CN116593700A - 一种用于鉴定抗mda5阳性皮肌炎患者的分子标记物 - Google Patents
一种用于鉴定抗mda5阳性皮肌炎患者的分子标记物 Download PDFInfo
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- G01N2800/10—Musculoskeletal or connective tissue disorders
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- G—PHYSICS
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- G01N2800/50—Determining the risk of developing a disease
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- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
涉及一种用于鉴定抗MDA5阳性皮肌炎患者的分子标记物,所述的分子标记物为GPX4(glutathione peroxidase 4,GPX4)蛋白,具体的,当患者外周血CD3+T细胞中的GPX4蛋白表达量降低时,鉴定所述患者为抗MDA5阳性皮肌炎患者,并且,当GPX4蛋白表达量低于0.74时,所述患者合并真菌感染风险高。
Description
技术领域
本发明属于生物医药技术领域,具体的,涉及一种用于鉴定抗MDA5阳性皮肌炎患者的分子标记物。
背景技术
皮肌炎(dematomyositis,DM)是临床上最为常见的特发性炎性肌病(idiopathicinflammatory myopathy,IIM),主要表现为四肢近端肌无力,特征性的皮疹以及包括肺在内的多脏器受累[1]。抗MDA5(抗黑色素瘤分化相关基因5)抗体阳性DM是一类独特的DM亚型,以高发间质性肺病(interstitial lung disease,ILD)、低淋巴细胞血症、高死亡率为特征[2]。约40%的抗MDA5阳性DM患者发生快速进展性ILD(rapidly progressive ILD,RP-ILD)[3],临床上这类患者进展迅速,病情凶险,治疗困难[4]。然而,目前抗MDA5抗体阳性DM的病因和发病机制尚未完全清楚,缺乏特异的治疗靶点。
临床上,外周血淋巴细胞数量减少是抗MDA5阳性DM患者最显著的免疫学特征。有研究报道淋巴细胞数量减少与抗MDA5抗体阳性DM患者RP-ILD的发生相关[5]。申请人最近发表于Journal of Internal Medicine杂志的一项研究证实,外周血淋巴细胞数量显著减少是抗MDA5抗体阳性DM患者独特的特征,并且外周血淋巴细胞数量可以对抗MDA5抗体阳性DM进行临床分型和预后预测,从而为临床诊疗提供一种简单可信的生物标志物[6]。基线水平外周血淋巴数量减少,提示更为严重的疾病程度和更差的预后[6]。
然而,抗MDA5阳性DM外周血淋巴细胞数量显著减少的原因仍不清楚。
首先淋巴细胞数量减少不是造血系统功能障碍导致,国内外学者均报道抗MDA5阳性DM-ILD患者骨髓淋巴细胞呈正常分布[6,7]。
其次,淋巴细胞迁移至肺组织可能导致外周淋巴细胞数量减少,但抗MDA5抗体阳性DM患者是否存在这种淋巴细胞迁移还有待明确,申请人发现外周血淋巴细胞严重减少的抗MDA5阳性DM患者,其支气管肺泡灌洗液中淋巴细胞百分比明显低于外周血淋巴细胞正常和轻度减少患者,与淋巴细胞迁移导致外周血淋巴细胞减少的假说不符[6]。
因此,抗MDA5阳性DM淋巴细胞数量减少更可能是外周血中细胞死亡机制异常而导致。
