CN103409372A - 一种稳定表达mDNA的细胞 - Google Patents
一种稳定表达mDNA的细胞 Download PDFInfo
- Publication number
- CN103409372A CN103409372A CN2013101085036A CN201310108503A CN103409372A CN 103409372 A CN103409372 A CN 103409372A CN 2013101085036 A CN2013101085036 A CN 2013101085036A CN 201310108503 A CN201310108503 A CN 201310108503A CN 103409372 A CN103409372 A CN 103409372A
- Authority
- CN
- China
- Prior art keywords
- mdna
- cell
- antibody
- cytolemma
- sle
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Peptides Or Proteins (AREA)
Abstract
本发明提供了一种稳定表达mDNA的Rd细胞，该细胞可用于制备抗mDNA抗体试剂盒，抗mDNA抗体对于系统性红斑狼疮的诊断具有重要意义。
Description
技术领域
本发明提供了一种稳定表达mDNA的Rd细胞，该细胞可用于制备抗mDNA抗体试剂盒，抗mDNA抗体对于系统性红斑狼疮的诊断具有重要意义。
一、技术背景
系统性红斑狼疮(systemic lupus erythematosus，SLE)是以多系统损害伴多种自身抗体为特征的自身免疫性疾病。SLE免疫学检查主要以抗核抗体、抗dsDNA抗体和抗Sm抗体为主。这些自身抗体的发现对SLE发病机制的研究及临床诊断均具有重要意义。
膜DNA(mDNA)是近年来人们新发现的一种存在于细胞膜上的DNA，mDNA表达于一些B淋巴细胞、单核细胞和人白细胞的细胞膜上，用mDNA阳性细胞涂片，利用免疫荧光法可检测到SLE特异性荧光图形——细胞膜连续的环状强荧光，该环状荧光用DNase酶处理后可使细胞膜连续的环状强荧光消失，但是用RNase酶或蛋白酶处理后却不能使细胞膜连续的环状强荧光消失。抗mDNA抗体在SLE患者中敏感性高，特异性强。然而，目前已报道的细胞膜上有mDNA表达的细胞，存在表达不稳定、表达量不高的缺陷，直接限制了mDNA抗体检测在SLE诊断中的应用。
二、发明概述
本发明的目的之一是提供能够稳定表达mDNA的Rd细胞。
本发明的目的之二在于阐明Rd细胞可用制备mDNA抗体检测试剂盒，用于系统性红斑狼疮的诊断。
自1971年Lerner等人首次报道在人B淋巴细胞株Wil2细胞膜上发现DNA分子以来，国外及我们实验室先后报道了，HL60、Raji、Priess、Daudi、BJAB、3D5、Jurkat、K562、PAa及Hela细胞膜上存在mDNA。以Raji细胞膜上mDNA表达最强，用Raji细胞作为底物，利用间接免疫荧光法检测mDNA，SLE患者中抗mDNA抗体的敏感性和特异性高达90％以上。然而，Raji细胞存在mDNA表达不稳定的弊端，限制了利用Raji细胞开发检测试剂盒。本小组近来研究，发明了一种高表达mDNA的细胞系Rd细胞，用Rd细胞作为底物可检测抗mDNA抗体，在SLE患者中具有很高的阳性率和特异性。
附图说明
图1.间接免疫荧光检测原理示意图。
图2.本发明所述Rd细胞用于检测抗mDNA抗体，抗体阳性表现为细胞膜连续的环状强荧光。mDNA阴性表现为细胞膜未见环状荧光或未见连续的环状荧光。
具体实施方式
实施例1 Rd细胞的建立
在Raji细胞生长对数期时，利用转染试剂lipofect2000转染mDNA重组质粒(载体为pCDM8)，每代均检测mDNA重组质粒的携带情况，最终获得带有mDNA重组质粒的细胞系，该细胞系命名为Rd细胞。
实施例2 抗mDNA抗体检测玻片的制备
将Rd细胞(Raji细胞作为对照)用磷酸盐缓冲液(PBS)(10mmol/L，pH7.4)洗2次，以0.5x106/ml悬浮细胞，在分隔的玻片上点成每孔20ul的点，每张玻片可点成1、3、5、10不同等份。37℃，干燥10分钟，待液体蒸干后，每孔加入4％甲醛固定3min。蒸馏水洗1次，吹干。铺有Rd细胞和Raji的玻片用于检测抗mDNA抗体。
实施例3 Rd细胞与Raji在检测抗mDNA抗体的比较
将实施例2制备好的玻片检测20例SLE和20例正常人血清，比较Rd细胞与Raji细胞在检测mDNA抗体中的阳性率。血清以缓冲液PBS1∶40稀释，加入M细胞孔点20ul，室温下孵育30min，PBST洗2次.加入FITC-IgG，室温下孵育30min，PBS洗2次后用甘油封固玻片，荧光显微镜下观察。沿细胞膜分布的连续的环状强荧光为阳性，不连续或无荧光为阴性。检测原理示意图见图2。
实施例4 抗mDNA抗体测定对系统性红斑狼疮患者诊断的意义
为评价用本发明Rd细胞用于检测抗mDNA抗体及抗mDNA抗体在各种疾病中的分布，本研究分别利用Rd细胞作为底物制备的玻片共检测200例系统性红斑狼疮，63例类风湿关节炎(RA)，72例原发干燥综合征(pSS)，32例系统性硬化及82例正常人血清中的抗mDNA抗体。将血清以缓冲液PBS1∶40稀释，加入细胞孔20ul，室温下孵育30min，PBST洗2次.加入20ul FITC-IgG，室温下孵育30min，PBS洗2次后用甘油封固玻片，荧光显微镜下观察。沿细胞膜分布的连续的环状强荧光为阳性，不连续或无荧光为阴性。
本实施例主要通过以下几个方面阐明本发明Rd细胞的意义：
1.Rd细胞与Raji细胞检测抗mDNA抗体的比较
以Rd细胞为底物的玻片共检测到17例SLE患者mDNA阳性，20例正常人中无一例阳性；而以Raji细胞为底物的玻片检测到13例SLE患者mDNA阳性，20例正常人中有2例阳性。由此可知，Rd在检测mDNA抗体中的敏感性和特异性明显优于Raji细胞。见表1。
表1.Rd细胞与Raji细胞检测抗mDNA抗体的比较
2.Rd细胞检测抗mDNA抗体敏感性和特异性在系统性红斑狼疮、类风湿关节炎、干燥综合征、系统性硬化及正常人的比较
200例SLE患者中有152例抗mDNA抗体阳性，阳性率为76％。而疾病对照组167例中仅有9例阳性，阳性率5.4％.其中SS6例，RA3例。32例系统性硬化患者和82名正常对照抗mDNA抗体均为阴性。抗mDNA抗体在SLE患者的敏感性76％，特异性97％。SLE组的抗mDNA抗体阳性率明显高于疾病对照组和正常对照组.差异有统计学意义(P均＜0.01)，见表2。
