,et al. Costimulatory molecules as adjuvants for immunotherapy [J].Front Biosci.2006,11:788-803.
[11]Ishida Y, Agata Y, Shibahara K, et al.Induced expression of PD-1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death [J]. EMBO.1992, 11:3887-3895.
[12]Greenwald R J,Freeman G J, Sharpe A H. The B7 family revisited [J].Annu Rev Immunol 2005,23:515-548.
[13]Agata Y, Kawasaki A, Nishimura H, et al. Expression of the PD-1 antigen on the surf ice Of stimulated mouse T and B lymphocytes [J].Int Immunol,1996,8:765.
[14]Totsutka T, Kanai, T, Makaita S, et al. Regulation of murine chronic colitis by CD4+D25-Programmed death-l+T cells [J]. Eur J Immunol.2005:35(6):1773.
[15]Augello A, Tasso R, Negrini SM, et al. Bone marrow mesenchymal Progenitor cells inhibit lymphocyte Proliferation by activation of the Programmed death 1 Pathway [J].Eur J Immunol.2005:35(5):1482.
[16]Petrovas C, Casazza JP, Brenchley JM, et al. PD-1 is a regulator of virus-specific CD8+T cell survival in HIV infection [J].J. Exp.Med.2006,203:2281-2292.
[17]Latehman, Y, Wood CR, Chernova T, Chaudhary D, Borde M, Chernova I, I wai Yet al. PD-L2 is a Second ligand for PD-1 and inhibit T ceel activation [J].Nat Immunol,2001, 2(3):261-267.
[18]Dong HD, Zhu GF, Tamada K, et al. B7-H1,a third member of the B7 family, costimulates T-cell Proliferation and interleukin-10 secretion [J].Nat Med,1999:5:1365.
[19]Koga N.Blockade of the interaction between PD-1 and PD-L1 accelerates graft arterialdisease in cardiac allografts[J]. Arterioscler Thromb Vasc Bio,1 2004,24: 2057-2062.
[20]Arlene H S. The function of programmed cell death 1 and its ligands in regulating autoimmunity and infection [J]. Nature Immunology,2007(8):239-245.
[21]Riley JL. PD-1 signaling in primary T cells. [J] Immunol Rev.2009,229:114-125.
[22]Nurieva R, Thomas S, Nguyen T,et al.T-cell tolerance or function is determined by combinatorial costimulatory signals [J]. EMBO.2006,25:2623-2633.
[23]Okazaki T, Maeda A, Nishimura H, et al.PD-1 immunoreceptor inhibits B cell receptor-mediated signaling by recruiting src homology 2-domain-containing tyrosine phosphatase 2 to phosphotyrosine [J]. Proc. Natl. Acad. Sci. USA.2001,98:13866-13871.
[24]Latchman Y, Wood CR, Chernova T, et al. PD-L2 is a second ligand for PD-1 and inhibits T cell activation [J].Nat Immunol.2001,2:261-268.
[25]Coyle AJ, Gutierrez-Rmaos JC. The expanding B7 superfamily:increasing complexity in costimulatory signals reg μ Lating T cell function [J].Nat Immunol,2001,2 (3):203.
[26]Latchman Y. PD-L2 is a second ligand forPD-1 and inhibitsT cell activation[J]. Nat Immuno,2001(2):261-268.
[27]KuipersH. Contribution of the PD-1 ligands/PD-1 signaling pathway to dendritic cell-mediated CD4+ T cell activation [J].Eur J Immuno,1 2006,36:2472-2482.
[28]BlockiF A. Induction of a gene expression program in dendritic cells with a crosslinking IgM antibody to the costimulatory molecule B7-DC [J].FASEB J,2006,20:2408-2410.
[29]Van Keulen V P.Immunomodulation using the recombinantmonoclonalhuman B7-DC cross-linking antibody rHIgML2[J]. Clin Exp Immuno,1 2006,143:314-321.
[30]Dong H, Zhu G, Tamada K, et al. B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion [J].Nat Med,1999,5(12):1365-1369.
[31]Tamura H, Dong H,Zhu GF, et al.B7-H1 costim μ Lation preferentially enhances CD28-independent T-helper function.Blood.2001,97:1809-1816.
[32]Laura L, Carter, Lynette A. Fouser, Jason jussif,et al.PD-1:PD-L inhibitiory pathway affects both CD4+ and CD8+T cells and is overcomed by IL-2 [J]. Eur. J. Immunol,2002, 32:634.
[33]Freeman GJ, Long AJ, Iwai Y, et al. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation [J].J. Exp. Med.2000,192:1027-1034.
