探讨益髓颗粒对免疫性血小板减少性紫癜动物模型的免疫网络调控机制

【作者】 吴晓勇；

【导师】 陈信义；

【作者基本信息】 北京中医药大学 ， 中西医结合临床， 2010， 博士

【摘要】 1.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和皮肤粘膜出血为特征的自身免疫性疾病。其发病与免疫失调关系密切,但导致免疫功能异常的调控因素仍未完全阐明。临床上应用糖皮质激素、脾切除术等治疗可使70%以上病例血小板数量维持在安全范围,但还有约30%病例此方案治疗无效,需进一步采用其它治疗措施来维持血小板数量在安全水平,防止严重出血。应用糖皮质激素和脾脏切除术治疗无效的病例为难治性ITP (refractory immune thrombocytopenic purpura, RITP)。本研究用外源性抗体注入小鼠体内免疫的方法建立ITP小鼠模型,用益髓颗粒进行干预研究,观察ITP小鼠外周血血小板、骨髓巨核细胞、细胞因子、脾脏和胸腺细胞凋亡指数及凋亡基因表达、脾脏调节性T细胞等指标进行研究,试图从免疫网络调控角度阐明“益髓颗粒”治疗ITP效应机制。2.方法制备豚鼠抗Balb/C小鼠血抗小板血清,予红细胞吸附后备用。造模前将20只Balb/C小鼠依据外周血血小板数随机分正常组、模型组。模型组按照100μ1／20g剂量向小鼠腹腔内注入1：4稀释的GP-APS,隔日一次,造模两周后,检测外周血小板计数、骨髓巨核细胞数量及形态分类,观察胸腺及脾脏病理学改变,以判定疾病模型是否成功。ITP疾病动物模型建立成功后,按血小板计数将70只Balb/C小鼠随机分为正常组、模型组、醋酸泼尼松治疗组、升血小板胶囊治疗组、益髓颗粒大、中、小剂量治疗组,于造模第8天开始各组均按0.2m1／10g体重分别予生理盐水及治疗药物灌胃,每日1次,共7天。通过观察造模小鼠外周血小板计数、骨髓巨核细胞数量及形态分类、血小板相关抗体、脏器指数及免疫组织器官病理学变化等指标,评价益髓颗粒治疗效果。在确定疗效后,我们按上述造模方法建立ITP,用大剂量益髓颗粒为治疗药进行干预,用ELISA检测与免疫密切相关的Th1/Th2细胞因子(IFN-y/IL-4)及TGF-β1, TUNEL法及免疫组化法分别检测脾脏和胸腺细胞凋亡指数及凋亡基因Bcl-2、Bax的表达,用流式细胞仪检测脾脏调节性T细胞等指标进行研究,探讨益髓颗粒治疗ITP可能的免疫调控机制。3.结果3.1 ITP小鼠模型的建立GP-APS稀释倍数为1：128时仍成阳性,说明豚鼠血清中已产生了特异性抗体,即豚鼠抗Balb/C小鼠血小板抗体。与正常组及造模前比较,ITP小鼠模型外周血血小板明显降低,差异显著有统计学意义(P<0.05,P<0.01),注射APS后24小时模型小鼠外周血小板数最低。模型组小鼠骨髓巨核细胞总数明显增加,而产板巨核细胞减少,与正常组比较,差异显著有统计学意义(P<0.05,P<0.01)。模型组凝血时间明显延长,与正常组比较,差异显著有统计学意义(P<0.001)。模型组小鼠脾脏指数明显大于正常组,差异显著有统计学意义(P<0.001),胸腺指数与正常组比较差异不显著无统计学意义。3.2益髓颗粒对ITP小鼠模型治疗作用的实验研究与模型组比,各治疗组血小板数均比模型组有所升高,差异显著有统计学意义(P<0.01,P<0.001),以益髓颗粒大剂量组和泼尼松组血小板升高明显。益髓颗粒大剂量组不成熟的骨髓巨核细胞增生受到抑制,产板巨核细胞增多,与模型组比较,有统计学意义(P<0.01,P<0.001)；模型组PAIgG水平明显升高低,与正常组比差异显著有统计学意义(P<0.001),经治疗后,益髓颗粒组PAIgG水平明显低于模型组,与正常组比差异不明显无统计学意义。与模型组比,各治疗组脾脏均有所缩小,脾脏指数降低,差异显著有统计学意义(P<0.001),以益髓颗粒大剂量组效果明显。3.3益髓颗粒对ITP小鼠免疫网络调控机制的实验研究与正常组相比,模型组小鼠IFN-y水平明显升高,IL-4水平明显降低,IFN-γ/IL-4比值升高,Th1/Th2细胞间失衡,提示ITP小鼠存在细胞免疫功能低下及T细胞亚群漂移,表现为Thl细胞优势；Treg细胞比例、TGF-β1水平明显降低,脾脏淋巴细胞凋亡减少,Bcl-2蛋白表达增多,Bax蛋白表达减少,各组胸腺淋巴细胞凋亡及Bcl-2、Bax蛋白差异不明显无统计学意义。而经治疗后ITP小鼠血清INF-y水平降低,IL-4水平长高,IFN-y/IL-4比值降低,与模型组比较,益髓颗粒治疗组的IFN-y降低,IL-4升高,IFN-γ/IL-4比值接近正常；Treg细胞比例及TGF-β1水平均有所提高,但Treg仍未达到正常水平,与正常组比仍存在差异,而TGF-β1与正常组比差异不明显无统计学意义；益髓颗粒组脾脏淋巴细胞凋亡增多,Bcl-2蛋白表达减少,Bax蛋白表达增多。4.结论4.1成功制备了GP-APS,IPT小鼠疾病模型建立成功,模型小鼠外周血小板下降、骨髓巨核细胞增多并伴成熟障碍,与ITP临床表现基本相符。4.2益髓颗粒能有效恢复ITP小鼠的血小板计数,并能促进骨髓巨核细胞向成熟方向分化,与醋酸泼尼松疗效相当。4.3 ITP存在细胞免疫功能低下及T细胞亚群漂移,表现为Thl细胞优势,其发病可能与Th1/Th2细胞间细胞因子失衡,Treg细胞及TGF-β1含量降低,淋巴细胞凋亡及Bcl-2、Bax蛋白表达异常等相关。益髓颗粒可能通过调节Th1/Th2细胞间的平衡,促进Treg细胞增殖及TGF-β1分泌,并能调节脾脏淋巴细胞凋亡及凋亡基因Bcl-2、Bax的异常表达,进而调节ITP免疫异常,降低血小板相关抗体,减少血小板破坏,提高血小板数量,促进骨髓巨核细胞向成熟方向分化而达到治疗效果。更多还原

