KIR4.1-293A细胞模型的构建及其在中枢神经系统脱髓鞘疾病中的研究
发布时间：2018-09-12 17:33
【摘要】：【背景】常见的中枢神经系统(central nervous system,CNS)炎性脱髓鞘疾病中包括有视神经脊髓炎谱系疾病(neuromyelitis optica spectrum disorders,NMOSD)、多发性硬化(multiple sclerosis,MS)、视神经炎(optic neuritis,ON)、急性播散性脑脊髓炎(acute disseminated encephalomyelitis,ADEM)等等。自从2004年研究发现水通道蛋白4(aquaporin 4,AQP4)抗体后,该抗体被公认为NMOSD最有意义的诊断指标,对疾病后期的治疗与预后具有重要价值。然而对于MS来说,目前尚缺乏相关的生物标记物来诊断MS,或者评估患者是复发-缓解型MS还是进展型MS。因此,学者们不停地试图寻找与CNS脱髓鞘疾病相关的其他自身抗体。内向整流钾离子通道4.1(inwardly rectifying potassium channel protein 4.1,KIR4.1)在中枢神经系统的神经胶质细胞中表达,主要功能包括有维持细胞内外钾离子体内平衡、维持胶质细胞静息膜电位以及调节星形胶质细胞清除谷氨酸等。有研究表明,在小鼠模型中敲除KIR4.1可导致严重的神经功能缺损,包括有共济失调、癫痫发作及神经性耳聋等。KIR4.1功能障碍与癫痫、脑外伤、脑缺血、炎症、肌萎缩性侧索硬化和阿尔茨海默症等神经系统疾病的发病过程密切相关,该膜蛋白离子通道在中枢神经系统中起着不可或缺的作用,这促使很多团队都加入到KIR4.1在人类疾病中潜在作用的研究中。在中枢神经系统内,AQP4主要分布在脑和脊髓组织的室管膜细胞及星形胶质细胞的细胞膜上,集中分布在星形胶质细胞的足突膜上,与KIR4.1的功能彼此耦联。二者通过介导中枢神经系统与血液间K+和水的转运实现神经细胞内外渗透平衡,是维持中枢神经系统正常功能的关键结构。AQP4抗体是NMOSD的生物标记物,参与了NMOSD的细胞免疫过程。目前研究发现MS患者免疫应答的病理性改变同时影响了B细胞和T细胞,检测出MS患者的脑脊液中免疫球蛋白合成增加、出现寡克隆带和补体沉积现象。由此可见,MS患者的中枢神经系统可能存在自身抗原成为了自发免疫潜在的靶点。KIR 4.1能否继APQ4抗体后,作为诊断、判断预后和监测脱髓鞘疾病的生物标记物?至今国外关于MS患者KIR 4.1抗体的报道各团队检测结果不一,阳性率差距较大,存在较大争议和分歧。抗KIR4.1抗体与多发性硬化发病是否相关,仍需进一步研究。第一部分:KIR4.1的质粒组成及293A细胞模型的构建【目的】构建KIR4.1质粒,通过克隆人KIR4.1基因,将基因转入人胚肾(human embryonic kidney,HEK)293A细胞中,构建细胞模型,用于血清KIR4.1抗体检测。【方法】1.pEnter-KIR4.1质粒的构建1.1.引物设计与合成使用引物设计软件Premier 5.0,参照Genebank中公布的人KIR4.1基因序列,分别设计KIR4.1基因的上游和下游引物,上游含ASCI引物酶,下游含Not I引物酶,18s rRNA为内参,送长沙博尚公司合成。1.2.人KIR4.1基因的获取使用提取试剂盒提取人体细胞的总核糖核苷酸(ribonucleic acid,RNA),然后并进行纯度检测。用上述合成的KIR4.1引物和人总RNA进行逆转录,从而得到它们的互补脱氧核糖核苷酸(complementary deoxyribonucleic acid,cDNA),再以KIR4.1-cDNA为模板进行聚合酶链式反应(polymerase chain reaction,PCR)扩增。然后将PCR产物进行凝胶电泳来鉴定目的基因是否扩增成功。1.3.酶切及质粒构建将KIR4.1-cDNA的PCR回收产物及pEnter载体分别用ASC I与Not I限制性核酸内切酶进行双酶切,并通过T7连接酶将KIR4.1基因与pEnter载体连接,连接后得到KIR4.1-pEnter质粒,再将质粒转化到JM09感受态细胞(克隆型)中,在含卡那霉素的LB平板上过夜培养,挑取单克隆细胞进行扩大培养,使用Endo-free Plasmid Mini Kit II 50试剂盒提取质粒用于鉴定阳性克隆,pEnter空载体做对照。1.4.pEnter-KIR4.1质粒的鉴定酶切后筛选阳性克隆质粒,并将其送至博尚公司进行基因测序,对GeneBank中人KIR4.1基因序列和博尚公司的测得结果进行Blast对比。2.KIR4.1-293A细胞模型的构建HEK 293A细胞复苏并进行培养,转染前1天对培养的293A细胞进行传代,至汇合率为70-80%时,通过转染试剂Lipofectamine 3000将KIR4.1质粒转染到293A细胞中。pEnter空载体做对照。3.嘌呤霉素筛选稳定表达KIR4.1的细胞株pEnter为真核细胞表达载体,自带嘌呤霉素抗性,所以可利用该抗性来筛选稳定表达目的蛋白的细胞株。用不同梯度浓度的嘌呤霉素处理293A细胞,细胞48h后全部死亡的嘌呤霉素的最小浓度,即确定为嘌呤霉素的最佳筛选浓度(0.3 ug/m L)。将转染48h后的KIR4.1-293A细胞培养皿里加入含0.3 ug/mL浓度嘌呤霉素的筛选培养基开始筛选,定期换液,直至没有转染的空白组细胞全部死亡。筛选后扩增培养已转染KIR4.1-pEnter质粒的存活细胞,即得稳定表达KIR4.1的细胞株。4.稳定表达KIR4.1的细胞株的表达鉴定转染成功的细胞通过western blotting和间接免疫荧光法(indirect immunofluorescence assay,IIFA)鉴定KIR4.1在293A细胞上的表达情况。成功构建的KIR4.1-293A细胞模型将用于血清KIR4.1抗体的检测。【结果】1.PCR扩增产物凝胶电泳鉴定:KIR4.1-cDNA的PCR产物进行凝胶电泳鉴定,结果图中可见与理论值一致的条带(KIR4.1:1142bp)。2.KIR4.1质粒的鉴定2.1.质粒酶切凝胶电泳鉴定:电泳图显示在相应理论值附近出现清晰的条带,证明KIR4.1质粒构建成功。2.2.基因测序分析鉴定:将基因测序结果与BLAST上的序列进行比对,原始序列与已知序列100%符合。3.KIR4.1在细胞上的表达:Western blotting显示KIR4.1-293A细胞表达蛋白在42kDa附近出现特异性条带。细胞经IIFA法检测发现与KIR4.1阳性抗体结合的细胞表面及细胞内激发绿色荧光,与4'6-二脒基-2-苯基吲哚(4'6-diamidino-2-phenylindole,DAPI)染核叠加。而pEnter空载细胞,293A细胞以及空白对照的细胞上未见相应荧光模式。【结论】成功克隆人KIR4.1基因,构建KIR4.1-293A表达质粒,并在293A细胞上稳定表达,筛选出稳定表达KIR4.1蛋白的稳定细胞系,提示KIR4.1-293A细胞模型构建成功。可为患者血清抗体检测提供一种可靠的实验方法,为进一步深入研究MS可能的疾病发病机理提供有用的实验依据。第二部分:CNS脱髓鞘疾病患者血清KIR4.1抗体检测【目的】通过构建的KIR4.1-293A细胞模型检测CNS炎性脱髓鞘疾病患者血清中的KIR4.1抗体,以探索KIR4.1抗体与该类疾病间的关系。【方法】1.病例及血清收集我们收集了2008年12月到2016年12月在广州医科大学附属第二医院诊断为MS的患者188例、NMOSD患者264例、其他炎症性神经系统疾病(other inflammatory neurological disease,OIND)患者209例、其他非炎症性神经系统疾病(other non-inflammatory neurological disease,OND)患者203例,另有健康人对照40人。所有患者均未使用免疫抑制剂或糖皮质激素治疗,并在清晨空腹状态下静脉取血。