[34]Barber DL, Wherry EJ, Masopust D,et al. Restoring function in exhausted CD8+T cells during chronic viral infection[J]. Nature,2006,439(7077):682-687.
[35]Latchman Y, Wood CR, ChernovaT, et al. PD-L2 is a second ligand for PD-1 and inhibits T eell activation [J].Nat Immunol,2001,2:261.
[36]Freeman GJ, Gribben JG, Boussiotis VA, etal. Cloning of B7.2:CTLA-4 counter-receptor that costimulate human T cell proliferation [J]. Science,1993,262(5135):909-911.
[37]MocizukiK, HayashiN, KatayamaK, et al. B7/BB-lexpression and hepatitis activity in liver tissues ofpatients with chronic hepatitis C. Hepatol [J].1997,25(3):713-71.
[38]NDEJEMBI M P, TEIJARO J R, PATKE D S, et al. Control of memory CD4 T cell recall by the CD28/B7 costimulatory pathway[J].J Immunol,2006,177:7698-7706.
[39]吴松泉.细胞免疫中共刺激分子研究进展[J].国外医学免疫学分册.2000,23：16-18.
[40]Jaume JC, Parry SL, Madec AM, et al. Suppressive effect of glutamic acid decarboxylase 65-specific autoimmune B lymphocytes on processing of T cell determinants located within the antibody epitope [J].J Immunol.2002,169:665-672.
[41]SANCHEZ-LOCKHART M, MILLER J. Engagement of CD28 outside of the immunological synapse resultsin up-regulation of IL-2 mRNA stability but not IL-2 transcription[J].J Immunol,2006,176:4778-4784.
[42]MARINARI B, COSTANZO A, MARZANO V, et al.CD28 delivers a unique signal leading to the selective recruitment of RelA and p52 NF-kappa B subunits on IL-8 and Bcl-xL gene promoters[J].PNAS,2004,101:6098-6103.
[43]FIELDS P E, FINCH R J, GRAY G S, et al. B7.1 is aquantitatively stronger costimulus than B7.2 in the activation of naive CD8+TCR-transgenic T cells[J].J Immunol,1998,161:5268-5275.
[44]Kuchroo VK, Das MP, Brown JA,et al. B7-land B7-2costimulatory molecules activate differentiallytheThl/Th2 developmental pathways:application to autoimmune disease therapy [J].Cell,1995,80:707-718.
[45]Jacqueline MS, Jill EH, Barbara EB. CD80 and CD86 are not equivalent in their ability to induce the tyrosine phosphorylation of CD28 [J]. J. Biol. Chem.1999,274 (5):3116-3126.
[46]Azuma M, Yssel H, PhilliPs JH, et al. Functional expression of B7/BBI on activated T lymphocytes [J]. J EXP Med.1993 Marl,177(3):845-50.
[47]Jens L, BirgitK, Shuwei J, et al. The inhibitory function of B7 costimulators in T cell responses to foreign and self-antigens [J].Nature Immunology,2003,4(7):664-669.
[48]Helmut J, Edgar S, Michael S, et al. Identification and functional characterization of human CD4+CD25+T cells with regulatory properties isolated from peripheral blood [J].J Exp Med,2001,193(11):1285-1294.
[49]Luis G,Stephen P,Cobbold HW. Identification of regulatory T cells in tolerated allografts [J].J Exp Med,2002,195(12):1641-1646.
[50]Yong Z,Claire NM, Michael L, et al. CD80and CD86 differentially modulate the suppressive function of human regulatory T cells [J]. J Immunol,2004,172(5):2778-2784.
[51]McMillan R.Autoantibodies and autoantigens in chronic immune thrombocytopenic purpura [J].Semin Hemato,1 2000,37(3):239-248.
[52]MocizukiK,HayashiN, KatayamaKetal. B7/BB-1 expression and hepatitis activity in liver tissues of patientswith chronic hepatitis C [J]. Hepato.11997,25(3):713-710.
[53]李立元.Th1/Th2细胞因子与移植免疫耐受的研究进展[J].心血管病学进展.2010,31(1)：141.
[54]Roncarolo MG, Levings MK. The role of different subsets of T regulatory cells in controlling autoimmunity[J]. CurrOpin Immuno,1 2000,12(6):676-683.
[55]Lucey DR,Clerici M, Shearer G M. Type-1 and type-2 cytokine dysregulation in human infectious, neopastic, and in flammatory diseases [J].Clin Microbiol Rev,1996,9(4):532-562.
[56]Panitsas F P, The odoropoulou M, Kouraklis A, et al. Adult chronic idiopathic thrombocytopenic purpura (ITP) is the manifestation of a type-1 polarized immune response[J]. Blood,2004,103 (7):2645-2647.
