【作者】 周方；

【导师】 陈胜喜；

【作者基本信息】 中南大学 ， 心胸外科学， 2009， 博士

【摘要】 研究背景重症肌无力主要是由乙酰胆碱受体抗体介导的,补体参与,细胞免疫依赖性,针对神经肌肉接头处突触后膜上乙酰胆碱受体的自身免疫性疾病。目前,重症肌无力的确切发病机制尚未完全明确,但普遍认为该病的发生与乙酰胆碱受体抗体的产生密切相关。B淋巴细胞是唯一能够分泌抗体的细胞,在体液免疫中发挥着重要作用,尤其是在血清抗体阳性的重症肌无力的发病过程中扮演了重要角色。趋化因子是能使细胞发生趋化作用的细胞因子的总称,为一类结构功能相似,分子量在8～10kD的小分子蛋白质,其主要作用是趋化细胞迁移。根据多肽链一级结构的不同将趋化因子分为CXC亚家族和CC亚家族。其中CXC亚家族分子靠近N一端两个半胱氨酸之间有一个任意氨基酸,主要作用于中性粒细胞,并对淋巴细胞有趋化作用。趋化因子受体是一类介导趋化因子行使功能的GTP蛋白耦联的跨膜受体,通常表达于免疫细胞、内皮细胞等细胞膜上。趋化因子能与其受体相互作用,通常一种趋化因子受体能与多种趋化因子相结合,而一个趋化因子可能有多个高亲和性受体,他们共同构成复杂的网络系统。CXCL13是CXC趋化因子家族成员之一,已经被证实在B淋巴细胞寻靶和淋巴组织中B细胞区的形成中有着重要作用,但目前尚无明确的关于CXCL13与重症肌无力发病机制相互关系的报道。实验性自身免疫性重症肌无力动物模型是研究重症肌无力发病机制的重要工具之一。目前,建立实验性自身免疫性重症肌无力动物模型有主动免疫和被动免疫两种方法,但是各有优缺点。一直以来,人类也在不断地探索,寻求建立完美的实验动物模型的方法,但尚未达成这一目标。目的通过检测CXCL13在重症肌无力患者血清中的水平以及CXCL13 mRNA在胸腺组织表达水平,来探讨CXCL13在重症肌无力的发病中的地位和作用,并阐述其在治疗研究中的价值。本研究拟通过被动移植重症肌无力患者增生的胸腺的方法建立实验动物模型,为研究重症肌无力的发病机制提供了一种新的实验工具。方法收集湘雅医院胸外科、神经内科重症肌无力患者治疗前及治疗后一周、一个月的血液标本各54份及健康体检者血液标本30份,用ELISA方法检测CXCL13在血清中的浓度。收集20例胸腺增生合并重症肌无力住院手术患者的胸腺标本及10例先天性心脏病手术患者胸腺组织标本,同时进行胸腺组织培养,用ELISA方法检测CXCL13在胸腺组织培养液上清中的水平,同时用RT-PCR的方法检测CXCL13mRNA在胸腺组织的表达水平,分析CXCL13、胸腺与重症肌无力三者之间的关系。将重症肌无力患者增生的胸腺组织被动移植到BALB/c裸小鼠腹腔建立重症肌无力动物模型,并通过症状观察、电生理检测、免疫组化、透射电镜和乙酰胆碱受体计数的方法来评价该动物模型,分析其用于重症肌无力发病机制研究中的意义。结果54份重症肌无力患者血清中CXCL13的浓度水平明显高于正常对照组(p＜0.05),在接受了肾上腺皮质激素治疗或胸腺切除手术的一个月之后,患者血清中CXCL13浓度水平较治疗之前有明显下降(p＜0.01),而CXCL13在眼肌型重症肌无力和全身型重症肌无力患者血清中的浓度水平无明显差异(p＞0.05);CXCL13在重症肌无力患者胸腺组织培养上清液中浓度水平显著高于正常对照组(p＜0.01),而在培养液中加入地塞米松后,其浓度水平会明显下降,并且与地塞米松的剂量呈负相关,而且在增生的胸腺组织中,CXCL13 mRNA的表达水平也显著高于正常胸腺组织(p＜0.01);在建立重症肌无力实验动物模型的研究中,25只实验组小鼠有9只(36%)出现了肌无力的表现,并通过电生理检测、免疫组化、透射电镜和乙酰胆碱受体计数的方法检测证实其组织改变与人类重症肌无力组织改变相近。结论CXCL13在重症肌无力患者血清中的浓度较正常人群明显增高,但在经过激素治疗和胸腺切除之后,其在血清中的浓度会缓慢下降,说明其可能可以作为重症肌无力患者血清指标,动态监测其水平变化可作为患者对治疗的反应性检测指标;CXCL13有可能成为评价重症肌无力患者预后的指标之一;在重症肌无力患者的增生胸腺组织中,存在着CXCL13 mRNA的大量表达,从而导致了CXCL13在组织培养上清液中及血清中水平增加,胸腺CXCL13的产生中扮演了重要角色。;地塞米松可直接抑制胸腺产生CXCL13;CXCL13有可能成为重症肌无力治疗研究的新靶点;通过将重症肌无力患者的胸腺组织被动转移至BALB/c裸小鼠腹腔的方法,可以成功建立实验性自身免疫性重症肌无力动物模型,此模型可作为研究胸腺与MG发病机制关系的实验工具。创新点1、首次通过ELISA方法和PT-PCR方法测定血清中CXCL13浓度水平及胸腺组织中CXCL13 mRNA的表达,从而评价了CXCL13、胸腺、重症肌无力三者之间的关系。2、通过胸腺组织块培养模拟胸腺在体内生长的环境,并加用地塞米松进行干预,从而证明地塞米松可抑制胸腺产生CXCL13,为将来重症肌无力治疗的研究提供一个新的切入点。3、首次采用将MG患者增生胸腺组织被动转移到BALB/c裸小鼠腹腔的方法成功建立了重症肌无力动物模型,该模型可更好地用于胸腺与MG发病关系的进一步研究,为MG发病机制的研究工作提供了新的研究工具。展望1、对MG患者进行血清CXCL13水平的长期动态监测,可能进一步评价其与症状波动之间的相关性。2、CXCL13水平与胸腺瘤合并MG患者的相关性有待于进一步研究。3、对于免疫抑制剂、放射治疗等干预措施,对CXCL13水平的影响尚需要深入探讨。4、本研究中的动物模型还有待于进一步完善,以进一步提高建模成功率、缩短建模周期。更多还原

