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重症肌无力患者胸腺组织蛋白4.1R的鉴定及表达研究

The Studies of Identification and Expression of Protein 4.1R in Thymus Patients of Myasthenia Gravis

作者: 专业:外科学 导师:张清勇 年度:2010 学位:硕士  院校: 郑州大学

Keywords

MyastheniaGravis, Protein4.1R, RT-PCR, Immunohistochemistry, Peptide mass fingerprint

        背景及目的重症肌无力(Myasthenia gravis,MG)是以横纹肌反复收缩乏力为特征,主要累及神经-肌肉接头处突触后膜乙酰胆碱受体的器官特异性自身免疫性疾病。发病原因尚不清楚,但与胸腺异常有关已成共识。为了深入研究MG的发病机理和胸腺异常在MG发生、进展中的作用,课题组在对MG患者胸腺提取液研究中发现,部分患者SDS-PAGE存在相对分子质量<11KD的差异(异常)蛋白带,并且该组患者在胸腺摘除术后疗效明显优于其他MG患者,表明这些差异(异常)蛋白可能与MG症状持续有关。于是在课题组前期建立的MG胸腺组织蛋白质组学筛选出差异表达蛋白基础上,选择其中的差异蛋白点进行质谱分析获得到每个蛋白点的肽质量指纹图(peptide mass flngerprint,PMF)图谱,而后根据所获得的PMF和串联质谱图各肽段的质荷比(m/z)数据进行检索,得到细胞骨架蛋白4.1R(Protein 4.1R)。Protein4.1R作为重要的膜骨架成分,它参与细胞增殖分裂的调节,可能在细胞粘附因子和细胞内信息传导中处于“信号中转站”的关键性作用。复习国内外文献均未见有与MG的相关报道,而后运用RT-PCR和免疫组化技术,对MG胸腺组织中蛋白4.1R表达情况进行了研究。通过蛋白鉴定、检测蛋白4.1R在MG患者胸腺组织中的表达水平,并探讨其在MG中的作用,从而为其在MG中进一步研究奠定了基础,同时也为MG的治疗提供新的思路和实验理论依据。材料和方法1、实验组为郑州大学第二附属医院2007年9月至2009年7月住院确诊MG病例,经知情同意后手术切除的胸腺标本20例,其中男8例,女12例,平均年龄(30.5±18.1)岁。根据临床病情严重程度参照Osserman改良分型,其中Ⅰ型8例,Ⅱ型5例,Ⅲ型4例,Ⅳ型3例;根据病理类型分为:增生型7例,萎缩型6例,伴胸腺瘤型7例。对照组7例,为先天性心脏病行矫治术时为暴露术野而部分切除的胸腺(病理检查证实为正常胸腺),男3例,女4例,平均年龄(21.5±19.5)岁,患者HIV(-),无MG及其他自身免疫性疾病。2、在课题组前期建立的MG胸腺组织蛋白质组学筛选出差异表达蛋白基础上,选择其中的16个差异蛋白点进行质谱分析获得到每个蛋白点的PMF图谱,而后根据所获得的PMF和串联质谱图各肽段的质荷比(m/z)数据登陆互联网http://www.matrixscience.com用Mascot程序和http://prowl.rockefeller.edu用Profound程序进行检索NCBlnr数据库,鉴定得到差异蛋白4.1R,而后利用RT-PCR和免疫组化方法,检测胸腺瘤、增生型、萎缩型胸腺MG组与正常对照组胸腺组织中差异蛋白4.1R的表达情况。3、所有数据用SPSS11.5统计软件处理,数据均以x±s表示,进行方差齐性检验和正态性检验,符合条件成组设计两样本用t检验和多组比较用单因素方差分析(one-way ANOVA),检验水准a=0.05。结果1、获得到2号蛋白PMF和串联质谱图,运用Mascot和Profound两种程序检索得到2号Protein 4.1R,基因序列号:gi/5353740,理论分子量8.136KDa、等电点(PI)4.76与表观分子量8.9KDa、PI 4.3相近且种属相符。2、免疫组化结果显示:MG组(胸腺瘤、增生型和萎缩型,n=20)胸腺组织中,蛋白4.1R的平均光密度(Mean optical density, MOD)为2067.500±2280.874,与对照组(n=7)1485.227±1182.164相比,差异无统计学意义(P=0.569>0.05);胸腺瘤(n=7)、增生型(n=7)和萎缩型(n=6)MG组胸腺组织中蛋白4.1R的MOD分别为1395.898±2651.899、4403.023±3629.256、3007.742±1734.527,经比较三者组间的差异无统计学意义(P=0.399>0.05)3、胸腺瘤(n=7)、萎缩型(n=6)MG组胸腺组织中蛋白4.1R的MOD分别与对照组(n=7)比较P=0.587>0.05、P=0.086>0.05差异无统计学意义,而增生型(n=7)与对照组(n=7)比较P=0.038<0.05差异有统计学意义。4、MG组(胸腺瘤和萎缩型,n=13)胸腺组织中蛋白4.1R mRNA的表达水平为0.76±0.33,与对照组(n=7)0.43±3.69相比明显增高,差异有统计学意义(P=0.033<0.05);而胸腺瘤(n=7)和萎缩型(n=6)MG组胸腺组织中蛋白4.1R mRNA的表达水平分别为0.84±0.46、0.67±0.10,经比较二者之间的差异无统计学意义(P=0.386>0.05)。结论1、成功鉴定出Protein 4.1R,为MG发病潜在分子机理的阐明提供了重要方法和线索。2、Protein 4.1R在MG患者胸腺组织中mRNA和蛋白水平表达异常,提示Protein 4.1R可能参与了MG的发生。3、Protein 4.1R是MG相关蛋白,是胸腺异常的物质基础,但与重症肌无力的发病及发展机制尚不清楚。
    Background and ObjectiveMyasthenia gravis (myasthenia gravis,MG) is an organ-specific autoimmune disease characterized by weak contraction in striated muscle,which mainly involved the nerve-muscle joints at postsynaptic membrane acetylcholine receptor.Its nosogenesis relate to thymic abnormalities but the etiology is not clear.In order to study the pathogenesis of MG and understand the role of thymus abnormalities in the occurrence and development