[57]Zhou YM, Huang ZQ, Hu MH, et al. Clinical study on the effect Shengxueling on idiopathic thrombocytopenic purpura[J]. Chin J Integr Med,2005,11(1):60-64.
[58]Freeman GL, Long AJ, Iwai Y, et a.l Engagement of the PD-1 immnoinhibitory receptorby a novel B7 familymember leads to negative regulation of lymphocyte activation[J].J Exp Med,2000,192:1027-1034.
[59]Loke P, Allison J. PD-L1 and PD-L2 are differentially regulated by Thl and Th2 cells[J]. Proc Natl Acad Sci U S A,2003,100:5336-5341.
[60]Skapenko A, Lipsky PE, Kraetsch HG, et al Antigen-independent Th2 cell differentiation by stim μ Lation of CD28:regulation via IL-4 gene expression andmitogen-activated protein kinase activation[J]. J Immunol 2001,166:4283-4292.
[61]何广胜,周玲,吴德沛.Thl和Th2细胞的分化调节机制[J].中华血液学杂志,2005：26(2)：125.
[62]Webber NP,Mascarenhas JO, Crow MK, etal. Functional properties of Lymphocytes in idiopathic thrombocytopenic purpura[J]. Hum Immunol,2001,62 (12):1346-1355.
[63]范颖,马骥,陶淑春.细胞因子对ITP发病机理与治疗的研究[J].中医药学刊.2002,20(1)：31-33
[64]Garcia-Suarez J, Prieto A, Reyes E, et al. Abnormal y IFN and a IFN secretion in Purified CD2+cells from autoimmune thrombocytopenic Purpura(ATP) Patients:Their implication in the clinicalCourse of the disease[J]. AM J Mematol,1995,49(4):271-276.
[65]Olsson B, Andersson PO, Jemas M, et al. T-cell-mediated cytotoxicity toward Platelets in Chronic idopathtic thrombocytopenic purpura [J].Nat Med,2003,9(9):1123-1124.
[66]Zhou Z,Yang L, Chen Z, et al. Health-related quality of life measured by the short form 36 in immune thrombocytopenic purpura:a cross-sectional survey in China [J]. Eur J Haematol,2007,78(6):518.
[67]杨仁池.特发性血小板减少性紫癜治疗中应该注意的几个问题[J].国际输血及血液学杂志,2007,30(6)：484.
[68]杨仁池.特发性血小板减少性紫癜的规范化治疗[J].中国实用内科杂志,2007,27(14)：1095.
[69]Portielje JA, Westendorp RGJ,Kluin-Nelemans HC,et al. Morbidity and mortality in Adults with with idiopathic thrombocytopenic purpura [J]. Blood,2001,97(9):2549.
[70]Zhao H, Li H,Zhang L, et al. Retrospective analysis of 472 Chineses Children with chronic idiopathic thrombocytopenic purpura:a single center experience[J]. Haematologica, 2005,90 (6):860.
[71]Li H, Zhang L, Zhao H, et al. Chronic idiopathic thrombocytopenic purpura in Adult Chinese patients:a retrospective single-centered analysis of 1791 cases [J].Chin Med J,2005,118(1):34.
[72]Cheng Y, Wong RSM, Soo YOY, et al. Initial treatment of immune thrombocytopenic purpura with high-dose dexamethasone [J]. N Engl J Med,2003,349(9):831.
[73]Zhou Z, Yang R. Rituximab treatment for chronic refractory idiopathic thrombocytopenic purpura [J]. Crit Rev Oncol Hematol,2008,65(1):21.
[74]杨菊妃,李娟,莫义玲,等.美罗华治疗难治性特发性血小板减少性紫癜的护理[J].护理学杂志,2010,25(15)：89-90.
[75]Zaja F, Baccarani M, Mazza P, et al. Dexamethasone plus rituximab yields higher sustained response rates than dexamethasone monotherapy in Adults with primary immune thrombocytopenia [J]. Blood,2010,115(14):2755.
[76]隋涛,张磊,周泽平,等.两种小剂量利妥昔单抗治疗方案原发免疫性血小板减少症疗效比较[J].中华血液学杂志,2011,32(9)：583.
[77]隋涛,薛峰,赵海丰,等.小剂量利妥昔单抗治疗特发性血小板减少性紫癜疗效分析[J] 中华血液学杂志,2010,31(3)：161.
[78]Stasi R, Pagano A, Stipa E, et al. Rituximab chimeric anti-CD20 monoclonal antibody treatment for Adults with idiopathic thrombocyt openic purpura [J]. Blood,2001, 98(4):952-957.
[79]Nishimura S, Ishida T, Imai K, et al. Monoclonal antibody therapy for disorders of hemostasis and coag μLation [J].Nippon Rinsho,2002,60(3):525-530.