【Abstract】 Background Myasthenia gravis(MG) is a neuromuscular disease with an autoimmune etiology.It is mediated by acetylcholine receptor antibody,which directed against nicotinic acetylcholine receptor. Although progress has been made in studying for MG,it is still unknown by us about its real pathogenesis.B-lymphocyte is known to be the only kind of cell that could secrete antibody,and it plays a key role in humoral immunity,especially in the patients with seropositive myasthenia gravis (SPMG).Chemotatic factor is a group of protein,which can stimulate cells migrating to the target tissue.Chemotatic factors can be divided into CXC subfamily and CC subfamily according to the difference of primary structure in polypeptide chain.Chemotatic factors in CXC subfamily can effect neutrophilic granulocyte and stimulate lymphocyte obtaining chemotaxis.Chemotatic factors could bind with their specified receptors to produce a marked effect of chemotaxis.CXCL13 is confirmed to be important for the formation and targeting of B-lymphocyte.It is reported that CXCL 13 plays a key role in several autoimmune disease,but there is no report about the relationship between CXCL13 and MG disease.Experimental autoimmune myasthenia gravis animal model is an important tool to study pathogenesis of MG.There are several methods to establish the animal model at the moment,but none of these methods is perfect.Objective To elucidate the relationship among CXCL13,thymus and MG,the concentration in serum and thymus tissue of CXCL13 in MG patients have been tested by way of ELISA.And we also test the level of expression of the CXCL13 mRNA in thymus tissues by way of RT-PCR. At the same time,we transfer the thymus tissue to the nude mice in order to establish a kind of animal model about experimental autoimmune myasthenia gravis(EAMG) for the further study in MG.Methods 54 serum samples of patients with MG complicated with thymic hyperplasia were respectively collected in Xiangya hospital in one week post-treatment,one month post-treatment and pretherapy. The concentration in serum of CXCL13 is tested by ELISA.20 resected tissue samples of thymus of MG patients are examined by RT-PCR to observe the expression level of CXCL13.And the concentration in supernatant of thymic tissue culture of CXCL13 is also tested by ELISA. The fragments of hyperplasia thymic tissue were transferred into abdominal cavity of BALB/c nude mice in order to establish a kind of animal model about experimental autoimmune myasthenia gravis.And the methods of immunohistochemistry,transmission electron microscope, electromyogram and symptom observation were used to evaluate the animal model.Results The CXCL13 concentration in serum of MG patients in pretherapy is thicker than the normal people(p＜0.05),but it will decrease obviously one month after the treatment of glucocorticoid or thyectomy (p＜0.01).There is no difference of CXCL13 concentration between ocular MG patients and generalized MG patients(p＞0.05).The CXCL13 concentration in supernatant of hyperplasia thymic tissue culture of MG patients is thicker than that in normal thymic tissue(p＜0.01).And the level of CXCL13 mRNA expression in hyperplasia thymic tissue is also higher than the normal one.If we add dexamethasone into the culture fluid,we can observe that the CXCL13 concentration in supernatant becomes thinner(p＜0.01).The more the dexamethasone we add,the thinner the CXCL13 concentration will be.In the experiment of establishing animal models of EAMG,we obtain 9 mice presenting the symptoms of MG(9/25),and it is confirmed to fit the morphological features of human MG by way of immunohistochemistry,transmission electron microscope and electromyogram.Conclusions Because the serum CXCL13 concentration of MG patients is thicker than that in normal people,in addition,it will decrease obviously after treatment,we think that CXCL13 may be the serum maker of MG patients,and we could evaluate the effect of treatment and the prognosis by monitoring CXCL13 concentration in MG patients’ serum.Because of over expression of CXCL13 mRNA in hyperplasia thymic tissue,which lead to increasing of CXCL13 concentration in supematant of hyperplasia thymic tissue culture,we think that the thymus may play a key role of MG.Dexamethasone could inhibit the expression of CXCL13 mRNA directly,and CXCL13 might be the new target of further research for the MG treatment.A new animal model of EAMG could be established by way of transplanting the fragment of hyperplasia thymic tissue into BALB/c nude mice,which might be a kind of new tool for the further research of MG,especially for the research of relationship between thymus and MG.更多还原