【摘要】：【背景】常見的中樞神經(jīng)系統(tǒng)(central nervous system,CNS)炎性脫髓鞘疾病中包括有視神經(jīng)脊髓炎譜系疾病(neuromyelitis optica spectrum disorders,NMOSD)、多發(fā)性硬化(multiple sclerosis,MS)、視神經(jīng)炎(optic neuritis,ON)、急性播散性腦脊髓炎(acute disseminated encephalomyelitis,ADEM)等等。自從2004年研究發(fā)現(xiàn)水通道蛋白4(aquaporin 4,AQP4)抗體后,該抗體被公認為NMOSD最有意義的診斷指標,對疾病后期的治療與預后具有重要價值。然而對于MS來說,目前尚缺乏相關(guān)的生物標記物來診斷MS,或者評估患者是復發(fā)-緩解型MS還是進展型MS。因此,學者們不停地試圖尋找與CNS脫髓鞘疾病相關(guān)的其他自身抗體。內(nèi)向整流鉀離子通道4.1(inwardly rectifying potassium channel protein 4.1,KIR4.1)在中樞神經(jīng)系統(tǒng)的神經(jīng)膠質(zhì)細胞中表達,主要功能包括有維持細胞內(nèi)外鉀離子體內(nèi)平衡、維持膠質(zhì)細胞靜息膜電位以及調(diào)節(jié)星形膠質(zhì)細胞清除谷氨酸等。有研究表明,在小鼠模型中敲除KIR4.1可導致嚴重的神經(jīng)功能缺損,包括有共濟失調(diào)、癲癇發(fā)作及神經(jīng)性耳聾等。KIR4.1功能障礙與癲癇、腦外傷、腦缺血、炎癥、肌萎縮性側(cè)索硬化和阿爾茨海默癥等神經(jīng)系統(tǒng)疾病的發(fā)病過程密切相關(guān),該膜蛋白離子通道在中樞神經(jīng)系統(tǒng)中起著不可或缺的作用,這促使很多團隊都加入到KIR4.1在人類疾病中潛在作用的研究中。在中樞神經(jīng)系統(tǒng)內(nèi),AQP4主要分布在腦和脊髓組織的室管膜細胞及星形膠質(zhì)細胞的細胞膜上,集中分布在星形膠質(zhì)細胞的足突膜上,與KIR4.1的功能彼此耦聯(lián)。二者通過介導中樞神經(jīng)系統(tǒng)與血液間K+和水的轉(zhuǎn)運實現(xiàn)神經(jīng)細胞內(nèi)外滲透平衡,是維持中樞神經(jīng)系統(tǒng)正常功能的關(guān)鍵結(jié)構(gòu)。AQP4抗體是NMOSD的生物標記物,參與了NMOSD的細胞免疫過程。目前研究發(fā)現(xiàn)MS患者免疫應答的病理性改變同時影響了B細胞和T細胞,檢測出MS患者的腦脊液中免疫球蛋白合成增加、出現(xiàn)寡克隆帶和補體沉積現(xiàn)象。由此可見,MS患者的中樞神經(jīng)系統(tǒng)可能存在自身抗原成為了自發(fā)免疫潛在的靶點。KIR 4.1能否繼APQ4抗體后,作為診斷、判斷預后和監(jiān)測脫髓鞘疾病的生物標記物?至今國外關(guān)于MS患者KIR 4.1抗體的報道各團隊檢測結(jié)果不一,陽性率差距較大,存在較大爭議和分歧。抗KIR4.1抗體與多發(fā)性硬化發(fā)病是否相關(guān),仍需進一步研究。第一部分:KIR4.1的質(zhì)粒組成及293A細胞模型的構(gòu)建【目的】構(gòu)建KIR4.1質(zhì)粒,通過克隆人KIR4.1基因,將基因轉(zhuǎn)入人胚腎(human embryonic kidney,HEK)293A細胞中,構(gòu)建細胞模型,用于血清KIR4.1抗體檢測�！痉椒ā�1.pEnter-KIR4.1質(zhì)粒的構(gòu)建1.1.引物設計與合成使用引物設計軟件Premier 5.0,參照Genebank中公布的人KIR4.1基因序列,分別設計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為內(nèi)參,送長沙博尚公司合成。1.2.人KIR4.1基因的獲取使用提取試劑盒提取人體細胞的總核糖核苷酸(ribonucleic acid,RNA),然后并進行純度檢測。