[80]Bussel J B. Fc receptor blockage and immune thrombocytopenic purpura [J]. Semin Hematol,2000,37(3):261-266.
[81]Wang T,Wang Z,Yang R. Thrombopoietic growth factorsin the treatment of immune thrombocytopenic purpura [J].Crit Rev Oncol Hematol,2011,77(3):172.
[82]王书杰,杨仁池,邹萍,等.重组人血小板生成素治疗特发性血小板减少性紫癜的多中心随机对照临床试验[J].血栓与止血学,2010,15(4)：149.
[83]Goto H,Kikuta T, Ota A, et al. Successful teratment of refractory idiopathic thrombocytopenic purpura by eradiciation helicobacter pylori [J]. Rinsho Ketsueki,2001, 42(12):1192-1194.
[84]Oyama Y, Papadopoulos EB,Miranda M, et al. Allogeneic stem cell transplantation for Evans syndrome [J].Bone Marrow Transplant,2001,28(9):903-905.
[85]Perotta A, Sunneberg JA, Scott V, et al. Rituxan in the treatment of chronic idiopathic thrombocytopenic purpur(ITP)[J]. Blood,1999,94(1):14.
[86]Tyndall A, Passweg J, Gratwohl A, et al. Haemopoietic stem cell transplantation in the treatment of severe autoimmune disease [J].Ann Rheum Dis,2001,60(7):702-707.
[87]Tao J, Huang Y, Li H, et al. Long-term outcomes of combination chemotherapy in chronic refractory idiopathic thrombocytopenic purpura in China [J].Chin Med J,2007, 120(18):1643.
[88]梁冰,血液病[M].北京：人民卫生出版社,2002,233.
[89]吴晓勇.傅汝林治疗特发性血小板减少性紫癜经验拾萃[J].辽宁中医杂志,2008,35(11)：1635.
[90]周永明,程军.原发性血小板减少性紫癜的病机特点和治疗对策[J].辽宁中医杂志,2008,12：714-715.
[91]李杨,曲道炜.特发性血小板减少性紫癜的中医病因病机探要[J].辽宁中医杂志,2007,12(5)：34.
[92]侯丕华.原发性血小板减少性紫癜的中医论治体会[J].中日友好医院学报,2002,16(1)：59.
[93]许毅,黄振翘.黄振翘教授调补泻火法治疗难治性特发性血小板减少性紫癜经验介绍[J].新中医,2005,37(9)：12-13.
[94]杨琳,蒋文明.蒋文明教授治疗特发性血小板减少性紫癜经验[J].湖南中医杂志,2007,23(2)：36-37.
[95]王延丰.生血消斑饮治疗特发性血小板减少性紫癜临床观察[J].天津中医,2001,18(4)：3.
[96]李扬,曲道炜,马骥.特发性血小板减少性紫癜的中医病因病机探要[J].辽宁中医杂志,2007,34(5)：574-575.
[97]田胜利.姚乃中运用温肾填精、凉血活血法治疗特发性血小极减少性紫癜经验[J].上海中医药杂志,2007,41(6)：12-13.
[98]裴明创,王兴丽,阮文让等.戴锡孟教授治疗原发性血小板减少性紫癜经验[J].天津中医 药,2007,24(1)：9-80.
[99]何文清.加减归脾汤治疗特发性血小板减少性紫癜疗效观察[J].辽宁中医杂志,2006,33(9)：1146.
[100]周永明,程军.原发性血小板减少性紫癜的病机特点和治疗对策[J].辽宁中医杂志,2001,28(12)：714.
[101]沈伟,徐旻,姚乃中.补肾方治疗难治性血小板减少性紫癜40例的临床疗效观察[J].黑龙江中医药,2007,6(4)：5-6.
[102]李海燕.紫癜冲剂治疗免疫性血小板减少性紫癜的临床研究[J].辽宁中医杂志,2002,29(1)：33.
[103]韩宁林.中西医结合治疗特发性血小板减少性紫癜疗效观察[J].安徽中医学院学报,2005,24(5)：11-12.
[104]徐瑞荣,李芮,焦中华,等.凉血消斑汤为主治疗特发性血小板减少性紫癜30例[J].四川中医,2002,6(1)：27.
[105]那丽,雍延礼,刘丽梅.孙伟正教授治疗原发性血小板减少性紫癜经验[J].中医药信息,2008,25(4)：-48.
[106]王忠武.从痰、从瘀、从虚论治难治性特发性血小板减少性紫癜[J].新中医,2004,36(5)：69-70.
[107]郭茜.难治性特发性血小板减少性紫癜辨治体会[J].中国中医急症,2008,17(6)：55-856.