外周血淋巴细胞主要分为T细胞、B细胞和NK细胞,其中T细胞占比最高。抗MDA5阳性DM患者外周血T细胞下降非常明显,并且与肺部感染和预后密切相关[8]。既往研究中初步探索发现DM患者T细胞RIG-1表达显著升高,且可能诱导细胞凋亡和抑制T细胞增殖[9],部分解释了DM外周血T细胞减少的原因。但是T细胞减少与抗MDA5抗体阳性DM亚型独特关联性及具体机制仍有待充分阐明。
铁死亡(ferroptosis)是一种铁依赖性的脂质过氧化驱动的、非细胞凋亡性的调节性细胞死亡形式,其主要特征是脂质过氧化物和活性氧(reactive oxygen species,ROS)的过量蓄积[10]。铁死亡的形态学主要表现为线粒体缩小、膜密度增加、线粒体嵴减少甚至消失以及线粒体外膜破裂,但细胞核形态不变。铁死亡受到多种细胞代谢途径的调控,包括氧化还原稳态、铁代谢、线粒体活性和氨基酸、脂质、糖的代谢,以及各种与疾病相关的信号途径。抗氧化系统system Xc-(胱氨酸/谷氨酸反向转运体)-谷胱甘肽(glutathione,GSH)-谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)被认为是铁死亡经典调控通路,在抑制铁死亡中发挥重要作用。其中,system Xc-由催化亚基溶质载体家族7成员11(solute carrier family 7member 11,SLC7A11)和调节亚基溶质载体家族3成员2(solutecarrier family 3member 2,SLC3A2)组成。抗氧化系统的调控核心酶GPX4作为铁死亡关键调控因子,利用GSH将脂质氢过氧化物还原为脂质醇,抑制铁死亡[11]。目前,已在多种自身 免疫性疾病中检测到铁死亡的激活,包括系统性红斑狼疮、类风湿关节炎、炎症性肠病等 [12],但是迄今为止,仍未有铁死亡在IIM发病机制中的研究报道。
基于此,提出本发明。
[参考文献]
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发明内容
本发明首先涉及GPX4(glutathione peroxidase 4,GPX4)蛋白在制备检测试剂盒中的应用,所述的检测试剂盒用于:
(1)检测抗MDA5(抗黑色素瘤分化相关基因5)抗体阳性皮肌炎(DM);或
(2)预测抗MDA5抗体阳性皮肌炎(DM)患者真菌感染的风险。
所述抗MDA5抗体阳性DM是一类DM亚型,以高发间质性肺病(interstitial lungdisease,ILD)、低淋巴细胞血症、高死亡率为特征。
所述的GPX4蛋白为患者外周血中的GPX4蛋白,优选为外周血CD3+T细胞中的GPX4蛋白。
所述的预测抗MDA5抗体阳性皮肌炎(DM)患者真菌感染的风险指,将抗MDA5抗体阳性皮肌炎(DM)患者区分为真菌感染高风险组和低风险组。
进一步的,当GPX4水平降低时,
(1)所述DM患者为抗MDA5抗体阳性DM患者;或
(2)抗MDA5抗体阳性DM患者真菌感染的风险高。
更进一步的,
(1)当外周血CD3+T细胞中的GPX4蛋白量低于0.785时(数值为GPX4相对表达量,指GPX4相对于GAPDH的表达量的比值)时,该患者为抗MDA5抗体阳性DM患者;或
(2)当外周血CD3+T细胞中的GPX4蛋白量低于0.74时(数值为GPX4相对表达量,指GPX4相对于GAPDH的表达量的比值),该患者真菌感染风险高。
本发明的有益效果在于,
通过分析了抗MDA5阳性DM患者外周血T细胞蛋白表达情况,阐明了及GPX4表达情况与抗MDA5阳性DM疾病之间的关联,并通过进一步的数据解析,揭示了GPX4蛋白的表达情况与抗MDA5阳性DM患者继发真菌感染之间的关联。
附图说明
图1.抗MDA5阳性DM患者外周血CD3+T细胞中GPX4蛋白表达降低。
1A、通过蛋白质印迹分析IIM患者和HC中GPX4的蛋白质水平;
