MPO-ANCA表位识别特异性与其相关性血管炎临床损害间关系探究

发布时间：2018-03-10 17:44

本文选题：蛋白重组　切入点：抗中性粒细胞胞浆髓过氧化物酶抗体　出处：《安徽医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：背景MPO-ANCA相关性血管炎(myeloperoxidase-specific antineutrophil cytoplasmic autoantibody associated vasculitis,MPO-AAV)在我国并不少见,主要包括显微镜下多血管炎(Microscopic polyangiitis,MPA)、肉芽肿性多血管炎(Granulomatosis with polyangiitis,GPA)及嗜酸性肉芽肿性多血管炎(Eosinophilic granulomatosis with polyangiitis,EGPA)。此类疾病临床表现复杂多样、进展快,未得到及时诊治的患者常因病情延误而死亡。临床报道、动物实验以及体外实验证据均显示了MPO-ANCA具有致病性,而针对其表位识别特性的研究报道显示,MPO-ANCA不同的表位识别在AAV的不同疾病中,可能发挥不同的作用。但MPO-ANCA不同表位在MPO-AAV不同临床损害中的作用尚未见系统性研究报道。目的:研究MPO-AAV患者ANCA的表位识别分布情况,探讨其与临床损害之间的相关性。方法:本研究通过构建重组质粒、原核表达及蛋白纯化的分子生物学方法以获得7个覆盖全部MPO线性抗原的多肽片段,包括轻链(aa 165-272)、重链6个肽段:H4肽段(aa279-409)、H5肽段(aa341-474)、H6肽段(aa410-537)、H7(aa512-598)、H8肽段(aa538-745)及H9(aa651-745),并鉴定它们的抗原性。临床收集初诊活动期MPO-AAV患者173例(其中,原发性MPO-AAV患者165例、PTU继发MPO-AAV患者8例)的血清。分析MPO-AAV患者临床损害情况。分别用所合成肽段包被ELISA反应孔后与患者血清反应,检测患者及健康对照血清ANCA识别MPO抗原表位肽段的特异性,分析并探讨其与患者临床多系统损害之间的关系。结果1.获得人MPO蛋白及七个多肽片段的c DNA克隆,其表达的蛋白多肽具有MPO抗原性。2.正常人血清可识别H5、H6、H8、H9表位。3.8例继发于PTU的MPO-AAV患者中,4例识别H8肽段,其中1例识别H7肽段。4.MPO-AAV患者中,有肺损较无肺损者轻链肽段抗体的识别阳性率、ELISA检测OD值高,且轻链肽段抗体阳性患者比阴性患者的肺BVAS积分高;5.有肾损害(包括有肾功能损害、仅血尿蛋白尿)的患者和无肾损害患者H4、H5肽段抗体的阳性率差异有统计学意义,均为前者高于后者,6.有肾功能损害的患者H9肽段抗体阳性率以及OD值水平均高于仅尿隐血/或尿蛋白阳性患者,其余各肽段抗体阳性率比较差异均无统计学意义。7.有肾功能损害患者中,急性进展性肾小球肾炎、慢性肾功能损伤患者之间比较,慢性肾功能损害患者的H4、H5、H8、H9肽段抗体OD值均高于急性肾功能损害患者。8.急性进展性肾小球肾炎、慢性肾功能损伤患者之间比较H4、H5肽段抗体阳性率差异有显著性,后者均较前者阳性率高。9.在急性肾功能损害患者(30例)中,轻链肽段抗体ELISA值和肾脏BVAS积分之间存在正相关;10.有关节损伤较无关节损伤的患者H7、H8肽段抗体检测OD值高,各肽段抗体阳性率差异无统计学意义。结论1.获得MPO7个不同多肽的片段,他们保留有MPO的抗原性,可用于MPO-AAV相关的进一步研究。2.正常人血清存在的少量ANCA识别MPO的抗原表位分散:H5、H6、H8、H9肽段(aa341-754)。3.MPO-AAV患者体内的ANCA主要识别MPO轻链和重链两端:轻链、H4、H5、H8、H9(aa165-272,aa279-474,aa538-745)的抗原表位4.相对于原发性MPO-AAV,PTU继发性的MPO-AAV患者血清ANCA识别的MPO抗原表位比较局限,主要位Hf(aa538-598),且患者年龄相对小,病情较轻。5.MPO轻链检测阳性的ANCA可能有MPO-AAV患者的肺侵害相关。6.MPO H4、H5(aa279-474)肽段检测阳性的ANCA可能与MPO-AAV患者的肾损害相关。在肾累及的患者中,识别H9(aa538-745)的ANCA可能与肾功能损害有关,在肾功能损害的MPO-AAV患者中,识别Ha、Hb、Hc(aa279-474)、H8(aa538-745)段表位的ANCA导致慢性肾损害可能性大,在急性进展性肾功能损伤患者中,出现MPO-轻链(aa165-272)抗体的患者,病情更加严重。7.识别H7、H8肽段的ANCA抗体水平与MPO-AAV患者的关节损害有关。
[Abstract]:The background of MPO-ANCA associated vasculitis (myeloperoxidase-specific antineutrophil cytoplasmic autoantibody associated vasculitis, MPO-AAV) is not uncommon in China, mainly including the microscopic polyangiitis (Microscopic polyangiitis, MPA), granulomatous vasculitis (Granulomatosis with multiple polyangiitis, GPA) and eosinophilic granulomatous polyangiitis (Eosinophilic granulomatosis with polyangiitis, EGPA). This kind of clinical disease performance is complicated, progresses quickly, did not receive timely treatment of patients due to illness delays and death. Clinical reports, animal experiments and in vitro experiments showed evidence of MPO-ANCA pathogenicity, and the epitope recognition properties of research reports show that MPO-ANCA different epitope recognition in different diseases in AAV, may to play a different role. But different MPO-ANCA epitopes in MPO-AAV damage in different clinical The research is not yet reported. Objective: To study the MPO-AAV of patients with ANCA epitope recognition distribution, and explore the correlation between clinical damage. Methods: This study through the construction of recombinant plasmid, polypeptide fragment molecular biology method of prokaryotic expression and protein purification to obtain 7 cover all MPO linear antigens, including the light chain (AA 165-272), heavy chain 6 peptides: H4 peptide (aa279-409), H5 peptide (aa341-474), H6 peptide (aa410-537), H7 (aa512-598), H8 peptide (aa538-745) and H9 (aa651-745), and identify their antigenicity. 