[108]何煜舟,徐璎.徐志瑛辨治难治性特发性血小板减少性紫癜经验[J].浙江中医杂志,2009,44(11)：783.
[109]王蔚然,杨文华.从脾胃论治成人难治性特发性血小板减少性紫癜初探[J].辽宁中医杂志,2005,32(11)：1139-1140.
[110]赵伟,张飞宇,武君.特发性血小板减少性紫癜的中医药治疗概况[J].辽宁中医药大学学报,2008,10(4)：56.
[111]钱会南.张景治十三法[J].浙江中医学院学报1992,16(6)：28.
[112]王伟涛.吴澄治血证八法评析[J].中医药学报,2010,38(4)：11.
[113]陈永辉.特发性血小板减少性紫癜分期论治[J].中医药研究,2001,2(17)：29.
[114]吴维海,刘清池,武大勇,等.凉血解毒法治疗慢性难治性血小板减少性紫癜38例[J].中医杂志,2005,46(7)：515.
[115]唐旭东,麻柔.特发性血小板减少性紫癜的中医病机和治疗[J].内科急危重症杂志,2006,12(6)：262-263.
[116]陈洪洲.中西医结合治疗原发性血小板减少性紫癜60例总结[J].湖南中医杂志,2008,24(3)：15-16.
[117]李克煦.清热凉血法治疗特发性血小板减少性紫癜[J].四川中医,2005,23(3)：61.
[118]周永明,黄振翘,胡明辉,等.生血灵治疗特发性血小板减少性紫癜的临床研究[J].中国中西医结合杂志,2005,11(1)：60-64.
[119]周永明,周韶红,黄振翘,等.泻火宁血健脾滋肾法治疗难治性原发性血小板减少性紫癜35例[J].上海中医药杂志,2002,36(1)：22-23.
[120]何文清.加减归脾汤治疗特发性血小板减少性紫癜疗效观察[J].辽宁中医杂志,2006,33(9)：1146.
[121]曾建平.扶正化癜汤治疗原发性血小板减少性紫癜[J].中国中医药信息杂志,2004,11(4)：336.
[122]李海燕,陶淑春.治疗原发性血小板减少性紫癜的经验[J].辽宁中医杂志,2003,30(1)：16-17.
[123]陆东亮.中医中药治疗特发性血小板减少性紫癜[J].医药产业资讯,2005(17)：96.
[124]熊德上.宁血饮干预治疗特发性血小板减少性紫癜临床观察[J].实用中医药杂志,2011,27(8)：530-531.
[125]杨淑莲,杜昊,周振环,等.柴莲生血颗粒治疗慢性难治性特发性血小板减少性紫癜50例[J].河北中医,2010,32(8)：1145.
[126]吴雪琴,白玉盛.益气活血方治疗难治性特发性血小板减少性紫癜的临床观察[J].新疆中医药,2010,28(2)：31-33.
[127]李树年,刘志,丁建华.“生精化血饮”治疗原发性血小板减少性紫癜43例临床研究[J].江苏中医药,2009,41(12)：23.
[128]陈志雄,戴嫩,于天启,等.祛风凉血补肾法治疗难治性特发性血小板减少性紫癜的临床观察[J].广州中医药大学学报,2008,25(6)：495-497.
[129]赵武能,蔡锐,李珊,等.门冬清肺饮加味治疗慢性难治性血小板减少性紫癜30例临床观察[J].湖南中医药大学学报,2008,28(3)：62-65.
[130]王志慧,窦晶晶.仙连升板汤治疗慢性特发性血小板减少性紫癜30例[J].河南中医,2007,27(3)：47-48.
[131]周全良,陈学达.陈学达运用柴平生血汤治疗特发性血小板减少性紫癜经验[J].中医药临床杂志,2006,18(3)：219-220.
[132]宋振邦等.自拟羊枣黄芪汤加味治疗特发性血小板减少性紫癜36例[J].辽宁中医杂志,2006,33(3)：316-317.
[133]潘迎英.益气升板汤治疗特发性血小板减少性紫癜65例[J].中华临床医学杂志,2005,18(22)：1606.
[134]赵海东.益气生血汤治疗特发性血小板减少性紫癜45例[J].山西中医,2005,21(4)：17-18.
[135]周清华,魏自敏.消斑饮口服液治疗慢性原发性血小板减少性紫癜100例[J].实用中医内科杂志,2004,18(8)：2.
[136]宋华.紫癜宁治疗特发性血小板减少性紫癜48例小结[J].甘肃中医,2004,17(3)：26.
[137]张晓琳,彭军,侯明.特发性血小板减少性紫癜中协同刺激信号的作用研究[J].中国实验血液学杂志,2006,14：1053-1055.