of MG,our research group analyzed the thymus extract and found that there were molecular weight<11KD different (abnormal) protein bands existed in some patients by SDS-PAGE.The therapeutic effect with removal of the thymus were more significant in patients than in other MG patients,which indicated that these different (abnormal) protein bands may be related to the duration of symptoms related to MG. On the basis of the methods using MG thymus tissue proteomics to screening difference protein which we had established in our early study,the differences protein spots were selected,and analyzed by mass spectrometry to obtain peptide mass fingerprint (PMF) for each protein spots,and then retrieved data according to the PMF and tandem mass spectra of all peptide mass charge ratio (m/z),finally we found that it was cytoskeletal protein 4.1R (Protein 4.1R).As an important component of membrane skeleton,which participate in the regulation of cell division,protein 4.1R may be play a key role as "signal transfer station" during the signal transduction process between cell adhesion molecule and intra-cellular.There were no literatures or relevant reports about MG were reviewed, we investigated the expression of protein 4.1R in MG thymus tissue by RT-PCR and immunohistochemistry.After protein identification,we detected the expression level of protein 4.1R in MG patients’thymus tissue,and explored their function in MG.It established foundation for further study and provided new ideas and experimental evidence for MG therapy.Materials and methods1.We choose the hospitalized patients diagnosed MG from September 2007 to July 2009 in the Second Affiliated Hospital of Zhengzhou University as the Experimental group.We resected thymus from 20 patients as the specimen after the patient’s consent.Among them,there are 8 males and 12 females.their mean age is 30.5±18.1 years.According to Osserman’s improved classification and the severity of disease,we divided these cases into 4 types,includingⅠtype:8 cases,Ⅱtype: 5 cases,Ⅲtype:4 cases,Ⅳtype:3 cases.In terms of pathological type,they can be divided into three types,including hyperplasia type:7cases,atrophic type:6 cases, thymic tumor type:7cases.The control group includes 7 cases of patients with congenital heart disease.We perforn surgery to treat such disease.We resected the part of thymus in order to make exposure to operative field more clear (the thymus resected is confirmed to be normal by pathology).The 7 cases of patients includes 3 males and 4 females.Their mean age is 21.5±19.5 years.These patients have no MG and other autoimmune diseases,HIV(-).2.Based on the thymus different expression selected by Proteomics,we use Mass Spectrometry to analyze 16 different protein spots chosen randomly to acquire the PMF map for the every protein spot.Then,according to the PMF obtained and each peptide’mass to charge ratio (m/z) data in the Series