摘要
研究背景 多发性硬化(multiple sclerosis，MS)是一种中枢神经系统自身免疫性疾病，其病因尚未完全清楚，遗传和环境因素均能影响对该病的易感性。其中遗传因素在MS易感性方面的作用虽已被肯定，但目前还没有发现任何一个基因对于MS的发病是起唯一决定作用的，即未发现任何单一基因可致MS，因而寻找MS与基因的相关性已成为人们试图揭示其发病机制的重要研究方向之一。Sp3(Specificity protein，Sp3)基因是一种核转录因子，属Sp家族成员之一，是继Sp1后发现的具有与之相同的DNA结合区的转录调节因子，近年来发现它对众多基因均具有调节作用，它通过与GC/GT box结合，激活许多病毒和哺乳动物细胞的启动子，决定启动子的转录效率和特异性。还发现Sp3在基因转录激活中既可有抑制作用，也可有激活作用，甚至对同一基因的不同时期也可发挥不同的作用，因此对人体内众多基因的表达有着重要意义。1996年Grekova发现79％MS患者存在Sp3基因表达缺失，此现象已开始引起国内外同行的普遍关注。
目的 通过对核转录因子Sp3基因表达的研究，了解中国人多发性硬化Sp3基因表达缺失的特点及与临床的关系，为临床诊断及评判预后提供帮助，并探讨该基因在MS表达缺失的可能原因，有助于MS发病机制的进一步阐明。
方法 本研究的第一部分采用RT-PCR方法，通过收集临床确诊的MS病例，提取总RNA，设计Sp3特异性引物，观察Sp3在MS中表达缺失现象，对其与临床的关系进行揭示，并对Sp3在正常人不同组织中广泛表达现象进行了观察。第二部分采
中南人学湘雅【院博1：学位论上 中义搞公
用半定量 RTPCR方法对 Sp3与 MS外周血单个核细胞 ILJ 表达关系进行初
步观察。第三部分利用已公布的Sp3全长CDNA序列，经同源性比较分析，对该基因
进行染色体定位及基因的结构预测，并对Sp3表达缺失的原因进行了二方面的探讨。
首先采用SSCP方法，扩增2～5号外显子的编码序列，对病人和正常人进行比较，以
判明 Sp3基因全长编码序列基因突变与 Sp3不表达的关系。其次对 MS病人 ZI～2065fit
（根据 Genbank登录号 gi：13162672序歹）约 2000hp cDNA序歹进行 了坝序，探讨
MS病人 Sp3基因是否存在 m RNA水平的改变。
结果 实验的第一部分以CDNA为模板进行PCR，56例MS患者有33例Sp3为
阳性结果，另外23例为阴性结果，缺夫率为41．l％；正常对照组35例30例为阳性
结果，缺失率为8石％；OD组27例23例为阳性结果，缺失率为14．3％。经统计学处
理，OD组与正常对照组之间缺失率差异无显著性意义，但＊S组与两组对照组之间
的差异具有显著性意义（P＜0．of）。此外，还对＊D组，如 RA、SLE、MG、GBS等
作了对照研究，结果显示在这类疾病中Sp3的表达缺失为15％，比正常人稍高，也无
统计学意义。对该组MS的研究显示有5例先后2次抽血复查Sp3表达，其间接受了
激素治疗、病程有的经历了急性期到稳定期的变化，但其Sp3表达与否并未改变。从
EDSS评分来看，SP3缺失组与表达组比较急性期无明显差别，稳定期则有较显著差
别，经统计学处理显示P（0刀5。本组视神经脊髓炎有15例，表达缺失率为26厂％，
其缺失率与MS组比较，经统计学处理P值为0卜此外还观察到Sp3在正常人8个
不同组织均有广泛表达的现象。第二部分初步观察到25例MS中Sn3阳性组几一10
表达较高，阴性组表达较低，但非显著性意义的差别（P＞0．2）。第三部分利用已公布
的Sp3全长CDNA序列，经同源性比较，将该基因定位在2号染色体上，它包含5个
外显子和 4个内含子。首先采用 SSCP方法，对病人和正常人2～5号外显子的编码序
3
中南大学湘雅医院博卜学位论文 中文摘要
列进行筛查，未筛检到单个核着酸序列的改变，同时排除了大片段DNA缺失。其次
对MS病人对～2065lit（根据 Genbank登录号i：13162672序列）约2000hp cDNA
序列进行了测序，仅发现239处存在G—A单核着酸的改变，导致氨基酸序列相应的
改变，即苏氨酸（Thr）一丙氨酸W。该结果说明了 MS患者 Sp3在 mRNA水平的
结构也大致正常。
结论 Sp3与MS免疫学发病机制之间可能存在一定的相关性，通过对MS Sp3
基因表达的研究，观察到中国人MS存在该基因表达缺失，但其缺失率与西方MS人
群的分布存在较明显差异；Sp3表达缺失对于MS病人是比较特异的一种现象，其临
床价值在于①可供临床作为判断的一个辅助资料，如可疑MS的病人，遇到Sp3阴性，
对本病的诊断考虑可能有一定帮助。②Sp3基因表达与否与 MS临床表现之间存在一
定的关系，MS病人中SP3表达或不表达和年龄、性别、复发次数、病灶数目、病程、
疾病活动性或是否激素治疗等并无关系：③…不表达可能与＊S稳定期的残废程度
有关，Sp3阴性可能提示预后欠佳；④过去认为视神经脊髓炎是一个独立的疾病，近
年来多数学者认为它是MS的一个?