[138]王忠武,于天启,杨宏光.祛风凉血法治疗特发性血小板减少性紫癜的理论基础[J].广州中医药大学学报,2004,21(2)：91-93.
[139]王忠武,孙莺,杨宏光,等.祛风凉血法对慢性特发性血小板减少性紫癜患者T细胞亚群及NK细胞的影响[J].广州中医药大学学报,2005,22(5)：344-347.
[140]曾英坚.祛风凉血补肾法对CITP骨髓巨核细胞分化成熟及TGF_β1的调节.博士论文.广州中医药大学,45,2008.
[141]戴媺,陈志雄,于天启,等.祛风法对凉血补肾中药治疗慢性特发性血小板减少性紫癜疗效的影响[J].广州中医药大学学报.2009,26(2)：128-130.
[142]郑其进,于天启.祛风凉血补肾法治疗难治性ITP15例疗效观察[J].河南中医,2010,(30)6：64-66.
[143]刘俊忠,姚大莉,姚雯.益气补肾活血法治疗原发性血小板减少性紫癜25例[J].国医论坛,1999,14(5)：36.
[144]朱珊.血得安颗粒治疗特发性血小板减少性紫癜的临床研究[J].河南中医.2000,20(3)：333-34.
[145]徐旻,沈伟,姚乃中.补肾填髓活血方对慢性特发性血小板减少性紫癜患者T细胞亚群的影响[J].陕西中医,2010,31(6)：680-681.
[146]计磊,刘方军,王玉萍,等.蕾苏茵陈汤治疗难治性特发性血小板减少性紫癜[J].中国中医药信息杂志,2000,7(1)：56-57.
[147]俞亚琴,孙伟正,孙风.补肾化瘀解毒方药对ITP患者血浆β-血小板球蛋白和血小板第4因子影响的研究[J].云南中医学院学报,2003,26(4)：1.
[148]武君紫癜圣愈散对小鼠CITP模型外周血共刺激分子CD86影响的实验研究.硕士论文.黑龙江中医药大学硕士学位论文,42,2008.
[149]尹艳,刘耒,李喜伟.桑椹三仙胶对慢性特发性血小板减少性紫癜患者共刺激分子CD80和CD86表达的影响[J].中华中医药学刊,2010,28(11)：2403-2405.
[150]杨宇飞,周霭祥,麻柔.免疫性血小板减少性紫癜动物模型的建立[J].中华血液学杂志,1994,15(3)：160-161.
[151]富琦,范颖,王家辉,等.免疫性血小板减少性紫癜病证结合动物模型建立与评价[J]中国中医基础医学杂志,2004,10(2)：30-32.
[152]曲道炜,林庶茹,范颖.免疫法建立特发性血小板减少性紫癜动物模型过程的改进及其体会[J].中医药学刊,2005,23(5)：928.
[153]宋崇顺,周立孝.医用中药药理学[M].人民卫生出版社,1996,106.
[154]中华医学会编著.临床诊疗指南血液学分册[M].人民卫生出版社,2006,106.
[155]张之南.血液病诊断及疗效标准[M].北京：科学出版社,2007,3：172-174.
[156]武大勇,庞宇慧,崔永芳,等.再生11号胶囊为主治疗特发性血小板减少性紫癜96例临床观察[J].中西医结合学报,2004,2(5)：332.
[157]张爱军,侯明.免疫性血小板减少性紫癜动物模型的研究进展[J].中国实验血液学杂志,1991,12(2)：623-626.
[158]Harrington W J, M innichV, Hollingseorth JW, etal. Demonstration of a thrombocytopenic factor in the blood of patients with thrombocytopenic purpura [J]. J Lab Clin Med,1951, 38:1-10.
[159]杨宇飞,麻柔,周霭祥,等.免疫性血小板减少性紫癜实验动物模型研究进展[J].中国中医基础医学杂志,1995,1(3)：43.
[160]Stenberg. Susained thromboeytopenia in mice serial studies of megarkaryoetes and plate [J]. Experimantal Hematology,1990,18:124.
[161]杨宇飞,周霭祥,麻柔,等.免疫性血小板减少性紫癜动物模型建立[J].中华血液学杂志,1994,15(4)：160-162.
[162]富琦,范颖,王家辉,等.免疫性血小板减少性紫癜病证结合动物模型建立与评价[J].中国中医基础医学杂志,2004,10(2)：30-32.
[163]曲道炜,林庶茹,范颖.免疫法建立特发性血小板减少性紫癜动物模型过程的改进及其体会[J].中医药学刊,2005,23(5)：928.