1B、β-actin用作蛋白质印迹分析的内部参照,IIM患者和HC GPX4的相对表达量;
1C、抗MDA5阳性DM患者中GPX4和外周血CD3+T细胞数目的相关性;
图中,***表示p<0.001。
图2.流式细胞分析IIM患者外周血CD4和CD8 T细胞GPX4蛋白表达水平。
2A.流式细胞分析IIM患者和健康对照的GPX4表达水平(平均荧光强度表示)。
CD3 T细胞(2B),CD4 T细胞(2C)和CD8 T细胞(2D)的GPX4表达水平在抗MDA5阳性合并T细胞数量减少的DM患者中均显著降低。
抗MDA5阳性DM者中GPX4和外周血CD3(2E),CD4(2F)和CD8(2G)T细胞数目显著正相关;图中,*、**、***分别表示p<0.05、p<0.01和p<0.001。
图3.抗MDA5阳性DM合并T细胞减少患者及健康人T细胞形态学分析。
3A.透射电镜显示抗MDA5阳性DM合并T细胞减少患者相对健康人,外周血T细胞线粒体空泡形成增加,线粒体膜密度增加,线粒体嵴消失。比例尺,1μm。箭头,线粒体。
3B.计算线粒体面积。每个点代表一个细胞的平均线粒体面积。***表示p<0.001。
图4.抗MDA5阳性DM患者T细胞脂质过氧化物沉积。
4A.抗MDA5阳性DM合并T细胞减少患者及健康对照的外周血T细胞Liperfluo染色代表图像。比例尺,100μm。
4B.计算平均荧光强度。
4C.抗MDA5阳性DM患者与健康对照的血清MDA水平。*、**分别表示p<0.05、p<0.01。
图5.抗MDA5阳性DM患者T细胞铁代谢检测。
5A.蛋白印迹分析患者和健康对照(HC)T细胞TFRC的蛋白质水平;
5B.患者和健康对照T细胞TFRC的相对表达量比较;
5C.抗MDA5阳性DM合并T细胞减少患者及健康对照的外周血T细胞FerroOrange染色代表图像。比例尺,100μm。
5D.计算平均荧光强度。*、**分别表示p<0.05、p<0.01。
具体实施方式
材料与方法
1.研究对象
本研究纳入了2021年11月至2022年8月中日友好医院风湿免疫科收治的114例IIM患者,其中包括97例DM患者,9例抗合成酶综合征(ASS)患者,8例免疫介导的坏死性肌病(IMNM)患者。
此外纳入了57名与患者年龄性别相匹配的健康对照。DM的诊断符合2017年欧洲风湿病联盟及美国风湿病学会(European LeagueAgainst Rheumatism ExecutiveCommittee and American College of Rheumatology,EULAR/ACR)标准[13]。IMNM的诊断符合第224届欧洲神经肌肉中心国际研讨会的病理标准进行的分类[14]。ASS诊断符合2010年Connor标准[15]。排除重叠综合征,肌营养不良,重症肌无力,药物相关性肌病,神经肌肉病,运动神经元疾病,代谢相关性肌病等。
2.临床数据资料收集
通过医院的电子病历,获取患者的临床表现和实验室数据(包括患者基本资料、主要临床表现和体格检查、既往病史、实验室数据和肺功能检查等)。
3.实验方法
3.1、人外周血CD3+T细胞分离
抽取新鲜的外周血20mL使用美国Sigma公司Histopque-1077分离液采用密度梯度离心法分离PBMC。随后使用美国Thermo Fisher公司的Invitrogen FlowComp Human CD3试剂盒对PBMC中的CD3+T细胞进行分选。将上述提取的PBMCs用500ul分离缓冲液重悬,加入25ul人源CD3抗体,4℃孵育10分钟。加入2ml分离缓冲液,室温下离心,离心条件为400xg,10分钟。用移液枪移除上清液,1ml分离缓冲液重悬细胞沉淀。在细胞重悬液中加入75ul清洗过的磁珠,轻轻吹打混匀,在室温下于摇床上倾斜滚动孵育15分钟。加入1ml分离缓冲液,移液枪吹打5次轻轻混匀,将离心管置于磁力架上3-5分钟。