表2.抗mDNA抗体在结缔组织性疾病中的敏感性
注：P值为各组抗mDNA抗体的敏感性与系统性红斑狼疮组敏感性的比较
3、抗mDNA抗体与SLE患者临床表现及与其他实验室指标的相关性：
抗mDNA抗体阳性的患者在皮疹、脱发、口腔溃疡及关节痛的发生率高于抗mDNA抗体阴性组患者，但差异无统计学意义。SLE患者的白细胞、C3和C4减低，IgG、IgA和IgM增高在抗mDNA抗体阳性组较为常见，差异无统计学意义。(结果未显示)。
Claims (4)
1.Rd细胞。
2.权利要求1的Rd来源于Raji细胞。
3.权利要求1的Rd细胞的细胞膜高表达mDNA。
4.权利要求1的Rd细胞可用于制备抗mDNA抗体检测试剂盒。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013101085036A CN103409372A (zh) | 2013-04-01 | 2013-04-01 | 一种稳定表达mDNA的细胞 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013101085036A CN103409372A (zh) | 2013-04-01 | 2013-04-01 | 一种稳定表达mDNA的细胞 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103409372A true CN103409372A (zh) | 2013-11-27 |
Family
ID=49602414
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2013101085036A Pending CN103409372A (zh) | 2013-04-01 | 2013-04-01 | 一种稳定表达mDNA的细胞 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103409372A (zh) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1059033A (zh) * | 1991-08-15 | 1992-02-26 | 中山医科大学附属第一医院 | 一种新型的ana、抗dna联合检测法 |
| CN101165491A (zh) * | 2006-10-19 | 2008-04-23 | 陕西西大北美基因股份有限公司 | 以金磁微粒为载体检测抗双链dna抗体的方法 |
| CN102818890A (zh) * | 2011-06-10 | 2012-12-12 | 苏州卫生职业技术学院 | 一种检测系统性红斑狼疮患者自身抗体的方法 |
-
2013
- 2013-04-01 CN CN2013101085036A patent/CN103409372A/zh active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1059033A (zh) * | 1991-08-15 | 1992-02-26 | 中山医科大学附属第一医院 | 一种新型的ana、抗dna联合检测法 |
| CN101165491A (zh) * | 2006-10-19 | 2008-04-23 | 陕西西大北美基因股份有限公司 | 以金磁微粒为载体检测抗双链dna抗体的方法 |
| CN102818890A (zh) * | 2011-06-10 | 2012-12-12 | 苏州卫生职业技术学院 | 一种检测系统性红斑狼疮患者自身抗体的方法 |
Non-Patent Citations (4)
| Title |
|---|
| HAI-YING CHEN ET AL.: "Significance of anti-cell membrane-associated DNA (mDNA) antibodies in systemic lupus erythematosus", 《CLIN RHEUMATOL》, 31 December 2008 (2008-12-31), pages 183 - 187 * |
| ILSE WECKER ET AL.: "RD Cells in the Laboratory Diagnosis of Enteroviruses", 《MED. MICROBIOL. IMMUNOL.》, 31 December 1977 (1977-12-31) * |
| JULONG CHENG ET AL.: "Plasma membrane associated transcription of cytoplasmic DNA", 《PNAS》, 3 July 2012 (2012-07-03), pages 10827 - 10831 * |
| 赵越等: "以人B淋巴细胞株Raji、人早幼粒白血病细胞株HL60为底物检测抗细胞膜DNA抗体在系统性红斑狼疮中的诊断价值", 《中华微生物学和免疫学杂志》, 30 April 2011 (2011-04-30) * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wildner et al. | B cell analysis in SARS-CoV-2 versus malaria: increased frequencies of plasmablasts and atypical memory B cells in COVID-19 | |
| Myles et al. | Expression of Toll-like receptors 2 and 4 is increased in peripheral blood and synovial fluid monocytes of patients with enthesitis-related arthritis subtype of juvenile idiopathic arthritis | |
| Szodoray et al. | Altered Th17 cells and Th17/regulatory T-cell ratios indicate the subsequent conversion from undifferentiated connective tissue disease to definitive systemic autoimmune disorders | |
