【作者】 唐志宇；

【导师】 陈志雄；

【作者基本信息】 广州中医药大学 ， 中医内科学， 2012， 博士

【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(P<O.05),疏风凉血补肾法(方)各剂量组之间无显著性差异(P>0.05),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<O.05)：与模型组比较,疏风凉血补肾法(方)高剂量组和强的松组IL-4水平降低有极显著性差异(P<0.01)。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<O.05),和祛风凉血补肾法(方)加强的松组疗效相似。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<O.01)；疏风凉血补肾法(方)加强的松组治疗后,CITP患者外周血IL-4水平明显低于强的松组和疏风凉血组(P<O.05),体现出协同作用。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(P<O.05),治疗后,疏风凉血补肾法(方)组和疏风凉血补肾法(方)加强的松组INF-γ明显升高,与对照组比较无明显差异(P>0.05),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<O.05)。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。更多还原

【Abstract】 ObjectiveImmune 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.Methods1Animal Experiment StudyTwenty 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 Study39CITP (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.Results1Animal Experiment Study results1.1The 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 results2.1With 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).ConclusionThe 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.更多还原