血清AQP4-M23-IgG的檢測及其對NMOSDs患者的胃、唇腺、骨骼
發(fā)布時間：2018-09-10 14:07
【摘要】：【背景】視神經(jīng)脊髓炎譜系疾病(neuromyelitis optica spectrum disorders,NMOSDs)是一種中樞神經(jīng)系統(tǒng)(central nervous system,CNS)炎性脫髓鞘性自身免疫性疾病。隨著Lennon等[1]對血清水通道蛋白4抗體(aquaporin-4 antibody,AQP4抗體)即AQP4-IgG的發(fā)現(xiàn),大大提高了對視神經(jīng)脊髓炎(neuromyelitis optica,NMO)的診斷特異性。然而,部分AQP4-IgG陽性的患者有多種不同的臨床特征,出現(xiàn)廣泛的綜合征證實與NMO密切相關(guān),于是延伸出另一個擴展性概念NMOSDs[2]。研究顯示20-30%的NMO/NMOSDs患者血清抗AQP4抗體為陰性[3]。各種檢測方法顯示AQP4-IgG對NMOSDs診斷的高度特異性(91-100%)和高靈敏度(83-91%),但目前最推薦的檢測方法是基于細胞的檢測法(cell based assay,CBA)[4]。同時,有報道稱在外周血發(fā)現(xiàn)的AQP4-IgG參與了NMO/NMOSDs的發(fā)病機理中,證實AQP4-IgG在CNS中是致病性的[5]。然而在同樣表達有AQP4的外周器官、組織如胃、腎、骨骼肌中,小鼠模型的研究顯示血清AQP4-IgG可快速結(jié)合特異性抗原靶點,但不產(chǎn)生實質(zhì)的外周器官、組織免疫損傷效應(yīng)[6]。有趣的是,也有報道稱血清AQP4-IgG陽性的NMOSDs患者可伴有流產(chǎn)風(fēng)險的胎盤炎[7]、內(nèi)耳炎[8]和胃炎[9]等。這些研究結(jié)果表明,AQP4-IgG可能對表達AQP4的外周器官、組織有免疫損傷效應(yīng)。因此,本研究采用基于轉(zhuǎn)染M23-AQP4的人胚胎腎細胞(human embryonic kidney293 cells,HEK293)的CBA法檢測血清AQP4-IgG,并對AQP4-M23-IgG陽性的NMOSDs患者外周器官、組織如胃、唇腺、骨骼肌、腎區(qū)域免疫效應(yīng)作進一步的研究和機制探討。【目的】采用基于M23-AQP4-HEK293細胞的CBA法檢測血清AQP4-IgG,明確研究對象NMOSDs、MS以及非脫髓鞘性疾病患者的血清AQP4-IgG結(jié)果,以便比較AQP4-M23-IgG陽性的NMOSDs患者外周器官、組織(胃、唇腺、骨骼肌及腎)是否存在類似CNS中AQP4脫失等病理損傷改變或局部免疫炎癥效應(yīng)�！痉椒ā恳弧⒒贛23-AQP4-HEK293細胞的CBA法檢測血清AQP4-IgG將pcDNA3.1+-M23-AQP4質(zhì)粒通過Lipofectamine 3000轉(zhuǎn)染接種于HEK293細胞中,經(jīng)CBA法鑒定M23-AQP4是否在HEK293細胞中表達。采用CBA法檢測21例NMOSDs、13例MS以及17例非脫髓鞘性疾病患者血清AQP4-IgG,明確研究對象的血清抗AQP4抗體的結(jié)果。二、基于血清AQP4-IgG陽性的NMOSDs患者外周器官、組織免疫效應(yīng)的研究在上述研究對象的知情同意下,收集56例患者活檢組織:包括39例胃、10例唇腺、6例骨骼肌以及1例腎臟組織,其中有3例患者同時有胃、唇腺的活檢組織,1例患者同時有胃、骨骼肌的活檢組織,1例患者同時有唇腺、骨骼肌的活檢組織。對相應(yīng)的活體組織進行石蠟包埋、切片以及AQP4抗體、CD138抗體免疫組織化學(xué)染色等實驗操作�！窘Y(jié)果】1.CBA法鑒定M23-AQP4在HEK293細胞中的表達M23-AQP4-HEK293細胞膜表面出現(xiàn)明顯的綠色熒光染色,部分細胞的細胞質(zhì)也有熒光表達。而未轉(zhuǎn)染質(zhì)粒的HEK293細胞、空載體細胞、陰性對照組細胞的膜表面、胞質(zhì)等均未見綠色熒光表達。經(jīng)鑒定可用于后續(xù)實驗對血清AQP4-IgG的檢測。2.CBA法檢測血清AQP4-IgGNMOSDs組AQP4-IgG陽性率達100%(21/21),MS組與非脫髓鞘性疾病對照組陽性率均為0(0/13,0/17)。結(jié)果認為差異有統(tǒng)計學(xué)意義(p0.001)。3.AQP4、CD138抗體在不同組別胃的表達3.1NMOSDs、MS、非脫髓鞘性疾病三組胃AQP4抗體陽性率分別為55.3%(8/15)、88.9%(8/9)、66.7%(10/15),三組間AQP4抗體陽性率差異無統(tǒng)計學(xué)意義(p0.05)。3.2NMOSDs、MS、非脫髓鞘性疾病三組胃CD138抗體陽性率分別為86.7%(13/15)、66.7%(6/9)、73.3%(11/15),三組間CD138抗體陽性率差異無統(tǒng)計學(xué)意義(p0.05)。4.AQP4、CD138抗體在不同組別唇腺的表達4.1NMOSDs、MS兩組唇腺AQP4抗體陽性率分別為28.6%(2/7)、66.7%(2/3),兩組間AQP4抗體陽性率差異無統(tǒng)計學(xué)意義(p0.05)。4.2NMOSDs、MS兩組唇腺CD138抗體陽性率分別為71.4%(5/7)、33.3%(1/3),兩組間CD138抗體陽性率差異無統(tǒng)計學(xué)意義(p0.05)。5.AQP4、CD138抗體在不同組別骨骼肌纖維的表達:3例NMOSDs與3例AQP4-M23-IgG陰性的非脫髓鞘疾病對照組的AQP4、CD138抗體陽性纖維密度值比較,差異均無統(tǒng)計學(xué)意義(p0.05)。6.僅1例NMOSDs的腎AQP4、CD138抗體在腎集合管上皮細胞中均呈陽性表達。【結(jié)論】血清AQP4-IgG可能不引起NMOSDs患者表達AQP4的外周器官、組織胃、唇腺、骨骼肌及腎由自身免疫抗體介導(dǎo)的免疫炎癥損傷效應(yīng),其機制仍需進一步研究。