2.血清中KIR4.1抗体的检测基于细胞法(cell based assay,CBA)的间接免疫荧光方法检测患者血清中的KIR4.1抗体,所用二抗为异硫氰酸荧光素(fluorescein isothiocyanate,FITC)标记羊抗人IgG。为了鉴定患者抗体为KIR4.1抗体,使用患者血清和小鼠源抗KIR4.1单克隆抗体为一抗,FITC标记羊抗人IgG和CY3标记羊抗小鼠IgG作为二抗进行双标鉴定。3.统计分析所有结果经过社会科学统计软件包(statistical package for social sciences,SPSS)16.0进行分析处理。本实验结果数据为分类变量资料,组间阳性率进行X2检验,p0.05认为差异无统计学意义。【结果】不同疾病组的KIR4.1抗体检测阳性率细胞免疫荧光法检测188例MS患者血清,23例阳性(12.2%);264例视神经脊髓炎谱系疾病(NMOSD),阳性42例(15.9%);209例其他炎症性神经系统疾病(OIND),阳性32例(15.3%);203其他非炎症性神经系统疾病(OND),阳性24例(11.8%);40例健康人对照,阳性2例(5%)。采用卡方检验评估各组阳性率,结果差异无统计学意义(p=0.279)。【结论】KIR4.1抗体暂时不支持成为区别MS与其他中枢神经系统自身免疫性疾病的生物标志物,该抗体与多发性硬化发病是否相关,仍需进一步研究。第三部分:血清KIR4.1(+)的MS患者脑组织抗原的表达【目的】对比检测血清KIR4.1(+)的MS患者与健康对照脑组织的KIR4.1的表达差异,探讨KIR4.1抗体与MS发病机制的关系。【方法】选取血清抗KIR4.1抗体阳性的典型MS患者2例,取得疾病早期病灶活检标本(糖皮质激素治疗前),非神经系统疾病尸检脑组织1例作为对照,采用免疫组化方法检测3例标本中KIR4.1的表达情况。【结果】3例标本KIR4.1正常表达,皆未见KIR4.1抗原脱失。【结论】暂时不支持KIR4.1抗体参与了导致MS患者发病的过程,该抗体与多发性硬化发病是否相关,仍需进一步研究。
[Abstract]:[Background] Common inflammatory demyelinating diseases of the central nervous system (CNS) include neuromyelitis optica spectrum disorders (NMOSD), multiple sclerosis (MS), optic neuritis (ON), acute disseminated encephalomyelitis (ADE). Ephalomyelitis, ADEM, etc. Since the discovery of aquaporin 4 (AQP4) antibody in 2004, the antibody has been recognized as the most significant diagnostic marker of NMOSD and has important value in the treatment and prognosis of the later stage of the disease. However, for MS, there is still a lack of relevant biomarkers to diagnose MS, or to evaluate the patient's recovery. Therefore, scholars are constantly trying to find other autoantibodies associated with CNS demyelinating diseases. Inwardly rectifying potassium channel protein 4.1 (KIR4.1) is expressed in neuroglial cells of the central nervous system. Its main functions include maintaining intracellular and extracellular functions. Studies have shown that knockout of KIR4.1 in mouse models can lead to severe neurological deficits, including ataxia, seizures and neuronal deafness. KIR4.1 dysfunction and epilepsy, brain trauma, cerebral ischemia, inflammation, etc. Amyotrophic lateral sclerosis is closely related to the pathogenesis of neurological diseases such as Alzheimer's disease. The membrane protein ion channel plays an indispensable role in the central nervous system, which prompts many teams to join in the study of the potential role of KIR4.1 in human diseases. In the central nervous system, AQP4 is mainly distributed in the brain. The membranes of ependymal cells and astrocytes of spinal cord tissue are concentrated on the foot process membranes of astrocytes and are coupled with the functions of KIR4.1. They are involved in maintaining the normal function of the central nervous system by mediating the intracellular and extracellular osmotic balance of K + and water transport between the central nervous system and the blood. Bond structure. AQP4 antibody is a biomarker of NMOSD and participates in the cellular immune process of NMOSD. Current studies have found that the pathological changes of immune response in MS patients affect both B and T cells. Immunoglobulin synthesis in cerebrospinal fluid of MS patients has been detected to increase, oligoclonal bands and complement deposition have occurred. Whether KIR 4.1 can be used as a biomarker for the diagnosis, prognosis and monitoring of demyelinating diseases after APQ4 antibody? Up to now, the results of KIR 4.1 