当离心管仍置于磁力架中时,用移液枪小心吸出并丢弃含有CD3阴性细胞的上清液。用1ml释放缓冲液中重悬磁珠结合的细胞,室温下于摇床上倾斜滚动孵育10分钟,注意不要延长时间。移液枪轻柔吹打细胞20次以上,尽量避免气泡,放置于磁力架上2分钟。将含有无珠的CD3阳性细胞上清液移至新的无菌15mL离心管中,再次放在磁铁上1分钟以去除残留磁珠。含有无珠的CD3阳性细胞上清液移至新的无菌15mL离心管中,加入2ml分离缓冲液,室温下离心,离心条件为400xg,10分钟。移除上清液,细胞沉淀为CD3阳性T细胞。
3.2、外周血CD3+T细胞的培养
使用美国Biolegend公司的抗CD3抗体在gibico公司的1640培养基(不含血清)中稀释为5μg/ml。300μl/孔(24孔板),37℃培养箱包被4小时。磁珠分选得到T细胞,将细胞以1*106/ml的密度继续加入下列细胞因子与抗体:抗CD3抗体(5ug/mL),美国Biolegend公司的抗CD28抗体(2.5ug/mL),美国Perprotech公司的IL-2(20ng/mL)。洗去包被的孔板中剩余的抗CD3抗体的液体,每孔加入1ml的细胞悬液。48-72小时后进行细胞计数,如长的密度比较高,则稀释为1*106/ml的密度加入提前包好抗CD3抗体的孔中,补加抗CD3抗体(5ug/mL),抗CD28抗体(2.5ug/mL),IL-2(20ng/mL)刺激继续培养2-3天。达到足量细胞后进行后续实验。
3.3、蛋白质印迹分析
使用北京碧云天公司的RIPAbuffer和美国Sigma公司的蛋白酶抑制剂在冰上裂解细胞。使用BCA蛋白测定试剂盒根据制造商的说明书测定裂解产物的蛋白浓度。细胞裂解物在95℃用1X蛋白上样缓冲剂煮沸5min,通过12%的SDS-PAGE胶进行蛋白分离,然后转移到NC膜。用美国Abcam公司的抗GPX4抗体、抗TFRC抗体和抗β-actin抗体在4℃孵育过夜。用美国Abcam公司抗兔或小鼠IgG-HRP孵育1h后,用蛋白质印迹检测系统Image lab(Bio-Rad,美国)观察蛋白条带。
3.4、流式细胞分析
使用以下抗体对细胞进行染色:PerCP/Cyanine5.5 anti-human CD3抗体,PE/Cyanine7 anti-human CD8抗体,APC/FireTM750anti-human CD4抗体,均购自美国BioLegend公司。V500-C抗人CD45抗体购自BD Biosciences(Franklin Lakes,NJ,USA)。
对200μL全血用BD公司的溶红素对红细胞裂解后剩余的细胞进行分析。根据制造商的说明,配置抗体Mix对细胞进行膜标记染色(在室温下避光15分钟)。再加入固定液100ul,避光室温孵育15分钟。使用瑞士Coulter公司的胞浆抗原染色对细胞膜固定15分钟后破膜加入GPX4抗体1ul,避光室温孵育15分钟。再加入Biolegend公司的PE驴抗兔抗体5ul,室温避光孵育15分钟。PBS溶液洗涤重悬,上机分析。使用Canto II流式细胞仪(BDBiosciences,Franklin Lakes,NJ,USA)处理样本。结果使用Kaluza软件(BeckmanCoulter,california,USA)进行分析。
3.5血浆丙二醛(MDA)水平的检测
使用美国Abcam公司生产的MDA定量ELISA试剂盒检测患者及健康对照血浆MDA水平,具体操作方案参照说明书。
3.6Liperfluo活细胞胞内脂质荧光染色
将分离得到的T细胞用HBSS溶液重悬,密度为1*106/mL。加入DOJINDO公司的Liperfluo溶液,浓度为7umol/L,在37℃5%的CO2培养箱中培养1h。用激光共焦荧光显微镜观察(激发波长:488nm,发射波长:500-550nm)。
3.7FerroOrange活细胞胞内Fe2+荧光染色
