抗PLA2R抗体检测方法的建立及在膜性肾病中的应用

发布时间：2018-04-18 01:42

本文选题：膜性肾病 + 抗磷脂酶A2受体抗体　；参考：《第二军医大学》2014年硕士论文

【摘要】：目的：构建真核重组质粒,体外表达PLA2R蛋白；运用双抗夹心原理建立抗PLA2R抗体的间接免疫荧光检测方法(IIFT)；通过检测MN患者血清和尿液抗PLA2R抗体,观察血清和尿液抗体与患者疾病严重程度和组织病理改变的相关性。方法： 1.PLA2R真核重组质粒的构建和蛋白表达基因库查找PLA2R全长mRNA序列,合成全长基因,构建pcDNA3.1/Hygro-PLA2R-IRES2-eGFP和pcDNA3.1/Hygro-PLA2R-His重组质粒。通过Lipofectamine2000介导质粒瞬时转染HEK293T细胞,进行目的蛋白表达,运用流式细胞技术、免疫荧光、免疫组化和WB方法对表达蛋白进行鉴定。 2.抗PLA2R抗体IIFT的建立与验证用表达目的蛋白的HEK293T细胞作为捕获抗原与MN患者血清中的抗PLA2R抗体进行结合,再用二抗进行荧光标记,建立抗PLA2R抗体IIFT。用自建的抗体检测方法对150例肾小球疾病患者和30例健康成人血清进行检测,同时用国外检测试剂盒作为对照,评价自建检测方法的有效性,明确抗体对原发性MN患者的诊断特异性。 3.抗PLA2R抗体在MN中的临床研究收集长征医院114例原发性MN和18例存在继发因素MN患者的血清、尿液,用自建的抗PLA2R抗体IIFT对患者血清和尿液进行定性检测,记录患者的临床资料和病理报告,采用聚合免疫散射法对患者血清IgG和IgG4进行定量检测。观察原发性MN患者血清抗体阳性组和阴性组的临床指标和病理的差异性,并对血清IgG和IgG4值进行分析；将血清抗体阳性的原发性MN患者分为尿液阳性组和阴性组,观察两组患者临床指标和病理分期的差异性。对存在继发因素MN患者的血清和尿液抗体进行检测,观察抗体的阳性率。结果： 1.选取基因库中PLA2R的1型转录体全长mRNA(NCBI,NM_007366.4)作为目的基因,合成全长碱基序列,通过酶切手段构建PLA2R的真核重组质粒pcDNA3.1/Hygro-PLA2R-IRES2-eGFP和pcDNA3.1/Hygro-PLA2R-His,运用Lipofectamine2000将重组质粒瞬时转染HEK293T细胞,通过免疫组化、流式细胞学技术、免疫荧光和WB方法鉴定PLA2R蛋白的表达。 2.用表达PLA2R蛋白的HEK293T细胞作为捕获抗原,建立抗PLA2R抗体的IIFT。用该方法对150例肾小球疾病患者和30例健康成人血清进行检测,结果抗体仅出现在原发性MN患者血清中,其它肾小球疾病和健康成人均为阴性,自建检测方法与国外试剂盒的检测结果一致。 3.114例原发性MN患者有84例(73.68%)血清抗体阳性,阳性组血清白蛋白、甘油三酯、肌酐、IgG、24h蛋白尿、尿TRF、IGU、eGFR和组织病理分期与阴性组存在差异(p0.05),而血清胆固醇、IgG4及IgG4/IgG值间差异无统计学意义(p0.05)；18例存在继发因素的MN患者有6例血清抗体阳性。血清抗体阴性患者的尿液抗体均阴性,84例血清阳性患者有60例(71.42%)尿液抗体阳性,占总例数的52.63%；尿液阳性组血清白蛋白、肌酐、尿TRF、IGU和eGFR与阴性组存在差异(p0.05),而血脂、24h蛋白尿、血清IgG、IgG4、 IgG4/IgG和组织病理无统计学差异(p0.05)；6例存在继发因素、血清抗体阳性的MN患者有4例尿液抗体阳性。结论：本课题构建的PLA2R真核重组质粒能表达PLA2R蛋白,自建的抗PLA2R抗体IIFT能有效检测MN患者的抗PLA2R抗体。检测抗PLA2R抗体可以作为诊断原发性MN的一种方法,其在原发性MN血清中的阳性率为73.68%,在尿液中阳性率为52.63%；抗PLA2R抗体在存在继发因素的MN患者血清中有部分阳性,但未在其他肾小球疾病和健康人群血清中检测到。血清抗体与患者的临床指标显著相关,而尿液抗体可能与患者肾功能损伤程度有关,抗体的出现提示患者临床病情和肾脏损伤程度加重。
[Abstract]:Purpose :