【Abstract】 1.ObjectiveImmune thrombotopenic purpura is an autoimmune (ITP) disorder characterized by a low platelet count and mucocutaneous bleeding.A number of features suggest this destruction is immune-mediated,But the exact mechanism of the immune dysfunction, however, is generally not known. More than 70% patients response to treatment with glucocorticoids and splenectomy,but there are about 30% of refractory patients (refractory immune thrombocytopenic purpura, RITP) are defined as those in whom treatment with standard-dose corticosteroids and splenectomy fails and who further therapy because of unsafe platelet counts or clinical bleeding.In this study,ITP mouse model were made by injected exogenous antibodies intraperitoneally guinea pig-antimouse platelet serum (GP-APS) and treated with YiSui granules.Observe the effect of Yisui granules on ITP mouse peripheral blood platelets,bone marrow megakaryocytes,cytokines,spleen and thymus apoptosis index and the apoptotic gene expression,regulatory T cells in the spleen and other factors.Trying to explore Yisui granules effective mechanism on the regulation of immune network in ITP.2.MethodsPreparation of guinea pig anti-Balb/C mice anti-platelet serum (GP-APS) and red blood cell adsorption and reserved.Twenty Balb/c mice were randomly divided into normal group,model group according to the counts of Peripheral platelet.ITP mouse model were made by injected intraperitoneally 1:4 guinea pig-antimouse platelet serum (GP-APS) at a dose of 100μl/20g every other day.Detect the peripheral platelet counts,bone marrow megakaryocyte counts and morphology classification,observe the pathological changes in thymus and spleen to after two weeks in order to judge whether the model was successful or not.After ITP mouse model was verified successful,seventy Balb/C mice were were randomly divided into normal group,model group,Prednisolone acetate group,ShengXue XiaoBan capsule and Yisui granules high,medium and low-dosege groups.The 8th day establishing the model,each group were intragastric administration at a dose of 0.2ml/10g·d-1 volume with normal saline and every treated medicine daily for additional one week respectively.Evaluate the therapeutic effect of YiSui granules according to detecting the peripheral platelet counts,blood coagulation time, bone marrow megakaryocyte counts and morphology classificatio,platelet-associated antibodies,the immune organ index and the pathological changes.After the therapeutic effect of YiSui granule had been determined,the ITP mouse model were established as the methods above-mentioned and treated with high dosage of YiSui granules.Two weeks latter,The levels of immune closely related to Thl/Th2 cytokines (IFN-y and IL-4) and TGF-β1 in peripheral blood serum were detected by ELISA.The spleen and thymus were embedded in paraffin and sliced 4μm,lymphocyte apoptosis index were detected by TUNEL,Bcl-2 and Bax protein expression were detected by immunohistochemistry.The Treg cells were detected by flow cytometry (FCM) in spleen single cell suspension.Explore the possible immune regulation mechanism of Yisui granule on ITP according to detect the factors above-mentioned.3.Results3.1 ITP mouse modelGP-APS is still positive when dilution 1:128,indicating guinea pig has generated a specific antibody in serum, that is guinea pig anti-Balb/C mice platelet antibodies. Compared with the normal group,the peripheral blood platelets counts of ITP mouse model decreased significantly,theer was statistically significant (P<0.05,P<0.01).The peripheral platelet counts was lowest after APS injected 24 hours. The counts of megakaryocytes in bone marrow of model group mice increased significantly,but the megakaryocyte which can produce platelet decreased (P< 0.05,P< 0.01).The blood coagulation time of model group was prolonged than normal group (P< 0.001), spleen index of model group was significantly higher than normal group.But the thymus index has no statistically significant as compared with