[Abstract]:[BACKGROUND] Neuromyelitis optica spectrum disorders (NMOSDs) is an inflammatory demyelinating autoimmune disease of the central nervous system (CNS). With the discovery of serum aquaporin-4 antibody (AQP4 antibody, AQP4 antibody) by Lennon et al., AQP4-IgG is greatly enhanced. However, some patients with positive AQP4-IgG have a variety of clinical features. Extensive syndromes have been confirmed to be closely related to NMO, thus extending the concept of NMOSDs [2]. Studies have shown that 20-30% of patients with NMO/NMOSDs have negative serum anti-AQP4 antibodies. Sex [3]. Various detection methods show that AQP4-IgG is highly specific (91-100%) and highly sensitive (83-91%) for the diagnosis of NMOSDs, but the most recommended method is cell-based assay (CBA)[4]. However, in peripheral organs, such as stomach, kidney and skeletal muscle, with the same expression of AQP4, mouse model studies have shown that serum AQP4-IgG binds to specific antigen targets rapidly, but does not produce parenchymal peripheral organs. Tissue immune damage effect [6]. Interestingly, there have also been reports of patients with NMODs with serum AQP4-IgG positive. These results suggest that AQP4-IgG may have immunological effects on peripheral organs expressing AQP4. Therefore, CBA based on human embryonic kidney 293 cells (HEK293) transfected with M23-AQP4 was used to detect AQP4-IgG in serum. To study the immunological effects of peripheral organs, tissues such as stomach, lip gland, skeletal muscle and kidney in patients with NMOSDs positive for AQP4-M23-IgG. [Objective] To detect serum AQP4-IgG by CBA method based on M23-AQP4-HEK293 cells, and to determine the results of NMOSDs, MS and serum AQP4-IgG in patients with non-demyelinating diseases. To compare the presence of AQP4-M23-IgG-positive NMOSDs in peripheral organs and tissues (stomach, lip gland, skeletal muscle and kidney) with pathological changes or local immune inflammation effects similar to AQP4 loss in CNS. [Methods] 1. Detection of serum AQP4-IgG by CBA based on M23-AQP4-HEK293 cells transfected pcDNA3.1 +M23-AQP4 plasmid through Lipofectamine 3000. The expression of M23-AQP4 in HEK293 cells was detected by CBA assay. Serum AQP4-IgG was detected in 21 NMOSDs, 13 MS and 17 patients with non-demyelinating diseases by CBA assay. The results of anti-AQP4 antibodies in the sera of the subjects were confirmed. 