将分离得到的T细胞用HBSS溶液重悬,密度为1*106/mL。加入DOJINDO公司的FerroOrange溶液,浓度为3umol/L,在37℃5%的CO2培养箱中培养30min。用荧光显微镜观察(激发波长:561nm,发射波长:570-620nm)细胞荧光染色。
3.8.透射电镜制备
收集分离得到的T细胞沉淀,体积约1-2mm3左右,加入2.5%戊二醛,于4度保存。锇酸固定:将用戊二醛固定的T细胞沉淀,经0.1M磷酸缓冲液(PH7.4)漂洗3次,每次15分钟用1%的锇酸*0.1M磷酸缓冲液(PH7.4)室温(20℃)固定2小时(固定时间因样品不同而适当调整);然后再经0.1M磷酸缓冲液(PH7.2)漂洗3次,每次15分钟。脱水:组织经30%,50%,70%,80%,85%,90%,100%(两次)酒精梯度上行脱水,每次15-20分钟。渗透:渗透剂依次为丙酮:环氧树脂(2:1),丙酮:环氧树脂(1:1),环氧树脂,37度温箱内,每次渗透8-12小时。包埋:将渗透好的样品放入包埋板中,加入包埋剂环氧树脂,在60度温箱中聚合48小时。切片:超薄切片机切片80-100nm。双染色:铀铅双染色(2%醋酸铀饱和水溶液,枸橼酸铅,室温染色15min切片室温干燥过夜,电镜观察。
3.9.RSL-3刺激人外周血T细胞
细胞增殖48h-72h后,取出细胞,设置空白对照组及不同浓度RSL3组(WB检测:浓度2,4,6,8umol/L;细胞活力测定:浓度0.1,0.3,0.5umol/L)。每组细胞均设置副孔。加入相应浓度的RSL3刺激剂。将培养皿转移至培养箱中,条件为37℃,5%CO2,培养相应时间(WB检测:8小时;细胞活力测定:12小时)后进行检测。
3.10T细胞活力测定
使用Promega公司的化学发光细胞活性检测试剂盒测定刺激后T细胞的活力。
4、统计分析
分类变量用计数和百分比表示,连续变量用平均值±SD或中位数(四分位距[IQR])表示。两组间比较采用Mann-Whitney U检验(非正态分布)或非配对t检验(正态分布)。多组间比较采用Kruskal-Waills H检验。采用错误发现率(false discovery rate,FDR)校正方法解决多组比较的问题。对于分类变量,数据比较采用卡方检验或Fisher精确检验。采用SPSS 26.0,GraphPad Prism8.0及Image J进行数据处理。P<0.05为差异有统计学意义。
实施例1、抗MDA5抗体阳性DM病人GPX4蛋白表达水平降低
1、抗MDA5抗体阳性DM病人GPX4蛋白水平表达降低
GPX4的功能主要为去除脂质过氧化物,是细胞铁死亡的关键负调节因子[16]。通过蛋白质印迹分析,我们发现GPX4在抗MDA5阳性皮肌炎合并T细胞减少的患者组相较T细胞 正常组、其他炎性肌病组以及健康对照组表达显著降低(P<0.05)(图1A和1B)。通过ROC曲线计算GPX4表达水平对抗MDA5阳性皮肌炎合并T细胞减少患者与对照组的区别能力,发现选择cut-off为0.785时(数值为GPX4相对表达量,指GPX4相对于GAPDH的表达量的比值),能够显著区分抗MDA5阳性皮肌炎合并T细胞减少患者与健康对照及其它皮肌炎患者(图1C,P<0.001),敏感性为72.73%,特异性为88.00%。在抗MDA5阳性DM患者中进一步分析GPX4表达量与患者外周CD3+T细胞数目的关系发现,GPX4表达量与患者外周CD3+T细胞数目呈现正相关性(图1D)。
我们进一步通过流式细胞术验证DM患者GPX4表达情况(图2A),发现抗MDA5阳性DM合并T细胞减少患者CD3+T细胞的GPX4平均荧光强度(MFI)较抗MDA5阳性DM且T细胞正常组、其他炎性肌病组以及健康对照组表达显著降低(图2B)。进一步将CD3+T细胞分为CD4和CD8T细胞,分析发现抗MDA5阳性DM合并T细胞减少患者CD4+和CD8+T细胞的GPX4平均荧光强度均显著降低(图2C和2D)。