用上述合成的KIR4.1引物和人總RNA進行逆轉(zhuǎn)錄,從而得到它們的互補脫氧核糖核苷酸(complementary deoxyribonucleic acid,cDNA),再以KIR4.1-cDNA為模板進行聚合酶鏈式反應(polymerase chain reaction,PCR)擴增。然后將PCR產(chǎn)物進行凝膠電泳來鑒定目的基因是否擴增成功。1.3.酶切及質(zhì)粒構(gòu)建將KIR4.1-cDNA的PCR回收產(chǎn)物及pEnter載體分別用ASC I與Not I限制性核酸內(nèi)切酶進行雙酶切,并通過T7連接酶將KIR4.1基因與pEnter載體連接,連接后得到KIR4.1-pEnter質(zhì)粒,再將質(zhì)粒轉(zhuǎn)化到JM09感受態(tài)細胞(克隆型)中,在含卡那霉素的LB平板上過夜培養(yǎng),挑取單克隆細胞進行擴大培養(yǎng),使用Endo-free Plasmid Mini Kit II 50試劑盒提取質(zhì)粒用于鑒定陽性克隆,pEnter空載體做對照。1.4.pEnter-KIR4.1質(zhì)粒的鑒定酶切后篩選陽性克隆質(zhì)粒,并將其送至博尚公司進行基因測序,對GeneBank中人KIR4.1基因序列和博尚公司的測得結(jié)果進行Blast對比。2.KIR4.1-293A細胞模型的構(gòu)建HEK 293A細胞復蘇并進行培養(yǎng),轉(zhuǎn)染前1天對培養(yǎng)的293A細胞進行傳代,至匯合率為70-80%時,通過轉(zhuǎn)染試劑Lipofectamine 3000將KIR4.1質(zhì)粒轉(zhuǎn)染到293A細胞中。pEnter空載體做對照。3.嘌呤霉素篩選穩(wěn)定表達KIR4.1的細胞株pEnter為真核細胞表達載體,自帶嘌呤霉素抗性,所以可利用該抗性來篩選穩(wěn)定表達目的蛋白的細胞株。用不同梯度濃度的嘌呤霉素處理293A細胞,細胞48h后全部死亡的嘌呤霉素的最小濃度,即確定為嘌呤霉素的最佳篩選濃度(0.3 ug/m L)。將轉(zhuǎn)染48h后的KIR4.1-293A細胞培養(yǎng)皿里加入含0.3 ug/mL濃度嘌呤霉素的篩選培養(yǎng)基開始篩選,定期換液,直至沒有轉(zhuǎn)染的空白組細胞全部死亡。篩選后擴增培養(yǎng)已轉(zhuǎn)染KIR4.1-pEnter質(zhì)粒的存活細胞,即得穩(wěn)定表達KIR4.1的細胞株。4.穩(wěn)定表達KIR4.1的細胞株的表達鑒定轉(zhuǎn)染成功的細胞通過western blotting和間接免疫熒光法(indirect immunofluorescence assay,IIFA)鑒定KIR4.1在293A細胞上的表達情況。成功構(gòu)建的KIR4.1-293A細胞模型將用于血清KIR4.1抗體的檢測�！窘Y(jié)果】1.PCR擴增產(chǎn)物凝膠電泳鑒定:KIR4.1-cDNA的PCR產(chǎn)物進行凝膠電泳鑒定,結(jié)果圖中可見與理論值一致的條帶(KIR4.1:1142bp)。2.KIR4.1質(zhì)粒的鑒定2.1.質(zhì)粒酶切凝膠電泳鑒定:電泳圖顯示在相應理論值附近出現(xiàn)清晰的條帶,證明KIR4.1質(zhì)粒構(gòu)建成功。2.2.基因測序分析鑒定:將基因測序結(jié)果與BLAST上的序列進行比對,原始序列與已知序列100%符合。3.KIR4.1在細胞上的表達:Western blotting顯示KIR4.1-293A細胞表達蛋白在42kDa附近出現(xiàn)特異性條帶。細胞經(jīng)IIFA法檢測發(fā)現(xiàn)與KIR4.1陽性抗體結(jié)合的細胞表面及細胞內(nèi)激發(fā)綠色熒光,與4'6-二脒基-2-苯基吲哚(4'6-diamidino-2-phenylindole,DAPI)染核疊加。而pEnter空載細胞,293A細胞以及空白對照的細胞上未見相應熒光模式�！