2. Immunological effects in peripheral organs and tissues of patients with NMOSDs positive for serum AQP4-IgG were evaluated. With the informed consent of the above-mentioned study subjects, biopsy tissues of 56 patients were collected, including 39 stomach, 10 lip glands, 6 skeletal muscles and 1 kidney. Three patients had biopsy tissues of stomach and lip glands, one patient had biopsy tissues of stomach and skeletal muscles, and one patient had biopsy tissues of labial glands and skeletal muscles. [Results] 1. CBA method was used to identify the expression of M23-AQP4 on the surface of HEK293 cell membrane, and some of the cytoplasm was also fluorescent. The positive rate of AQP4-IgG was 100% (21/21) in serum AQP4-IgGNMOSDs group and 0% (0/13, 0/17) in MS group and non-demyelinating disease control group. The positive rates of AQP4 and CD138 antibodies were 55.3% (8/15), 88.9% (8/9) and 66.7% (10/15), respectively. There was no significant difference in the positive rates of AQP4 antibodies among the three groups (p0.05). 3.2 NMOSDs, MS, and non-demyelinating diseases. The positive rates of anti-CD138 antibody in stomach were 86.7% (13/15), 66.7% (6/9) and 73.3% (11/15), respectively. There was no significant difference in the positive rates of anti-CD138 antibody among the three groups (p0.05). 4. AQP4, anti-CD138 antibody expression in labial glands of different groups was 4.1 NMOSDs. The positive rates of anti-AQP4 antibody in labial glands of MS group and MS group were 28.6% (2/7) and 66.7% (2/3) respectively. Significance (p0.05).4.2 NMOSDs, MS two groups of labial CD138 antibody positive rates were 71.4% (5/7), 33.3% (1/3), there was no significant difference between the two groups of CD138 antibody positive rate (p0.05). 5. AQP4, CD138 antibody expression in skeletal muscle fibers in different groups: 3 NMOSDs and 3 AQP4-M23-IgG negative control group of non-demyelinating disease AQP4, CD138 antibody positive. Only one case of NMOSDs showed positive expression of AQP4 and CD138 antibodies in renal collecting duct epithelial cells. [Conclusion] Serum AQP4-IgG may not induce AQP4 expression in peripheral organs, stomach, labial gland, skeletal muscle and kidney of patients with NMOSDs. The mechanism of wound effect still needs further study.
