【中文摘要】 研究目的:特发性血小板减少性紫癜(Idiopathic Thrombocvtopenic Purpura,ITP),也称为自身免疫性血小板减少性紫癜,是临床上常见的一种出血性疾病。临床上以出血及外周血血小板减少、骨髓巨核细胞数正常或增多伴有成熟障碍为主要表现。该病的发病率为50～100/106万人口,其中半数以上是儿童,小儿患者以急性型多见,多呈自限性。约有20%～25%患儿可发展成为慢性,其临床表现缓和,但呈慢性难治性。对于慢性ITP(CITP),其常规治疗包括肾上腺皮质激素、静脉免疫球蛋白(IVIG)、a—干扰素、脾切除、抗D抗体及其它免疫抑制治疗,疗效皆不理想。患者长年受出血的影响,往往需反复输注血小板,不仅费用高,维持时间短,还容易传播肝炎、艾滋病等疾病。近年来,与此病密切相关的白细胞介素-11(IL-11)成为大家研究和开发的新宠儿。IL-11是起源于骨髓基质细胞的一种造血生长因子,具有多种生物学活性。它可以诱导造血干细胞的生长和分化;协同白细胞介素-3(IL-3)、干细胞因子(SCF)促进原始细胞的增殖;促进巨核细胞和成熟血小板的生成,对巨核细胞系和血小板生成的不同阶段都...更多有刺激作用。此外,它能减轻免疫反应、抑制脂肪形成(故IL-11又称脂肪形成抑制因子)、刺激海马神经祖细胞的增殖、参与上皮细胞的正常生长调控、降低发热、还具有一定的抗炎作用。自1997年10月,美国FDA批准rhIL-11(重组人IL-11,商品名oprelvekin)上市,成为第一个在临床上应用于肿瘤病人放化疗后升高血小板的药物,针对其临床应用的研究逐渐增多。大量研究证明IL-11可用于治疗放化疗或骨髓衰竭引起的血小板减少,甚至对粒、红两系也有不同程度的提升作用。近年来,不少学者采用IL-11治疗CITP,取得了一定疗效。有研究证明,IL-11可直接影响T细胞及其受体的功能,在体内外均可抑制白细胞介素-2(IL-2)、干扰素-α(IFN-α)、白细胞介素-6(IL-6)等多种关键性免疫刺激因子的产生,促进CD4+T细胞增生、Th细胞(T辅助细胞)向Th2(T辅助细胞2)亚群分化,从而引起T辅助细胞1/T辅助细胞2(Th1/Th2)比例失调,增加了ITP的发病几率。另外有学者进行临床研究发现CITP患者血清中IL-11水平高于正常,提示IL-11可能在ITP的发病机制中有一定的作用。既然CITP患者血清中IL-11水平升高,为何还要用IL-11治疗且治疗有效?对于IL-11治疗无效的患者,是否存在IL-11受体(IL-11R)的不应答或者受体的缺失?CITP患者体内IL-11与IL-11R如何结合发挥作用以及两者在CITP发病中的具体机制如何?这些问题目前尚无定论。为此,我们在成功建立CITP小鼠模型的基础上,检测其外周血中IL-11及IL-11R的水平,了解两者之间的关系,进一步探讨ITP的发病机制,试图为CITP的临床治疗提供新的思路。研究方法:SPF级健康BALB/C小鼠共90只,雌雄各半,体重20±2g,随机分为2组:对照组(n=36);CITP组(n=54)。对照组:生理盐水5mL/kg/只,于第1,3,5,7,9,11,13,15,17d腹腔注射。CITP组:1:3稀释的豚鼠抗小鼠血小板血清(APS)5mL/kg/只,于第1,3,5,7,9,11,13,15,17d腹腔注射。分别观察以下指标:1.各组小鼠的发病及死亡情况。2.第0,8d每只小鼠均剪尾取血,测血常规。3.第8,11,14,17d随机抽取对照组小鼠8只,CITP模型组小鼠12只,分别称重,断头取血两管,肝素抗凝处理,一管用于测定IL-11R水平,另一管2000r/分离心20min(400G,离心半径11cm),制备血清,-20℃储存备用,以测定IL-11水平。4.将3中处理后的80只小鼠解剖(1)取脾脏称重,并以多聚甲醛固定、切片,观察造血情况。(2)取双侧股骨骨髓,缓慢离心后推片,瑞-姬氏复合染液染色后,光学显微镜下观察骨髓巨核细胞情况。5.剩余10只小鼠正常饲养1周,观察存活及死亡情况。研究结果:1.两组小鼠发病及死亡情况对照组小鼠进食、活动无明显改变,毛色光亮顺滑,无自发性出血及死亡;CITP组小鼠在实验中出现不同程度的尾部断端自发性出血,并有2只小鼠于实验后期死亡。2.两组小鼠用药前后血常规的变化两组小鼠用药前血常规各项指标WBC、Hb及PLT计数均无明显变化;用药后第8d,CITP组小鼠PLT计数较对照组明显下降(p＜0.05),WBC及Hb无明显变化,符合CITP血象。3.两组小鼠外周血中IL-11及IL-11R水平的比较CITP组小鼠第8,11,14,17d外周血中IL-11平均水平较对照组均升高(p＜0.05),且其升高水平呈逐步降低的趋势;IL-11R平均水平较对照组升高(p＜0.05),其升高水平无变化趋势,其中有11只小鼠IL-11R水平未出现升高而较对照组降低。4.两组小鼠脾脏重量的比较及脾脏造血情况脾脏/体重重量比两组之间无明显差异(p＞0.05),均未见髓外造血。5.两组小鼠骨髓巨核细胞的比较对照组小鼠骨髓巨核系增生活跃,血小板成堆易见;CITP组小鼠骨髓巨核细胞计数较对照组明显增多(p＜0.05),产板巨计数明显减少(p＜0.05),且存在成熟障碍,血小板少见,符合CITP骨髓象。6.CITP组小鼠IL-11及IL-11R水平与骨髓巨核细胞的相关性CITP组小鼠外周血中IL-11水平与骨髓巨核细胞计数及产板巨计数之间无相关性;IL-11R水平与骨髓巨核细胞计数及产板巨计数呈直线正相关(相关系数r巨核=0.77,r产板巨=0.84,p＜0.05)。研究结论:1.通过腹腔注射豚鼠抗小鼠血小板血清(APS)的方法能够成功建立CITP小鼠的模型,便于实验的顺利进行。2.CITP组小鼠外周血中IL-11水平升高,且升高水平呈逐步降低的趋势;IL-11R水平普遍升高,其升高水平无变化趋势,其中有11只小鼠IL-11R水平降低,提示IL-11在CITP的发病机制中其一定的作用。3.CITP组小鼠外周血中IL-11水平与骨髓巨核细胞计数及产板巨计数之间无相关性;IL-11R水平与骨髓巨核细胞计数及产板巨计数呈直线正相关。故通过检测骨髓中巨核细胞及产板巨的计数可以了解外周血中IL-11R水平的高低。4.IL-11与IL-11R在CITP时是如何结合发挥作用的,以及与CITP发病之间的关系尚需进一步的研究和探讨。 