窘Y(jié)論】成功克隆人KIR4.1基因,構(gòu)建KIR4.1-293A表達質(zhì)粒,并在293A細胞上穩(wěn)定表達,篩選出穩(wěn)定表達KIR4.1蛋白的穩(wěn)定細胞系,提示KIR4.1-293A細胞模型構(gòu)建成功�？蔀榛颊哐蹇贵w檢測提供一種可靠的實驗方法,為進一步深入研究MS可能的疾病發(fā)病機理提供有用的實驗依據(jù)。第二部分:CNS脫髓鞘疾病患者血清KIR4.1抗體檢測【目的】通過構(gòu)建的KIR4.1-293A細胞模型檢測CNS炎性脫髓鞘疾病患者血清中的KIR4.1抗體,以探索KIR4.1抗體與該類疾病間的關(guān)系�！痉椒ā�1.病例及血清收集我們收集了2008年12月到2016年12月在廣州醫(yī)科大學附屬第二醫(yī)院診斷為MS的患者188例、NMOSD患者264例、其他炎癥性神經(jīng)系統(tǒng)疾病(other inflammatory neurological disease,OIND)患者209例、其他非炎癥性神經(jīng)系統(tǒng)疾病(other non-inflammatory neurological disease,OND)患者203例,另有健康人對照40人。所有患者均未使用免疫抑制劑或糖皮質(zhì)激素治療,并在清晨空腹狀態(tài)下靜脈取血。2.血清中KIR4.1抗體的檢測基于細胞法(cell based assay,CBA)的間接免疫熒光方法檢測患者血清中的KIR4.1抗體,所用二抗為異硫氰酸熒光素(fluorescein isothiocyanate,FITC)標記羊抗人IgG。為了鑒定患者抗體為KIR4.1抗體,使用患者血清和小鼠源抗KIR4.1單克隆抗體為一抗,FITC標記羊抗人IgG和CY3標記羊抗小鼠IgG作為二抗進行雙標鑒定。3.統(tǒng)計分析所有結(jié)果經(jīng)過社會科學統(tǒng)計軟件包(statistical package for social sciences,SPSS)16.0進行分析處理。本實驗結(jié)果數(shù)據(jù)為分類變量資料,組間陽性率進行X2檢驗,p0.05認為差異無統(tǒng)計學意義。【結(jié)果】不同疾病組的KIR4.1抗體檢測陽性率細胞免疫熒光法檢測188例MS患者血清,23例陽性(12.2%);264例視神經(jīng)脊髓炎譜系疾病(NMOSD),陽性42例(15.9%);209例其他炎癥性神經(jīng)系統(tǒng)疾病(OIND),陽性32例(15.3%);203其他非炎癥性神經(jīng)系統(tǒng)疾病(OND),陽性24例(11.8%);40例健康人對照,陽性2例(5%)。采用卡方檢驗評估各組陽性率,結(jié)果差異無統(tǒng)計學意義(p=0.279)�！窘Y(jié)論】KIR4.1抗體暫時不支持成為區(qū)別MS與其他中樞神經(jīng)系統(tǒng)自身免疫性疾病的生物標志物,該抗體與多發(fā)性硬化發(fā)病是否相關(guān),仍需進一步研究。第三部分:血清KIR4.1(+)的MS患者腦組織抗原的表達【目的】對比檢測血清KIR4.1(+)的MS患者與健康對照腦組織的KIR4.1的表達差異,探討KIR4.1抗體與MS發(fā)病機制的關(guān)系�！痉椒ā窟x取血清抗KIR4.1抗體陽性的典型MS患者2例,取得疾病早期病灶活檢標本(糖皮質(zhì)激素治療前),非神經(jīng)系統(tǒng)疾病尸檢腦組織1例作為對照,采用免疫組化方法檢測3例標本中KIR4.1的表達情況�！窘Y(jié)果】3例標本KIR4.1正常表達,皆未見KIR4.1抗原脫失�！窘Y(jié)論】暫時不支持KIR4.1抗體參與了導致MS患者發(fā)病的過程,該抗體與多發(fā)性硬化發(fā)病是否相關(guān),仍需進一步研究。
[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.
【學位授予單位】：廣州醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R744.5

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