【學(xué)位授予單位】：廣州醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R744.52
本文編號：2234661
[Abstract]:[BACKGROUND] Neuromyelitis optica spectrum disorders (NMOSDs) is an inflammatory demyelinating autoimmune disease of the central nervous system (CNS). With the discovery of serum aquaporin-4 antibody (AQP4 antibody, AQP4 antibody) by Lennon et al., AQP4-IgG is greatly enhanced. However, some patients with positive AQP4-IgG have a variety of clinical features. Extensive syndromes have been confirmed to be closely related to NMO, thus extending the concept of NMOSDs [2]. Studies have shown that 20-30% of patients with NMO/NMOSDs have negative serum anti-AQP4 antibodies. Sex [3]. Various detection methods show that AQP4-IgG is highly specific (91-100%) and highly sensitive (83-91%) for the diagnosis of NMOSDs, but the most recommended method is cell-based assay (CBA)[4]. However, in peripheral organs, such as stomach, kidney and skeletal muscle, with the same expression of AQP4, mouse model studies have shown that serum AQP4-IgG binds to specific antigen targets rapidly, but does not produce parenchymal peripheral organs. Tissue immune damage effect [6]. Interestingly, there have also been reports of patients with NMODs with serum AQP4-IgG positive. These results suggest that AQP4-IgG may have immunological effects on peripheral organs expressing AQP4. Therefore, CBA based on human embryonic kidney 293 cells (HEK293) transfected with M23-AQP4 was used to detect AQP4-IgG in serum. To study the immunological effects of peripheral organs, tissues such as stomach, lip gland, skeletal muscle and kidney in patients with NMOSDs positive for AQP4-M23-IgG. [Objective] To detect serum AQP4-IgG by CBA method based on M23-AQP4-HEK293 cells, and to determine the results of NMOSDs, MS and serum AQP4-IgG in patients with non-demyelinating diseases. To compare the presence of AQP4-M23-IgG-positive NMOSDs in peripheral organs and tissues (stomach, lip gland, skeletal muscle and kidney) with pathological changes or local immune inflammation effects similar to AQP4 loss in CNS. [Methods] 1. Detection of serum AQP4-IgG by CBA based on M23-AQP4-HEK293 cells transfected pcDNA3.1 +M23-AQP4 plasmid through Lipofectamine 3000. The expression of M23-AQP4 in HEK293 cells was detected by CBA assay. Serum AQP4-IgG was detected in 21 NMOSDs, 13 MS and 17 patients with non-demyelinating diseases by CBA assay. The results of anti-AQP4 antibodies in the sera of the subjects were confirmed. 2. Immunological effects in peripheral organs and tissues of patients with NMOSDs positive for serum AQP4-IgG were evaluated. With the informed consent of the above-mentioned study subjects, biopsy tissues of 56 patients were collected, including 39 stomach, 10 lip glands, 6 skeletal muscles and 1 kidney. Three patients had biopsy tissues of stomach and lip glands, one patient had biopsy tissues of stomach and skeletal muscles, and one patient had biopsy tissues of labial glands and skeletal muscles. [Results] 1. CBA method was used to identify the expression of M23-AQP4 on the surface of HEK293 cell membrane, and some of the cytoplasm was also fluorescent. The positive rate of AQP4-IgG was 100% (21/21) in serum AQP4-IgGNMOSDs group and 0% (0/13, 0/17) in MS group and non-demyelinating disease control group. The positive rates of AQP4 and CD138 antibodies were 55.3% (8/15), 88.9% (8/9) and 66.7% (10/15), respectively. There was no significant difference in the positive rates of AQP4 antibodies among the three groups (p0.05). 3.2 NMOSDs, MS, and non-demyelinating diseases. The positive rates of anti-CD138 antibody in stomach were 86.7% (13/15), 66.7% (6/9) and 73.3% (11/15), respectively. There was no significant difference in the positive rates of anti-CD138 antibody among the three groups (p0.05). 4. AQP4, anti-CD138 antibody expression in labial glands of different groups was 4.1 NMOSDs. The positive rates of anti-AQP4 antibody in labial glands of MS group and MS group were 28.6% (2/7) and 66.7% (2/3) respectively. Significance (p0.05).4.2 NMOSDs, MS two groups of labial CD138 antibody positive rates were 71.4% (5/7), 33.3% (1/3), there was no significant difference between the two groups of CD138 antibody positive rate (p0.05). 5. AQP4, CD138 antibody expression in skeletal muscle fibers in different groups: 3 NMOSDs and 3 AQP4-M23-IgG negative control group of non-demyelinating disease AQP4, CD138 antibody positive. Only one case of NMOSDs showed positive expression of AQP4 and CD138 antibodies in renal collecting duct epithelial cells. [Conclusion] Serum AQP4-IgG may not induce AQP4 expression in peripheral organs, stomach, labial gland, skeletal muscle and kidney of patients with NMOSDs. The mechanism of wound effect still needs further study.
【學(xué)位授予單位】：廣州醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R744.52
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