173 cases of newly diagnosed activities MPO-AAV patients (including 165 patients with primary MPO-AAV were PTU, MPO-AAV patients 8 cases). The serum analysis of clinical impairment of patients with MPO-AAV. Respectively with the synthetic peptide coated by ELISA reaction hole after reaction with serum, detection of patients and healthy controls serum ANCA identification of MPO antigen. A specific peptide analysis, and to explore their relationships with patients with multisystem damage. Results in the 1. C DNA clone of MPO protein and seven peptide fragments, the expression of the protein polypeptide has the antigenicity of MPO.2. in normal human serum can be identified by H5, H6, H8, H9 epitope.3.8 cases after in PTU MPO-AAV patients, 4 cases of identification of H8 peptide, H7 peptide recognition including 1 cases of.4.MPO-AAV patients, the positive rate of loss is no recognition of pulmonary lung injury were light chain peptide antibody, ELISA staining, and the light chain peptide antibody positive patients than in patients with negative pulmonary BVAS integral 5.; kidney damage (including renal damage, only hematuria and proteinuria) in patients with and without renal damage in patients with H4, there were statistically significant differences in the positive rate of H5 peptide antibody, were higher, the positive rate of patients with H9 peptide antibody of 6. had renal damage and the OD value was higher than only urine occult blood or / Urine protein positive patients, the positive rate of the peptide antibody showed no significant difference.7. with renal damage in patients with acute progressive glomerulonephritis, comparison between patients with chronic renal injury, H4, patients with chronic renal damage in H5, H8, H9 peptide antibody OD value was higher than that of acute renal injury patients.8. in acute glomerulonephritis, comparison between H4 patients with chronic renal injury, there was significant difference in the positive rate of H5 peptide antibody, which were higher than the positive rate of patients with high.9. in acute kidney injury (30 cases), there is a positive correlation between BVAS and renal integral light chain peptide antibody of ELISA; 10. there is no joint injury of joint damage in patients with H7, H8 peptide antibody detection was high, there was no significant difference in the positive rate of peptide antibody. Conclusion MPO7 1. different peptide fragments, they retain antigenicity of MPO, can be used for A small amount of ANCA identification of MPO further study of MPO-AAV related.2. of normal human serum are epitopes: H5, H6, H8 dispersion, H9 peptide (aa341-754) in patients with.3.MPO-AAV ANCA recognition MPO light chain and heavy chain ends: light chain, H4, H5, H8, H9 (aa165-272, aa279-474, aa538-745) the antigen epitope of 4. relative to the primary MPO-AAV and secondary PTU MPO-AAV in serum of patients with ANCA to identify the epitopes of MPO is limited, mainly Hf (aa538-598), and the age of patients is relatively small, mild light chain.5.MPO ANCA may have MPO-AAV positive patients with lung damage related.6.MPO H4, H5 (aa279-474) peptide detection of renal damage in ANCA positive patients may be associated with MPO-AAV. The renal involvement in patients with H9 (aa538-745) on identification of ANCA may be associated with impairment of renal function in renal damage in patients with MPO-AAV, Hb, Hc recognition Ha (aa279-474), H8 (aa538-745) segment table a ANCA caused the possibility of chronic renal damage. In patients with acute progressive renal impairment, patients with MPO- light chain (aa165-272) antibodies were more severe..7. recognized H7 and H8 peptide ANCA antibody level was associated with joint damage in MPO-AAV patients.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R593.2

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