normal group (P< 0.001).3.2 Experimental study the therapeutic effect of Yisui granuls on ITP mouse modelCompare with model group,the platelet counts of each treatment groups were higher than those of model group,there was statistically significant difference (P<0.01,P<0.001).The counts of platelet in Yisui granules high-dosege group and prednisone group recovered obviously.Compare with model group, immature megakaryocyte proliferation in bone marrow of Yisui granules high-dosage group was inhibited, megakaryocyte which can produce platelet increased,there was statistically significant difference (P<0.01,P< 0.001); The levels of PAIgG of ITP model group were increased significantly than normal group.The levels of PAIgG decreased significantly after treatment with Yisui granules,it was significantly lower than model group (P< 0.001),there was no statistically significant different compared with the normal group.The spleen index of each treatment groups decreased than model group,the Yisui granuls high-dosage group decreased obviously,there was statistically significant difference (P< 0.001)3.3 Experimental study the immunological network regulation mechanism of Yisui granuls on ITP mouse modelCompared with normal group,the levels of IFN-γincreased in model group,the levels IL-4 decreased in model group,the ratio of IFN-γ/IL-4 increased. The balance between Thl and Th2 was brouken out. ITP has cellular immune function dereased and T cell subsets drift, manifestation of a type-1 polarized immune response.The proportion of Treg cells and the levels of TGF-β1 was significantly lower in model group as compared to normal group. Compared to normal group,spleen lymphocyte apoptosis decreased,Bcl-2 protein expression increased,Bax protein expression decreased in model group.But the thymus lymphocyte apoptosis and Bcl-2,Bax protein expression were no statistically significant difference in each group.After treatment,compared with model group,the levels of IFN-γand the ratio of IFN-γ/IL-4 decreased,the levels of IL-4 increased in YiSui Granules group. The proportion of Treg cells and the levels of TGF-β1 was higher in YiSui granules group as compared to model group.However,the proportion of Treg cells was lower in YiSui granules group than that in normal group, there is no significant difference of the levels of TGF-β1 in YiSui granules group as compared to normal group.Compared to model group,spleen lymphocyte apoptosis increased,Bcl-2 protein expression decreased,Bax protein expression increased in YiSui granules group.4.Conclusions4.1 Successfully preparation GP-APS and established ITP mouse models. The peripheral platelet counts decreased in ITP mouse model,and bone marrow megakaryocytes increased and ysmaturity.It’s basically consistent with the clinical manifestations of ITP4.2Yisui granules can increase the peripheral blood platelet counts of ITP mice effectively and stimulate the bone marrow megakaryocytes differentiation to maturation.Its curative effect was equal to prednisone4.3 ITP has cellular immune function dereased and T cell subsets drift, manifestation of a type-1 (INF-γ) polarized immune response.The morbidity of ITP may be related to Th1/Th2 cell cytokine imbalance, Treg cells and TGF-β1 was reduced, lymphocyte apoptosis and Bcl-2, Bax protein expression abnormality. Yisui granules may be by regulating the Th1/Th2 cell balance and promote Treg cell proliferation and TGF-β1 secretion, and can regulate lymphocyte apoptosis and apoptosis-regulating genes Bcl-2, Bax abnormal expression, thus regulating the immune abnormalities in ITP,reduceing the platelet-associated antibodies, reducing platelet destruction, increased platelet count, promoting bone marrow megakaryocyte differentiation to mature. surface to achieve therapeutic effectreduce the difference between platelet antibody to reduce platelet destruction, increased platelet count, the promotion of bone marrow megakaryocyte differentiation to mature to achieve therapeutic effect.更多还原