Mass spectrum,we entered into the internet http://www.matrixscience.com and http://prowl.rockefeller.edu in which we began searching the NCBInr database by using Mascot procedure and Profound procedure.We identify and aquire the difference protein:4.1R.Then,we probed the difference in the expression of difference protein 4.1R among thymoma,Hyperplasia type,Thymus of atrophic type in group MD and the normal control group.3.Statistical Methods:The statistical software SPSS 11.5 for windows was exploited to analyis data. All the data were expressed by (x±s) Deviation and difference were compared using the one way ANOVA and T test.The difference and significance were judged by P<0.05.Results1.The 2nd protein PMF and tandem mass spectra were obtained and the Protein 4.1R was obtained by using two procedures of the Mascot and Profound,gene sequence number:gi/5353740,apparent molecular weight 8.9KDa and isoelectric point (PI) 4.3 are similar and consistent on species with theoretical molecular weight 8.136KDa,PI 4.76.2.1mmunohistochemistry results showed that:There was no significant difference between the MG group (n=20,2067.500±2280.874) and control group(n =7,1485.227±1182.164) in the expression of protein 4.1R mean optical density(P= 0.569>0.05).But the expressions in different pathological classificition of MG thmic tissues were1395.898±2651.899,4403.023±3629.256 and 3007.742±1734.527, respectively,which was no significant difference(P=0.399>0.05).3.There is no significant difference between the control group(n=7) and group of thymoma(n=7,P=0.587> 0.05),atrophictype (n=6,P=0.086> 0.05)in the expression of protein 4.1R mean optical density,but it is significant different between the control group and the group of proliferative (n=1,P=0.038<0.05).4.The expression of protein 4.1R mRNA in the thymus tissues of MG (n=13, 0.76±0.33)was significantly higher than that of the control group(n=7,0.43±3.69) (P=0.033<0.05).There was no significant difference between the thymoma group (n=7,0.84±0.46) and atrophictype group(n=6,0.67±0.10) in the expression of Protein 4.1R mRNA (P=0.386>0.05).Conclusions1.Protein 4.1R successfully identified has provided an important clarification of methods and clues for the potential molecular mechanism of MG.2.The abnormal expression of Protein 4.1R mRNA and protein in thymus tissue of MG indicates that Protein 4.1R may be involved in the occurrence of MG.3.As the MG-related protein,Protein 4.1R is the material basis of abnormal thymus,but it is unclear that Protein 4.1R played a role on the occurrence and development of mechanism in MG.
        

重症肌无力患者胸腺组织蛋白4.1R的鉴定及表达研究

摘要5-8
Abstract8-11
论文部分 重症肌无力患者胸腺组织蛋白4.1R的鉴定及表达研究13-49
    引言13-16
    材料与方法16-26
    结果26-36
    讨论36-42
    结论42-43
    附图43-46
    参考文献46-49
综述部分 重症肌无力相关基因及蛋白的研究49-66
    参考文献62-66
附录部分66-68
    英文缩略语表66-67
    个人简历67-68
    致谢68
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