【作者】 王晓；

【导师】 李兴福； David S. Pisetsky；

【作者基本信息】 山东大学 ， 内科学， 2015， 博士

【摘要】 研究目的系统性红斑狼疮(Systemic Lupus Erythematosus, SLE)是一种异质性自身免疫疾病,常累及机体多个组织器官,发病原因至今不明。SLE的病理特点是多种自身抗体过量表达和大量抗原-抗体免疫复合物沉积。DNA是SLE最重要的自身抗原之一,抗双链DNA (ds-DNA)抗体作为SLE标志性抗体,对诊断和治疗效果的观察具有重要意义。SLE抗双链DNA抗体与DNA的主要结合部位为保守的磷酸骨架,也有部分抗体特异性结合于DNA碱基区域,具有种类差异性。多胺(polyamines)作为一类小分子生物胺类化合物,广泛存在于真核生物细胞核及原核生物细胞内,在DNA构象及基因转录等方面发挥重要作用。本研究通过检测多胺对SLE患者及健康正常人群(Normal Human Subjects, NHS)血清抗双链DNA抗体与小牛胸腺DNA及藤黄微球菌DNA结合力的影响,对DNA与抗DNA抗体的结合作用机制做了初步探讨,揭示了不同抗DNA抗体之间的差异性,以及多胺对SLE抗DNA抗体的可能作用。研究方法取SLE患者血清5例,其中女性3例,男性2例,其诊断符合1997年美国风湿病学会(ACR)修订的SLE分类标准。健康对照组NHS血清5例,其中女性3例,男性2例。采用酶联免疫吸附试验(Enzyme-linked Immune Sorbent Assay, ELISA),分别以小牛胸腺(Calf Thymus, CT) DNA和藤黄微球菌(Micrococcus Luteus, MC) DNA作为哺乳动物抗原和细菌抗原的代表,测定三种多胺(精胺、亚精胺和腐胺)在不同浓度下(50-1000μM)对SLE及NHS抗CT DNA抗体及抗MC DNA抗体浓度的影响。以多胺存在条件下与无多胺存在条件下抗体浓度的比值判定多胺对抗体与抗原结合力的作用水平。为进一步测定精胺对不同类型抗DNA抗体的作用,SLE或者NHS血清先经CT-DNA纤维素膜(CT-DNA cellulose)吸附除去非特异性抗DNA抗体,再测定精胺存在条件下抗体浓度变化。以破伤风类毒素为抗原,用相同方法测定精胺对SLE血清抗破伤风类毒素抗体浓度的影响,以检验多胺对抗DNA抗体作用的特异性。用单样本方差分析及配对T检验进行数据统计分析,P<0.05被认为差异有统计学意义。结果1.50-1000μM精胺及100-1000μM亚精胺可显著减低5例SLE血清抗CT DNA抗体与CT DNA结合力(P<0.01)；腐胺对SLE抗CT DNA抗体结合力无影响(P>0.05)。同等浓度下,精胺对抗CT DNA抗体的作用水平强于亚精胺和腐胺(P<0.01)。2.精胺不但直接抑制SLE血清抗CT DNA抗体结合于抗原,还可以解离已经形成的DNA抗原抗体复合物(P<0.01)。3.精胺对5例NHS血清抗MC DNA抗体与MC DNA结合无影响(P>0.05)。4.精胺显著抑制2例SLE血清S1、S2抗MC DNA抗体与MC DNA结合(P<0.01)；对于另外3例SLE血清S3、S4和S5抗MC DNA抗体与MC DNA结合无影响(P>0.05)。5.经CT-DNA纤维素膜吸附之后,5例SLE血清抗MC DNA抗体浓度下降(P<0.05)。其中S1、S2抗MC DNA抗体浓度降低程度明显大于S3、S4和S5(P<0.01)。5例NHS血清经CT-DNA纤维素膜吸附之后,其抗MC DNA抗体浓度无改变(P>0.05)。6.经CT-DNA纤维素膜吸附之后,精胺仍可以降低SLE血清S1、S2抗MC DNA抗体与MC DNA结合力(P<0.05),但作用程度小于相同条件下经普通对照纤维素膜吸附组(P<0.01)7.精胺对5例SLE血清抗破伤风类毒素抗体与破伤风类毒素结合无影响(P>0.05)。结论SLE抗DNA抗体与DNA结合可分为非特异结合和特异性结合两种方式,SLE患者个体所含两种抗体的相对含量不同。精胺可显著抑制非特异性结合的抗DNA抗体与DNA结合,并可成为检验抗DNA抗体与DNA作用方式的工具。其可能机制是多胺以静电作用结合于DNA磷酸骨架,改变DNA抗原性及降低抗DNA抗体与DNA之间的电荷吸引力。对于特异性结合于DNA非磷酸骨架部位的抗DNA抗体和非DNA抗体,精胺无作用。精胺作为一种生物性小分子胺类化合物,有望在SLE诊疗中发挥更大作用。研究目的多胺是包括精胺、亚精胺和腐胺在内的小分子脂肪族化合物,广泛分布于生物体的组织和细胞,是核酸、蛋白质等生物大分子重要的调控物质,在细胞代谢及增殖分化中发挥重要作用。多胺的合成及分解代谢在体内保持相对平衡,并受到精密调节。鸟氨酸脱羧酶(ODC)、S-腺苷甲硫氨酸脱羧酶(SAMDC)是多胺合成的两个关键酶,精胺／亚精胺N1-乙酰转移酶(SSAT)是多胺分解的关键酶。应用高效液相色谱(HPLC)技术,能够准确分离并测量生物样本内游离态精胺、亚精胺和腐胺的浓度。本论文第一部分,我们发现精胺、亚精胺可以在体外环境下抑制部分SLE抗ds-DNA抗体与DNA抗原结合。本部分我们继续检测了SLE患者及健康正常人群(Normal Human Subjects, NHS)外周血单个核细胞(PBMCs)多胺代谢酶基因ODC、SAMDC和SSAT mRNA表达水平及血浆中三种多胺的浓度。旨在进一步探究多胺在SLE患者的表达情况及其在SLE发生发展中的可能作用。研究方法取SLE患者32例,其中女性患者28例,男性4例,其诊断符合1997年美国风湿病学会修订的SLE分类标准；NHS组30例,其中女性26例,男性4例,均为健康体检者。使用实时定量PCR(RT-PCR)技术比较SLE组和NHS组PBMCs ODC、SAMDC和SSAT mRNA表达水平：使用HPLC技术检测SLE组和NHS组血浆精胺、亚精胺和腐胺的浓度。用独立样本T检验和卡方检验进行数据统计分析,RT-PCR结果由Real Time PCR仪自动分析得出SLE组目的基因mRNA对于NHS组目的基因mRNA的相对表达倍数,以P<0.05被认为差异有统计学意义。结果1.SLE组PBMCs ODC mRNA相对表达量为0.723,显著低于NHS组1.000,两组差别具有统计学意义(P<0.05)。2.SLE组PBMCs SAMDC mRNA相对表达量为1.101,NHS组为1.000,但两组差别不具有统计学意义(P>0.05)。3.SLE组PBMCs SSAT mRNA相对表达量为0.664,显著低于NHS组1.000,两组差别具有统计学意义(P<0.05)。4.SLE组血浆腐胺浓度为1.172ug/ml,显著低于NHS组1.570ug/ml,两组差别具有统计学意义(P<O.01)。5.SLE组血浆亚精胺浓度为1.303ug/ml, NHS组为1.710ug/ml,但两组差别不具有统计学意义(P>0.05)。6.SLE组血浆精胺浓度为0.471ug/ml,显著低于NHS组0.603ug/ml,两组差别具有统计学意义(P<0.05)。结论SLE患者PBMCs多胺合成关键酶之一ODC和多胺分解关键酶SSAT mRNA表达水平显著低于健康对照组。SLE患者血浆精胺和腐胺的浓度显著低于健康对照组。多胺与其代谢酶基因及多个因素相互作用,共同调节体内多胺浓度稳定。多胺水平可能与SLE细胞凋亡增加、激素水平异常有关。更多还原