【英文摘要】 ObjectiveIdiopathic Thrombocvtopenic Purpura(ITP) or autoimmune thrombocvtopenic purpura,is a common hemorrhagic disease.The main manifestation includes hemorrhage,thrombocytopenia and dysmaduritic megakaryocyte of normal or increased amount in the bone marrow.Its incidence is 50～100/106 million,among which more than 50%are children.Children often present acute and self-limited cases.20%～25%patients developed into chromic cases,with mild and refractory clinical symptoms.The conventional therapy for chronic and refractory ITP(CITP) covers GCS,IVIG,IFN-,splenectomize,anti-D Ig and other immunosuppressants.But the curative effect is still unsatisfactory.Patients have to be transfused with PLT repeatedly because of hemorrhage.The expense is great but the duration is short.Meanwhile it brings the risk of hepatitis and HIV etc.IL-11,a key cytokine of ITP,has become a research focus recently.IL-11 is one of haematopoietic growth factors with pleiotropic effects on multiple tissues.It can indu...ce the growth ofhematopoietic stem cells.Combined with IL-3 and SCF,it stimulates directly the proliferation of hematopotietic stem cells and induces megakaryocytes maturation resulting in increased platelet production.It also has biological activity on other tissues,including brain,spinal cord nervous,fat cells, et al.Since October,1997,the Food and Drug Administration approved rhIL-11 to be the first drug for increasing platelet production of patients with tumor following chemotherapy and radiation.A lot of researches proved IL-11 can use to treat chemotherapy-induced thrombocytopenia arising from marrow hypoplasia.It also enhanced leucocyte and erythrocyte.Recent years,some scholars get several curative effects by using IL-11 to treat ITP.Some research attest IL-11 can work on T cell and its receptor and restrain the production of IL-2,IFN-α,IL-6.It also can promote the hyperplasia of CD4+T cells,induce the maladjustment of Th1/Th2 which increased the incidence rate of ITP.Moreover,some studies discover that the levels of IL-11 in the blood are higher in the patients with CITP,which suggest that IL-11 maybe take a certain action in the mechanism of ITP.While the levels of IL-11 in the blood are higher in the patients with CITP,why are they using IL-11 to treat ITP ? Whether absenting of IL-11 or IL-11R in patients useless of treatment with IL-11 ? Further studies are needed to discuss the role of IL-11 and its receptor in the pathogenesis of chronic idiopathic thrombocytopenic purpura.So we induced the animal model of CITP,tested the level of their IL-11 and IL-11R,In order to assess whether absenting of IL-11 or IL-11 receptor and which will provide new theory to treat CITP.MethodsNinety(20±2 grams body weight) BALB/C mice were randomly dividedinto two groups:Group control(n=36) and Group CITP(n=54).Group control:Intraperitoneally inject NS 5mL/kg per one at the 1,3,5,7, 9,11,13,15,17 day.Group CITP:Intraperitoneally inject diluted APS(1:3) 5mL/kg per one at the 1, 3,5,7,9,11,13,15,17 day.Observation these items separately:1.Observed the clinical manifestation of mice.2.Taken blood to test blood routine by cutting trails at the zero and eighth day.3.Taken out eight mice in group control and twelve mice in group CITP randomly at the 8,11,14,17 day.Taken two tubes of blood via decollation.Use heparin to prevent cruor.One tube was used to test IL-11 receptor,while the other was used to test IL-11.4.Anatomized the third dead mice.