[164]M izutaniH, Engelman RW, Kurata Y, et al. Development and characterization of monoclonal antiplatelet autoantibodies from autoimmune thrombocytopenic purpura-prone [J] (NZW x BXSB) F1 mice. Blood,1993,82:837-844.
[165]MusajiA, Vanhoorelbeke K, Deckmyn H, et al.New model of transient strain-dependent autoimmune thrombocytopenia in mice immunized with rat platelets [J]. ExpHemato,12004; 32:87-94.
[166]MeKenzie SE, Taylor SM, MalladiP, etal. The role of the human Fc receptor/Fc γ TR11A in the immune celearance of platelets a transgenic mouse model [J].J Immunol,1999, 162:4311-431.
[167]张爱军,侯明.免疫性血小板减少性紫癜动物模型的研究进展[J].中国实验血液学杂志, 1991,12(2)：623-626.
[168]Cines DB, McKenzie SE, Siegel DL. Mechanisms of action of therapeutics in idiopathic thrombocytopenic purpura [J]. J Pediatr Hematolonco,1 2003,25(Suppll):52-56.
[169]张艳霞,周红艳,李建生.病证结合模型的研究进展[J].河南中医学院学报,2008,9(5)：97.
[170]刘丽梅,王瑞海,陈琳,等.病证结合方证相应在证候动物模型研究中的应用[J].中国中医基础医学杂志,2010,1(1)：90.
[171]Chambers C.The expanding world of co-stimulation:the two-signal model revisited [J].Trends immunol,2001,22:217-223.
[172]Hodge JW, Greiner JW, Tsang K Y, et al. Costimulatory molecules as adjuvants for immunotherapy [J].Front Biosci.2006,11:788-803.
[173]Freeman GJ, Long AJ, Iwai Y, et al. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation [J]. J. Exp. Med.2000,192:1027-1034.
[174]Latchman Y, Wood CR, Chernova T, et al. PD-L2 is a second ligand for PD-1 and inhibits T cell activation [J].Nat Immunol.2001,2:261-268.
[175]骆和生,罗鼎辉.免疫中药学：中药免疫药理与临床[M].北京大学医学出版社,1999,15.
[176]骆和生,罗鼎辉.免疫中药学：中药免疫药理与临床[M].北京大学医学出版社,1999,,25.
[177]王忠武,孙莺,杨宏光,等.祛风凉血法对慢性特发性血小板减少性紫癜患者T细胞亚群及NK细胞的影响[J].广州中医药大学学报,2005,22(5)：344-347.
[178]戴媺,陈志雄,于天启,等.祛风法对凉血补肾中药治疗慢性特发性血小板减少性紫癜疗效的影响[J].广州中医药大学学报.2009,26(2)：128-130.
[179]曾英坚.疏风凉血补肾法对CITP骨髓巨核细胞分化成熟及TGF_ β 1的调节[D].广州中医药大学博士学位论文,2008.
[180]陈桂玉,项东宇.防风的有效成分及药理学研究进展[J].黑龙江医药,2011,24(4)：601.
[181]邹升产.蝉蜕提取物诱导活动期SLE患者淋巴细胞凋亡的实验研究[D].成都中医药大学,2004.
[182]戴岳,毕培曦,陈耀邦.苍耳子对速发型过敏反应的抑制作用[J].中国野生植物资源,2002,21(6)：61.
[183]李发胜,杨光,徐恒瑰.补骨脂多糖的提取及免疫活性的研究[J].中国药师,2008,11(2)：140-142.
[184]陈小娟,李爱华,陈再智.巴戟多糖免疫药理研究[J].实用医学杂志,1995,11(5)：348-349.
[185]吕世静,黄槐莲.巴戟天对淋巴细胞增殖及产生细胞因子的调节作用[J].中医药研究,1997,13(5)：46-48.
[186]张庭廷,夏晓凯,陈传平,等.黄精多糖的生物活性研究[J].中国实验方剂学杂志,2006,12(7)：42-45.
[187]吴喜凤,韩淑英,朱丽莎,等.女贞子提取物对小鼠免疫功能的影响[J].华北煤炭医学院学报,2008,10(3)：303-304.
[188]金若敏,陈长勋,范广平,等.犀角与水牛角药理作用的研究[J].中成药,1997,19(7)：34.
[189]VasilikiP, ProkopiosM, Ioanna C, et a.1 Volatileswith antimicrobial activity fromthe roots ofGreek Paeonia taxa [J].J E thnopharm acol,2002,81(1):101.
[190]李逢春,周晓玲,磨红玲,等.丹皮酚注射增强免疫功能的实验研究[J].中国中西医结合杂志,1994,14(1)：37-38.
[191]朱作金,蔡福盛,杨志平,等.丹皮酚雾化吸入对大鼠免疫功能的影响[J].广西医科大学学报,1994,11(4)：386-387.