【Abstract】 ObjectiveSystemic lupus erythematosus (SLE) is a prototypic autoimmune disease that caused tissue inflammation and organ injury. The characteristic of SLE is over expression of antibodies to components of the cell nucleus in association with deposition of antigen-antibody immune complex. DNA is one of the most important auto-antigen in SLE that serves as a prominent immunological feature of SLE with diagnostic and prognostic significance. SLE anti-ds DNA antibody primarily binds to DNA conserved phosphodiester backbone while some selectively binds to base determinants. These antibodies vary in specificity and pathogenicity among individuals and species. Polyamines are biogenic small molecular amines that are found abundantly in eukaryotic cell nucleus and prokaryotic cells. They are essential for DNA conformation and gene transcription. To elucidate the specificity of DNA-antibodies interactions as well as the possibility of new treatment to SLE, the effect of polyamines on the binding of anti-DNA antibodies from patients with SLE and normal human subjects (NHS) were tested.MethodsFive sera samples from SLE patients (3 females,2 males, fulfilling the American College of Rheumatology revised classification criteria of SLE in 1997) and five sera samples from normal human subjects (3 females,2 males) as a healthy control were collected. Three kinds of polyamines (spermine, spermidine and putrescine) for their effect on SLE and NHS anti-DNA antibodies binding were test by ELISA. The antibody concentration with polyamine/without polyamine was calculated for the efficiency of polyamine on antibody binding.In addition to examine binding of anti-DNA to calf thymus (CT) DNA as antigen, we also assessed the binding of antibodies to Micrococcus Luteus (MC) DNA. To detect further action of spermine on the binding of anti-DNA differing in specificity, sera were incubated with CT-DNA cellulose first to remove non-specific antibodies, then the antibody level were tested in the presence of spermine. Tetanus toxoid was selected as another antigen and the effect of spermine on anti-tetanus antibody binding of SLE sera was tested to determine the specific function of polyamine on anti-DNA antibody binding. One-way ANOVA and Paired T Test were used for data analysis, P<0.05 was seen as statistical significance.Results1. Spermine at 50-1000μM and spermidine at 100-1000μM can significantly reduce SLE anti-DNA binding to CT DNA (P<0.01); Putrescine has no effect on SLE anti-DNA binding (P>0.05). In the same concentration, the inhibitory activity of spermine is higher than spermidine and putrescine (P<0.01).2. Spermine can not only directly inhibit antibody binding to CT DNA in SLEsera, but also can disassemble pre-formed DNA-antibody immune complexes (P<0.01).3. Spermine does not influence anti-DNA antibody binding to MC DNA in NHS sera (P>0.05).4. Spermine obviously decreased 2 SLE sera S1, S2 anti-DNA antibody binding to MC DNA antigen (P<0.01) but failed to affect the other three SLE sera S3, S4 and S5 anti-DNA antibody binding to MC DNA (P>0.05).5. The anti-MC DNA antibody level was significantly decreased for 5 SLE sera after they absorbed by CT-DNA cellulose (P<0.05). For SLE sera S1, S2, the declined extent of anti-MC antibody was much more than SLE sera S3, S4 and S5 (P<0.01).6. For SLE sera S1, S2, spermine can still inhibit their anti-DNA binding to MC DNA after the sera was absorbed by CT-DNA cellulose (P<0.05), but the extent was less than that absorbed by control cellulose (P<0.01).7. Spermine failed to affect anti-tetanus antibody binding by SLE sera (P>0.05).ConclusionThe binding patterns of antibody to DNA are non-specific binding and specific binding and their ratio in SLE individual is deffer. Spermine can obviously inhibit non-specific anti-DNA antibody binding to DNA antigen in SLE patients and serve as a probe of anti-DNA specificity. The possible mechanism is that polyamines combine with DNA phosphate