Background Multiple sclerosis (MS) is a central nervous system autoimmune disease, but the cause of the disease is not yet complete clarified. Both genetic and environmental factors may influence susceptibility to the disease, the function of the heredity has already been affirmed. But even now people haven't discover a unique gene whichever to decide the development of MS or any one gene can cause MS, but Looking for the relativity of Sp3 and MS has become an important research direction to announce the mechanisms of the disease. Sp3 gene is one of the transcriptional factors, which belonging to Sp family. It has the same DNA binding site with Spl and is concerned with regulation of many genes. It activates many virus and mammalian cells' promotors through the binding with GC/GT box, deciding the efficiency and specificity of the transcription of promotor. Sp3 gene turning
out both activating and repressing function, even to the same gene, it can also develop the different function in different period, and therefore has got the important meaning to numerous genes of human body. In 1996, the researchers discovered the deficient express of Sp3 gene existed in MS when they studied with a monozygotic twin by differential display method. Succedent clinical research proved the conclusion. The mechanism of the phenomena is still ambiguity, but it has aroused people's abroad attention.
Objective To observe the existence of Sp3 gene deficient express in Chinese multiple sclerosis, and discuss the correlation between Sp3 and the clinical presentation and prognosis in MS. Try to find out the possible reasons that cause the deficient express of Sp3, and further reveal mechanism of MS.
Methods In the first part, we collected the patients with definite MS, and extracted the total RNA from PBMC. The RT- PCR method was used to observe Sp3 deficient expression phenomenon in the MS. In the second part, semi-quantitative RT-PCR method was used to investigate interleukine 10 express in patients' PBMC, we further study the relationship between Sp3 deficient expression and the level of interleukine 10 express. In the third part we BLAST in the GenBank using the published Sp3 cDNA sequences, to estimate Sp3 gene organization and location in the chromosome. SSCP method was used to screen the nucleutide mutation of coding sequences inorder to compare patients with normal controls. Direct sequencing was used to sequence a MS patient's cDNA sequences including 21 to 2065 nucleutides.
Results In the first part, in MS group, we found 33 cases of 56 got Sp3 positive results, the other 23 cases got negative results, the absent rate was of Sp3 was 41.1%; in normal control group, 5 cases of 35 got negative results, the absent rate was 8.6%; in OD group, 4 cases of 27 got negative results, the absent rate was 14.3%; in OID group, 3 cases of 20 got negative results, the absent rate was 15.0%. In OD/OID groups, the Sp3 express rate were a little above the normal control group, but without statistical significant (P>0.05). But there were significant difference between MS and control groups by
statistical analysis (P<0.01). Sp3 expresses or not in MS patients was irrelevant with the number of relapse times, the number of the focus and the course of the disease. 5 Cases with hormone treatment patients who underwent active and stable stage didn't change the express of Sp3 or not. Sp3 lack express group showed no different with express group in EDSS grade in active MS, but there was distinct different in stable MS according to statistical analysis(P<0.05). Devic disease is an independent disease, most of the doctors think it is subspecies of MS. It showed lower deficient rate of Sp3 than other patients. We included 15 cases of Devic disease in our research, 4 of 15 lose the express of Sp3; while other members in MS group, 19 of 41 lose the express of Sp3, we found that Devic disease had a lower deficient rate of Sp3, but when we compared them with other members in MS group, there were no significant different between the lose rate, the valu
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