antibody detection in MS patients are different from each other, and the positive rate of KIR 4.1 antibody is different from each other. The relationship between anti-KIR4.1 antibody and the pathogenesis of multiple sclerosis still needs further study. Part I: The plasmid composition of KIR4.1 and the construction of 293A cell model [Objective] To construct KIR4.1 plasmid, clone human KIR4.1 gene, transfer the gene into human embryonic kidney (HEK) 293A cells and construct cell model for serum KIR4.1. [Methods] 1. Construction of pEnter-KIR4.1 plasmid 1.1. Primer design and synthesis using primer design software Premier 5.0, according to the human KIR4.1 gene sequence published in Genebank, designed the upstream and downstream primers of KIR4.1 gene, including ASCI primer enzyme in the upstream, Not I primer enzyme in the downstream, 18S rRNA as internal reference, sent to Changsha Boshan Company. 1.2. Human KIR4.1 gene was synthesized. Total ribonucleic acid (RNA) was extracted from human cells by extraction kit, and purity was tested. Their complementary deoxyribonucleic acid (cDNA) was obtained by reverse transcription with the above-mentioned synthetic primers and human total RNA. KIR4.1-cDNA was used as a template for polymerase chain reaction (PCR) amplification. Then the PCR products were gel electrophoresis to determine whether the target gene was amplified successfully. 1.3. Enzyme digestion and plasmid construction were used to construct the PCR recovery products of KIR4.1-cDNA and pEnter vectors were digested by ASC I and Not I restriction endonuclease, respectively. KIR4.1-pEnter plasmid was obtained by ligation of KIR4.1 gene with pEnter vector by T7 ligase. The plasmid was transformed into JM09 competent cells (clonal type). Monoclonal cells were cultured overnight on the LB plate containing kanamycin. The monoclonal cells were selected for enlarged culture. The plasmid was extracted by Endo-free Plasmid Mini Kit II 50 kit and used for further culture. Identification of positive clones, pEnter empty vector as control. 1.4. pEnter-KIR4.1 plasmid identification enzyme digestion screening positive clone plasmids, and then sent to Boshan Company for gene sequencing, GeneBank and the results of the Boshan company's KIR4.1 gene sequence and Blast comparison. 2. KIR4.1-293A cell model construction of HEK 293A cell recovery and progress KIR4.1 plasmid was transfected into 293A cells by Lipofectamine 3000. pEnter empty vector was used as control. 3. Puromycin screened cell line pEnter stably expressing KIR4.1 as eukaryotic expression vector and carried purinomycin resistance. The optimal concentration of purinomycin (0.3 ug/mL) was determined when 293A cells were treated with purinomycin of different gradient concentrations for 48 hours. The KIR4.1-293A cell culture dish containing 0.3 ug/mL was added 48 hours after transfection. The screening medium of doxycycline began to be screened, and the liquid was changed regularly until all the cells in the blank group died without transfection. After screening, the surviving cells which had been transfected with KIR4.1-pEnter plasmid were amplified and cultured. 