血清AQP4-M23-IgG的检测及其对NMOSDs患者的胃、唇腺、骨骼
发布时间：2018-09-10 14:07
【摘要】：【背景】视神经脊髓炎谱系疾病(neuromyelitis optica spectrum disorders,NMOSDs)是一种中枢神经系统(central nervous system,CNS)炎性脱髓鞘性自身免疫性疾病。随着Lennon等[1]对血清水通道蛋白4抗体(aquaporin-4 antibody,AQP4抗体)即AQP4-IgG的发现,大大提高了对视神经脊髓炎(neuromyelitis optica,NMO)的诊断特异性。然而,部分AQP4-IgG阳性的患者有多种不同的临床特征,出现广泛的综合征证实与NMO密切相关,于是延伸出另一个扩展性概念NMOSDs[2]。研究显示20-30%的NMO/NMOSDs患者血清抗AQP4抗体为阴性[3]。各种检测方法显示AQP4-IgG对NMOSDs诊断的高度特异性(91-100%)和高灵敏度(83-91%),但目前最推荐的检测方法是基于细胞的检测法(cell based assay,CBA)[4]。同时,有报道称在外周血发现的AQP4-IgG参与了NMO/NMOSDs的发病机理中,证实AQP4-IgG在CNS中是致病性的[5]。然而在同样表达有AQP4的外周器官、组织如胃、肾、骨骼肌中,小鼠模型的研究显示血清AQP4-IgG可快速结合特异性抗原靶点,但不产生实质的外周器官、组织免疫损伤效应[6]。有趣的是,也有报道称血清AQP4-IgG阳性的NMOSDs患者可伴有流产风险的胎盘炎[7]、内耳炎[8]和胃炎[9]等。这些研究结果表明,AQP4-IgG可能对表达AQP4的外周器官、组织有免疫损伤效应。因此,本研究采用基于转染M23-AQP4的人胚胎肾细胞(human embryonic kidney293 cells,HEK293)的CBA法检测血清AQP4-IgG,并对AQP4-M23-IgG阳性的NMOSDs患者外周器官、组织如胃、唇腺、骨骼肌、肾区域免疫效应作进一步的研究和机制探讨。【目的】采用基于M23-AQP4-HEK293细胞的CBA法检测血清AQP4-IgG,明确研究对象NMOSDs、MS以及非脱髓鞘性疾病患者的血清AQP4-IgG结果,以便比较AQP4-M23-IgG阳性的NMOSDs患者外周器官、组织(胃、唇腺、骨骼肌及肾)是否存在类似CNS中AQP4脱失等病理损伤改变或局部免疫炎症效应。【方法】一、基于M23-AQP4-HEK293细胞的CBA法检测血清AQP4-IgG将pcDNA3.1+-M23-AQP4质粒通过Lipofectamine 3000转染接种于HEK293细胞中,经CBA法鉴定M23-AQP4是否在HEK293细胞中表达。采用CBA法检测21例NMOSDs、13例MS以及17例非脱髓鞘性疾病患者血清AQP4-IgG,明确研究对象的血清抗AQP4抗体的结果。二、基于血清AQP4-IgG阳性的NMOSDs患者外周器官、组织免疫效应的研究在上述研究对象的知情同意下,收集56例患者活检组织:包括39例胃、10例唇腺、6例骨骼肌以及1例肾脏组织,其中有3例患者同时有胃、唇腺的活检组织,1例患者同时有胃、骨骼肌的活检组织,1例患者同时有唇腺、骨骼肌的活检组织。对相应的活体组织进行石蜡包埋、切片以及AQP4抗体、CD138抗体免疫组织化学染色等实验操作。【结果】1.CBA法鉴定M23-AQP4在HEK293细胞中的表达M23-AQP4-HEK293细胞膜表面出现明显的绿色荧光染色,部分细胞的细胞质也有荧光表达。而未转染质粒的HEK293细胞、空载体细胞、阴性对照组细胞的膜表面、胞质等均未见绿色荧光表达。经鉴定可用于后续实验对血清AQP4-IgG的检测。2.CBA法检测血清AQP4-IgGNMOSDs组AQP4-IgG阳性率达100%(21/21),MS组与非脱髓鞘性疾病对照组阳性率均为0(0/13,0/17)。结果认为差异有统计学意义(p0.001)。3.AQP4、CD138抗体在不同组别胃的表达3.1NMOSDs、MS、非脱髓鞘性疾病三组胃AQP4抗体阳性率分别为55.3%(8/15)、88.9%(8/9)、66.7%(10/15),三组间AQP4抗体阳性率差异无统计学意义(p0.05)。3.2NMOSDs、MS、非脱髓鞘性疾病三组胃CD138抗体阳性率分别为86.7%(13/15)、66.7%(6/9)、73.3%(11/15),三组间CD138抗体阳性率差异无统计学意义(p0.05)。4.AQP4、CD138抗体在不同组别唇腺的表达4.1NMOSDs、MS两组唇腺AQP4抗体阳性率分别为28.6%(2/7)、66.7%(2/3),两组间AQP4抗体阳性率差异无统计学意义(p0.05)。4.2NMOSDs、MS两组唇腺CD138抗体阳性率分别为71.4%(5/7)、33.3%(1/3),两组间CD138抗体阳性率差异无统计学意义(p0.05)。5.AQP4、CD138抗体在不同组别骨骼肌纤维的表达:3例NMOSDs与3例AQP4-M23-IgG阴性的非脱髓鞘疾病对照组的AQP4、CD138抗体阳性纤维密度值比较,差异均无统计学意义(p0.05)。6.仅1例NMOSDs的肾AQP4、CD138抗体在肾集合管上皮细胞中均呈阳性表达。【结论】血清AQP4-IgG可能不引起NMOSDs患者表达AQP4的外周器官、组织胃、唇腺、骨骼肌及肾由自身免疫抗体介导的免疫炎症损伤效应,其机制仍需进一步研究。
[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.
【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】：R744.52
本文编号：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.
【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】：R744.52
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