(1) Weighed and observed the hemopoiesis of spleen.(2) Observe the megakaryocyte of marrow by light microscope.5.Observed the living or death of remain mice for one week.Results1.The clinical manifestation of miceGroup control was normal.Group CITP appeared different degree of spontaneous bleeding,and two mice died in anaphase.2.The change of blood routineTwo groups' blood routines had no change before injection.At the eighth day after injection,the platelet count of group CITP descended obviously,while the white blood cell and hemoglobin had no change.3.The level of IL-11 and IL-11RCompared with group control,the average level of IL-11 in group CITP increased at the 8,11,14,17 day,and was decreased step by step.The average level of IL-11R in group CITP also increased,but there were eleven mice had lower leverl of IL-11R.4.The weight and hemopoiesis of spleenThe weights of spleen had no difference in two groups and hemopoiesis deside marrow was unseen.5.The megakaryocyte of marrowThe megakaryocyte of marrow was normal in group control.The megakaryocyte of marrow in group CITP increased obviously and had maturation barrier.It's according with ITP marrow picture.6.The correlation between IL-11,IL-11R and bone marrow megakaryocyte in group CITP There was no correlation between the lever of IL-11 in the blood and bone marrow megakaryocyte counts.A significant positive correlation was found between the lever of IL-11 receptor in the blood and bone marrow megakaryocyte counts (r=0.77,r=0.84,p＜0.05).Conclusions1.Using APS to intraperitoneally inject into the mice could induce to set up the animal model of chronic idiopathic thrombocytopenic purpura(CITP).And its death rate was so lower that be convenient for experiment.2.The level of IL-11 in the blood of group CITP was increased and was decreased step by step.The level of IL-11R in group CITP also increased,but there were eleven mice had lower leverl of IL-11R.These suggested that IL-11 maybe take a certain action in the mechanism of CITP.3.There was no correlation between the lever of IL-11 in the blood and bone marrow megakaryocyte counts.A significant positive correlation was found between the lever of IL-11 receptor in the blood and bone marrow megakaryocyte counts.These suggested that we can get the message of the level of IL-11R in the blood by detecting bone marrow megakaryocyte counts.4.Further studies are needed to discuss the role of IL-11 and its receptor in the pathogenesis of chronic idiopathic thrombocytopenic purpura(CITP). 

【中文关键词】 白细胞介素-11; 白细胞介素-11受体; 慢性特发性血小板减少性紫癜
【英文关键词】 interleukin11; IL-11 receptor; chronic idiopathic thrombocytopenic purpura