4. The cells stably expressing KIR4.1 were identified by Western blotting. The expression of KIR4.1 on 293A cells was identified by G and indirect immunofluorescence assay (IIFA). The KIR4.1-293A cell model successfully constructed will be used for the detection of serum KIR4.1 antibody. [Results] 1. PCR amplification product gel electrophoresis identification: KIR4.1-cDNA PCR products were identified by gel electrophoresis, the results showed that the map can be seen. Identification of plasmids (KIR4.1:1142bp). 2. KIR4.1. Identification of plasmids by enzyme digestion gel electrophoresis: Electrophoresis showed that there were clear bands near the corresponding theoretical values, proving the success of KIR4.1 plasmid construction. 2. The expression of KIR4.1 was 100% consistent with that of KIR4.1. Western blotting showed a specific band of KIR4.1-293A cell expression protein near 42kDa. The cells were detected by IIFA to stimulate green fluorescence on the cell surface and cells bound with KIR4.1 positive antibody, and to 4'6-diamidino-2-phenylindole (DAP). [Conclusion] The human KIR4.1 gene was successfully cloned and the expression plasmid of KIR4.1-293A was constructed. The stable cell lines stably expressing KIR4.1 protein were screened out on 293A cells, suggesting that the KIR4.1-293A cell model was successfully constructed. It can provide a reliable experimental method for the detection of serum antibodies and provide useful experimental basis for the further study of the pathogenesis of MS. Part II: Detection of serum KIR4.1 antibodies in patients with CNS demyelinating disease [Objective] To detect the serum levels of patients with CNS inflammatory demyelinating disease by constructing a KIR4.1-293A cell model. KIR4.1 antibody in the Second Affiliated Hospital of Guangzhou Medical University from December 2008 to December 2016, 188 patients with MS, 264 patients with NMOSD and other inflammatory neurological diseases were collected. There were 209 cases of ase, 203 cases of other non-inflammatory neurological disease (OND) and 40 healthy controls. All patients were not treated with immunosuppressive agents or glucocorticoids, and blood samples were taken intravenously in the morning and on an empty stomach. 2. The detection of KIR4.1 antibody in serum was based on cell method (cytometry). Cell-based assay (CBA) indirect immunofluorescence assay was used to detect KIR4.1 antibody in serum of patients. Fluorescein isothiocyanate (FITC) was used to label sheep anti-human IgG. All the results were analyzed and processed by SPSS (statistical package for social sciences) 16.0. The experimental data were classified as variable data. The positive rate between groups was tested by X2 test, P0.05 thought that there was no significant difference. Results The positive rate of KIR4.1 antibody in 188 patients with MS was 12.2%. There were 264 patients with NMOSD, 42 patients with NMOSD, 209 patients with other inflammatory nervous system diseases (OIND), 32 patients with positive rate (15.3%). 203 patients with other non-inflammatory nervous system diseases (OND) were positive. [Conclusion] KIR4.1 antibody can not be used as a biomarker to differentiate MS from other central nervous system autoimmune diseases for the time being, and whether the antibody is related to multiple sclerosis. Part 3: The expression of KIR4.1 (+) antigen in brain tissues of MS patients with serum KIR4.1 (+) was compared with that of healthy controls, and the relationship between KIR4.1 antibody and the pathogenesis of MS was discussed. The expression of KIR4.1 was detected by immunohistochemistry in 1 case of non-neurological autopsy brain tissue as control. [Results] The expression of KIR4.1 was normal in 3 cases, and no loss of KIR4.1 antigen was found in all 3 cases. [Conclusion] KIR4.1 antibody was not supported for the time being. Whether the antibody is related to the pathogenesis of multiple sclerosis remains to be further studied in the pathogenesis of MS.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R744.5