backbone to alter DNA antigenicity and interfere with antibody and DNA charge-charge interaction. Spermine has no effect on anti-DNA antibody that selectively binds to DNA non-specific determinants and non-DNA antibody. As a small biologic polyamine molecule, spermine can be a new tool for SLE diagnosis and treatment.ObjectivePolyamines (spermine, spermidine and putrescine) are small molecular aliphatic compounds that widely distributed in biologic tissues and cells. They are essential for the regulation of nucleic acid and protein and play a vital role in cell metabolism, proliferation and differentiation. Polyamine’s synthesis and catabolism are homeostasis with precise regulation. Ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) are key enzymes of polyamine synthesis, as well as spermine/spermidine N1-acetyltransferase (SSAT) is the key enzyme of polyamine’s catabolism. Using the technology of high performance lipid chromatography (HPLC), we can depart and detect free polyamines from biologic samples. In the first part of this thesis, we found spermine and spermidine could inhibit some SLE anti-dsDNA antibodies bind to DNA antigen in vitro. In this part we continue tested the mRNA expression levels of ODC, SAMDC and SSAT gene in peripheral blood mononuclear cells (PBMCs) in addition to the concentration of polyamines in plasma of SLE patients and normal human subjects. The aim of this study is to explore the expression status of polyamine in SLE patients and its possible function in SLE occurrence and development.Methods32 SLE patients (28 females,4 males, fulfilling the American college of Rheumatology classification criteria of SLE in 1997) and 30 normal human subjects (26 females,4 males) were enrolled. The ODC, SAMDC and SSAT mRNA levels of PBMCs were determined by Real-Time PCR (RT-PCR); the concentrations of spermine, spermidine and putrescine in plasma were tested by HPLC. Independent T test and Chi square test were used for data analysis. The results of mRNA expression were showed with relative multiples of SLE to NHS group that automatically analyzed by Real-Time PCR software. P<0.05 was seen as statistical significance.Results1. The relative expression of ODC mRNA in SLE group was 0.723, lower than NHS group 1.000. The difference was statistically significant (P<0.05).2. The relative expression of SAMDC mRNA in SLE group 1.101 while the NHS group is 1.000. But the difference has no statistically significant (P>0.05).3. The relative expression of SSAT mRNA in SLE group was 0.664, lower than NHS group 1.000. The difference was statistically significant (P<0.05).4. The plasma putrescine concentration in SLE patients is 1.172ug/ml, lower than the NHS group 1.570ug/ml. The difference was statistically significant (P<0.01).5. The plasma spermidine concentration in SLE patients is 1.303ug/ml while the NHS group is 1.710ug/ml. But the difference has no statistically significant (P>0.05).6. The plasma spermine concentration in SLE patients is 0.471ug/ml, lower than the NHS group 0.603ug/ml. The difference was statistically significant (P<0.05).ConclusionThe expression levels of ODC and SSAT gene mRNA in SLE patients’PBMCs is obviously lower than that in NHS group. The spermine and putrescine concentrations in SLE plasma are significantly lower than NHS plasma, either. Polyamines and its metabolic enzyme genes interact with each other and other factors to keep homeostasis of polyamine. The concentration of polyamine in SLE patients may be related to cell over apoptosis and hormone imbalance.更多还原