[192]王朴.生地黄的现代药理研究与临床应用[J].中国中医药现代远程教育,2008,6(8)：986.
[193]王丽雯,袁昉,魏伟.重组人白细胞介素对人外周血淋巴细胞功能的影响及芍药苷的作用[J].中国临床药理学杂志,2009,25(2)：129-133.
[194]葛志东.白芍总甙、芍药苷和白芍总甙去除芍药苷对佐剂性关节炎大鼠的免疫调节作用[J].中国药理学通报,1995,11(4)：303-305.
[195]Dong TX, Cui XM, Song ZH, et al. Chemical assessment of roots of anax notoginseng in China:regional and seasonal variations in its active constituents[J].J Agric Food Chem, 2003,51(16):4617.
[196]杨胜利,刘发.茜草双酯的免疫抑制作用[J].中国药学杂志,1996,31(7)：425-426.
[197]魏建宇,曹德来.黄芪的药理作用及黄芪多糖产品鉴别的方法[J].兽药,2012(4)：21.
[198]邓曼,窦晓兵,史亦谦,等.黄芪多糖定向诱生脐血来源树突状细胞及其对T细胞增殖作用的研究[J].中国免疫学杂志,2007,23(6)：539-550.
[199]程安玮,金征字,万发春.甘草多糖对小鼠腹腔巨噬细胞的激活作用[J].食品科学,2007,28(12)：431-434.
[200]刘霞,谢建新,李艳,等.甘草多糖免疫功能研究[J].中国公共卫生,2004,20(5)：572-573.
[201]高清平,曾艳,王琼玉,等.特发性血小板减少性紫癜患者B淋巴细胞的特征[J].中华微生物学和免疫学杂志,2005(25)：247.
[202]潘建玲,关秀茹,马学华,等.特发性血小板减少性紫癜患者外周血淋巴细胞共刺激分子的表达及白介素18的作用[J].中国实验血液学杂志,2007,15(3)：578-580.
[203]张春梅,赵春亭,滕清良,等.ITP患者外周血淋巴细胞共刺激分子[J].山东医药,2004,44(16)：7.
[204]马肖容,陈银霞,张王刚,等.特发性血小板减少性紫癜患者外周血共刺激分子的表达及与血小板抗体关系的临床研究[J].中国实验血液学杂志,2009,17(2)：483-486.
[205]Ling Y, CaoXS, Yu ZQ. Alteration and biological significance of peripheral dendritic cells in patients with chronic idiopathic thrombocytopenic purpura [J].Journal of experimental Hematology,2008,29:187-191.
[206]Chakir H,Wang H, Lefebvre DE, et al. T-bet/GATA3 ratio as a measure of the TH 1/TH2 cytokine profile in mixed cell populations predominant role of GATA3[J].J Immunol Methods,2003,278:157-169.
[207]Panitsas FP, Theodoropoulou M,Kouraklis A,etal Adult chronic idiopathic thrombocytopenic purpura(ITP) is the manifestation of a type-1 polarized immune response[J]. Blood,2004,103(7):2645-2647.
[208]Webber NP, Mascarenhas J 0, CrowMK, etal Functionalproperties of lymphocytes in idiopathic thrombocytopenic purpura[J].Hum Immunol,2001,62 (12):1346-1355.
[209]陈小芳,杨勤.原发性血小板减少性紫癜患儿部分白介素血清水平的研究[J].海南医学院学报,2006,12(4)：298-299.
[210]苏丽萍,郝国平.慢性特发性血小板减少性紫癜患者细胞免疫功能的研究[J].中华血液学杂志,2000,21(3)：144-145.
[211]Scott C S,Wheeler R,Ford P,etal T lymphocyte subpopulations in idiopathic thrombocytopenic purpura(ITP) [J].Scand J Haematol 1983,30(5):401-406.
[212]Webber NP, Mascarenhas JO, Crow MK, et al. FunCtional properties of lymphocytes inidiopathie thrombocytopenic purpura[J]. Hum immunol,2001,62(12):1346-1355.
[213]张丽萍,沈柏均,时庆,等.特发性血小板减少性紫癜患者血清IL-3、IL-4、IL-6水平的测定[J].中华血液学杂志,2000,21(3)：153.
[214]Krarauer T. Differential inhibitory effect of interleukin-10 and Interleukin-4 and dexamethason on staphylococcal exterotoxin-induced cytokine production and T cell activation[J]. J Leuka Biol,1995,57(3):450-454.
[215]蔡力生,汪明春,游伟文,等.成人慢性ITP患者Thl/Th2极化改变[J].临床医学,2005,4：391.