本文编号：2239747
[Abstract]:[Background] Common inflammatory demyelinating diseases of the central nervous system (CNS) include neuromyelitis optica spectrum disorders (NMOSD), multiple sclerosis (MS), optic neuritis (ON), acute disseminated encephalomyelitis (ADE). Ephalomyelitis, ADEM, etc. Since the discovery of aquaporin 4 (AQP4) antibody in 2004, the antibody has been recognized as the most significant diagnostic marker of NMOSD and has important value in the treatment and prognosis of the later stage of the disease. However, for MS, there is still a lack of relevant biomarkers to diagnose MS, or to evaluate the patient's recovery. Therefore, scholars are constantly trying to find other autoantibodies associated with CNS demyelinating diseases. Inwardly rectifying potassium channel protein 4.1 (KIR4.1) is expressed in neuroglial cells of the central nervous system. Its main functions include maintaining intracellular and extracellular functions. Studies have shown that knockout of KIR4.1 in mouse models can lead to severe neurological deficits, including ataxia, seizures and neuronal deafness. KIR4.1 dysfunction and epilepsy, brain trauma, cerebral ischemia, inflammation, etc. Amyotrophic lateral sclerosis is closely related to the pathogenesis of neurological diseases such as Alzheimer's disease. The membrane protein ion channel plays an indispensable role in the central nervous system, which prompts many teams to join in the study of the potential role of KIR4.1 in human diseases. In the central nervous system, AQP4 is mainly distributed in the brain. The membranes of ependymal cells and astrocytes of spinal cord tissue are concentrated on the foot process membranes of astrocytes and are coupled with the functions of KIR4.1. They are involved in maintaining the normal function of the central nervous system by mediating the intracellular and extracellular osmotic balance of K + and water transport between the central nervous system and the blood. Bond structure. AQP4 antibody is a biomarker of NMOSD and participates in the cellular immune process of NMOSD. Current studies have found that the pathological changes of immune response in MS patients affect both B and T cells. Immunoglobulin synthesis in cerebrospinal fluid of MS patients has been detected to increase, oligoclonal bands and complement deposition have occurred. Whether KIR 4.1 can be used as a biomarker for the diagnosis, prognosis and monitoring of demyelinating diseases after APQ4 antibody? Up to now, the results of KIR 4.1 antibody detection in MS patients are different from each other, and the positive rate of KIR 4.1 antibody is different from each other. The relationship between anti-KIR4.1 antibody and the pathogenesis of multiple sclerosis still needs further study. Part I: The plasmid composition of KIR4.1 and the construction of 293A cell model [Objective] To construct KIR4.1 plasmid, clone human KIR4.1 gene, transfer the gene into human embryonic kidney (HEK) 293A cells and construct cell model for serum KIR4.1. [Methods] 1. Construction of pEnter-KIR4.1 plasmid 1.1. Primer design and synthesis using primer design software Premier 5.0, according to the human KIR4.1 gene sequence published in Genebank, designed the upstream and downstream primers of KIR4.1 gene, including ASCI primer enzyme in the upstream, Not I primer enzyme in the downstream, 18S rRNA as internal reference, sent to Changsha Boshan Company. 