| Gao et al. | Regulatory CD4+ and CD8+ T cells are negatively correlated with CD4+/CD8+ T cell ratios in patients acutely infected with SARS-CoV-2 | |
| Li et al. | Detection of circulating tumor cells from cryopreserved human sarcoma peripheral blood mononuclear cells | |
| Saad et al. | Lymphocyte populations and apoptosis of peripheral blood B and T lymphocytes in children with end stage renal disease | |
| Lv et al. | CD147-mediated chemotaxis of CD4+ CD161+ T cells may contribute to local inflammation in rheumatoid arthritis | |
| Elshal et al. | Reduced dendritic cells expressing CD200R1 in children with inflammatory bowel disease: correlation with Th17 and regulatory T cells | |
| Aranburu et al. | Age‐associated B cells expanded in autoimmune mice are memory cells sharing H‐CDR3‐selected repertoires | |
| Clottu et al. | Response of mucocutaneous lesions to rituximab in a case of melanoma differentiation antigen 5-related dermatomyositis | |
| Song et al. | Regulation of host immune cells and cytokine production induced by Trichinella spiralis infection | |
| Miao et al. | Percentages of CD4+ CD161+ and CD4− CD8− CD161+ T Cells in the Synovial Fluid Are Correlated with Disease Activity in Rheumatoid Arthritis | |
| Gündüz et al. | Regulatory T cells in Behçet’s disease: Is there a correlation with disease activity? Does regulatory T cell type matter? | |
| Nandi et al. | CD8+ CD28− T cells: key cytotoxic players impacting disease pathogenesis in chronic HBV infection | |
| Reijnders et al. | Unraveling the identity of FoxP3+ regulatory T cells in Granulomatosis with Polyangiitis patients | |
| Metenou et al. | Highly heterogeneous, activated, and short‐lived regulatory T cells during chronic filarial infection | |
| Yu et al. | Detection of anti-p155/140, anti-p140, and antiendothelial cells autoantibodies in patients with juvenile dermatomyositis | |
| CN103409372A (zh) | 一种稳定表达mDNA的细胞 | |
| Mugnaini et al. | The T cell receptor repertoire of CD8+ CD28− T lymphocytes is dominated by expanded clones that persist over time | |
| CN102818890A (zh) | 一种检测系统性红斑狼疮患者自身抗体的方法 | |
| Yi et al. | Circulating regulatory T cells in patients with severe fever with thrombocytopenia syndrome | |
| Samani et al. | Alteration in CD8+ T cell subsets in enterovirus-infected patients: an alarming factor for type 1 diabetes mellitus | |
| Abuwarwar et al. | A pathogenic hierarchy for synovial fibroblasts in rheumatoid arthritis | |
| Carsons et al. | Detection and quantitation of stem cell factor (kit ligand) in the synovial fluid of patients with rheumatic disease. | |
| Mezzetti et al. | Autoimmune Heart Disease: A Comprehensive Summary for Forensic Practice |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20131127 |