constructing eukaryotic recombinant plasmid and expressing PLA2R protein in vitro ;
The indirect immunofluorescence assay ( IIFT ) of anti - PLA2R antibody was established by using double anti - sandwich principle .
The correlation of serum and urine antibodies to the severity of disease severity and histopathological changes was observed by detecting anti - PLA2R antibodies in serum and urine of MN patients .

Method :

Construction of eukaryotic recombinant plasmid of 1 . PLA2R and expression of protein

The recombinant plasmid pcDNA3.1 / Hygro - PLA2R - IRES2 - eGFP and pcDNA3.1 / Hygro - PLA2R - His were constructed by Lipofectamine 2000 - mediated transfection .

2 . Establishment and Verification of Anti - PLA2R Antibody IIFT

The anti - PLA2R antibody was used as capture antigen to bind to the anti - PLA2R antibody in serum of MN patients . The anti - PLA2R antibody IIFT was established by means of self - established antibody detection method .

3 . Clinical study of anti - PLA2R antibody in MN

The serum and urine of patients with primary MN and 18 cases of primary MN and 18 patients with secondary factors were collected , and the patients ' serum and urine were qualitatively detected by self - constructed anti - PLA2R antibody IIFT . The clinical data and pathological report of patients were recorded . The clinical indexes and pathological differences of serum antibody positive group and negative group were observed in patients with primary MN .
The serum and urine antibody of MN patients with secondary factors were detected and the positive rate of antibody was observed .

Results :

1 . The full - length mRNA of the 1 - type transcripts of PLA2R in the gene bank was selected as the target gene , and the full - length base sequence was synthesized . The eukaryotic recombinant plasmid pcDNA3.1 / Hygro - PLA2R - IRES2 - eGFP and pcDNA3.1 / Hygro - PLA2R - His were constructed by restriction enzyme digestion . The recombinant plasmid was transiently transfected with pcDNA3.1 / Hygro - PLA2R - IRES2 - eGFP and pcDNA3.1 / Hygro - PLA2R - His , and the expression of PLA2R protein was identified by immunohistochemistry , flow cytometry , immunofluorescence and WB .

2 . The expression of PLA2R protein was used as capture antigen to establish IIFT of anti - PLA2R antibody . Using this method , 150 patients with glomerular disease and 30 healthy adult sera were tested . The results showed that the antibody only appeared in serum of primary MN patients , and other glomerular diseases and healthy adult sera were negative , and the self - built test method was consistent with the test results of foreign kits .

3 . There were 84 cases ( 73.68 % ) of patients with primary MN ( 73.68 % ) . The serum albumin , triglyceride , creatinine , IgG , 24h proteinuria , urine trf , IGU ( IGU ) were significantly different from those in the negative group ( P < 0.05 ) , but there was no significant difference between the serum cholesterol , IgG and IgG values ( p < 0.05 ) .
Among the 18 patients with secondary factors , there were 6 serum antibody positive . The urine antibody was negative in serum antibody negative patients , and there were 60 ( 71.42 % ) urine antibody positive in 84 patients with positive serum antibody , accounting for 52.63 % of the total number of patients .
There was no significant difference in serum albumin , creatinine , urine trf , IGU in urine positive group and negative group ( P < 0.05 ) .
Among the 6 cases , there were secondary factors , and there were 4 positive urine antibodies in the MN patients with positive serum antibody .

Conclusion :

The monoclonal anti - PLA2R antibody could be used as a method for the diagnosis of primary MN . The positive rate of anti - PLA2R antibody in primary MN serum was 73.68 % and 52.63 % in urine .
The anti - PLA2R antibody was partially positive in the serum of MN patients with secondary factors , but was not detected in other glomerular diseases and healthy population serum . The serum antibody was significantly correlated with the patient ' s clinical index , and the urine antibody may be related to the degree of renal impairment of the patient , and the presence of the antibody suggests that the patient ' s clinical condition and the degree of kidney injury are aggravated .

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R692

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