1.2. Human KIR4.1 gene was synthesized. Total ribonucleic acid (RNA) was extracted from human cells by extraction kit, and purity was tested. Their complementary deoxyribonucleic acid (cDNA) was obtained by reverse transcription with the above-mentioned synthetic primers and human total RNA. KIR4.1-cDNA was used as a template for polymerase chain reaction (PCR) amplification. Then the PCR products were gel electrophoresis to determine whether the target gene was amplified successfully. 1.3. Enzyme digestion and plasmid construction were used to construct the PCR recovery products of KIR4.1-cDNA and pEnter vectors were digested by ASC I and Not I restriction endonuclease, respectively. KIR4.1-pEnter plasmid was obtained by ligation of KIR4.1 gene with pEnter vector by T7 ligase. The plasmid was transformed into JM09 competent cells (clonal type). Monoclonal cells were cultured overnight on the LB plate containing kanamycin. The monoclonal cells were selected for enlarged culture. The plasmid was extracted by Endo-free Plasmid Mini Kit II 50 kit and used for further culture. Identification of positive clones, pEnter empty vector as control. 1.4. pEnter-KIR4.1 plasmid identification enzyme digestion screening positive clone plasmids, and then sent to Boshan Company for gene sequencing, GeneBank and the results of the Boshan company's KIR4.1 gene sequence and Blast comparison. 2. KIR4.1-293A cell model construction of HEK 293A cell recovery and progress KIR4.1 plasmid was transfected into 293A cells by Lipofectamine 3000. pEnter empty vector was used as control. 3. Puromycin screened cell line pEnter stably expressing KIR4.1 as eukaryotic expression vector and carried purinomycin resistance. The optimal concentration of purinomycin (0.3 ug/mL) was determined when 293A cells were treated with purinomycin of different gradient concentrations for 48 hours. The KIR4.1-293A cell culture dish containing 0.3 ug/mL was added 48 hours after transfection. The screening medium of doxycycline began to be screened, and the liquid was changed regularly until all the cells in the blank group died without transfection. After screening, the surviving cells which had been transfected with KIR4.1-pEnter plasmid were amplified and cultured. 4. The cells stably expressing KIR4.1 were identified by Western blotting. The expression of KIR4.1 on 293A cells was identified by G and indirect immunofluorescence assay (IIFA). The KIR4.1-293A cell model successfully constructed will be used for the detection of serum KIR4.1 antibody. [Results] 1. PCR amplification product gel electrophoresis identification: KIR4.1-cDNA PCR products were identified by gel electrophoresis, the results showed that the map can be seen. Identification of plasmids (KIR4.1:1142bp). 2. KIR4.1. Identification of plasmids by enzyme digestion gel electrophoresis: Electrophoresis showed that there were clear bands near the corresponding theoretical values, proving the success of KIR4.1 plasmid construction. 2. The expression of KIR4.1 was 100% consistent with that of KIR4.1. Western blotting showed a specific band of KIR4.1-293A cell expression protein near 42kDa. The cells were detected by IIFA to stimulate green fluorescence on the cell surface and cells bound with KIR4.1 positive antibody, and to 4'6-diamidino-2-phenylindole (DAP). [Conclusion] The human KIR4.1 gene was successfully cloned and the expression plasmid of KIR4.1-293A was constructed. The stable cell lines stably expressing KIR4.1 protein were screened out on 293A cells, suggesting that the KIR4.1-293A cell model was successfully constructed. It can provide a reliable experimental method for the detection of serum antibodies and provide useful experimental basis for the further study of the pathogenesis of MS. Part II: Detection of serum KIR4.1 antibodies in patients with CNS demyelinating disease [Objective] To detect the serum levels of patients with CNS inflammatory demyelinating disease by constructing a KIR4.1-293A cell model. KIR4.1 antibody in the Second Affiliated Hospital of Guangzhou Medical University from December 2008 to December 2016, 188 patients with MS, 264 patients with NMOSD and other inflammatory neurological diseases were collected. There were 209 cases of ase, 203 cases of other non-inflammatory neurological disease (OND) and 40 healthy controls. All patients were not treated with immunosuppressive agents or glucocorticoids, and blood samples were taken intravenously in the morning and on an empty stomach. 2. The detection of KIR4.1 antibody in serum was based on cell method (cytometry). Cell-based assay (CBA) indirect immunofluorescence assay was used to detect KIR4.1 antibody in serum of patients. Fluorescein isothiocyanate (FITC) was used to label sheep anti-human IgG. All the results were analyzed and processed by SPSS (statistical package for social sciences) 16.0. The experimental data were classified as variable data. The positive rate between groups was tested by X2 test, P0.05 thought that there was no significant difference. Results The positive rate of KIR4.1 antibody in 188 patients with MS was 12.2%. There were 264 patients with NMOSD, 42 patients with NMOSD, 209 patients with other inflammatory nervous system diseases (OIND), 32 patients with positive rate (15.3%). 203 patients with other non-inflammatory nervous system diseases (OND) were positive. [Conclusion] KIR4.1 antibody can not be used as a biomarker to differentiate MS from other central nervous system autoimmune diseases for the time being, and whether the antibody is related to multiple sclerosis. Part 3: The expression of KIR4.1 (+) antigen in brain tissues of MS patients with serum KIR4.1 (+) was compared with that of healthy controls, and the relationship between KIR4.1 antibody and the pathogenesis of MS was discussed. The expression of KIR4.1 was detected by immunohistochemistry in 1 case of non-neurological autopsy brain tissue as control. [Results] The expression of KIR4.1 was normal in 3 cases, and no loss of KIR4.1 antigen was found in all 3 cases. [Conclusion] KIR4.1 antibody was not supported for the time being. Whether the antibody is related to the pathogenesis of multiple sclerosis remains to be further studied in the pathogenesis of MS.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R744.5
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