另外,抗MDA5抗体阳性DM患者外周血T细胞数目与相应的GPX4平均荧光强度呈正相关(图2E,F和G),说明GPX4的定量差异,不是由细胞数的变化导致。
2、抗MDA5阳性DM患者T细胞存在铁死亡形态学改变
进一步通过透射电镜(TEM)观察抗MDA5阳性DM患者T细胞形态并进行超微结构分析,发现抗MDA5阳性DM合并T细胞减少患者的T细胞表现出典型的铁死亡形态学特征,包括线粒体空泡形成,线粒体膜密度增加,线粒体嵴消失(图3A)。
与健康人T细胞内平均线粒体面积相比,抗MDA5抗体阳性DM合并T细胞减少的患者线粒体面积明显变小(图3B)。
3、抗MDA5阳性DM患者T细胞脂质代谢异常
通过细胞荧光染色检测抗MDA5阳性DM患者的T细胞脂质过氧化物水平,发现抗MDA5阳性DM合并T细胞减少的患者脂质过氧化物水平相较于健康人显著升高(图4A和B)。
丙二醛(MDA)是细胞膜中的脂质过氧化后的产物,与铁死亡发生密切相关[17]。我们通过ELISA检测抗MDA5阳性DM患者与健康对照的MDA水平,发现相较于健康对照组及T细胞正常的抗MDA5阳性DM患者,抗MDA5阳性DM合并T细胞减少患者的血浆MDA水平明显升高(P<0.05,图4C)。
4.抗MDA5阳性DM患者T细胞铁代谢异常
铁死亡过程中,转铁蛋白通过转铁蛋白受体(TFRC)介导铁离子摄取,TFRC表达升高可以提高铁摄取增加细胞内铁水平,促进铁死亡[18]。
通过蛋白印迹分析发现抗MDA5阳性DM合并T细胞减少患者,T细胞TFRC水平显著高于健康对照(图5A和B)。另外,通过细胞荧光染色分析T细胞内Fe2+水平,发现抗MDA5阳性DM合并T细胞减少患者,T细胞内Fe2+水平显著高于健康对照(图5C和D)。
实施例2、抗MDA5阳性DM患者T细胞GPX4低表达与真菌感染有关
以15例健康对照样本GPX4相对表达水平的95%置信区间(非正态分布)作为cut-off值(cutoff value:0.74,数值为GPX4相对表达量,指GPX4相对于GAPDH的表达量的比值),将抗MDA5阳性DM患者分为两组:一组为T细胞GPX4低表达组(GPX4相对表达量<0.74),另一组为GPX4水平正常组(GPX4相对表达量>0.74)。
我们发现,与GPX4水平正常组相比,T细胞GPX4低表达患者被真菌感染几率显著升高(30.8%VS 68.8%,P<0.05)(表1)。而在细菌感染与病毒感染方面,T细胞GPX4低表达患者感染比例无统计学差异(表1)。此外,T细胞GPX4低表达患者血沉水平更高(P<0.05,表1)。
表1.抗MDA5阳性DM患者T细胞GPX4低表达和正常水平组的特征比较
以15例健康对照样本95%可信区间(非正态分布)计算Cutoff值,
Cutoff值为0.74(cutoff值为GPX4相对表达量,指GPX4相对于GAPDH的表达量的比值)。
最后需要说明的是,以上实施例,仅用于帮助本领域技术人员理解本发明的实质,不用于限定本发明的保护范围。
Claims (5)
1.GPX4(glutathione peroxidase 4,GPX4)蛋白在制备检测试剂盒中的应用,所述的检测试剂盒用于:
(1)检测抗MDA5(抗黑色素瘤分化相关基因5)抗体阳性皮肌炎(DM);或
(2)预测抗MDA5抗体阳性皮肌炎(DM)患者真菌感染的风险;
所述抗MDA5抗体阳性DM是一类DM亚型,以高发间质性肺病(interstitial lungdisease,ILD)、低淋巴细胞血症、高死亡率为特征。
2.根据权利要求1所述的应用,其特征在于,所述的GPX4蛋白为患者外周血中的GPX4蛋白,优选为外周血CD3+T细胞中的GPX4蛋白。
3.根据权利要求1或2所述的应用,其特征在于,所述的预测抗MDA5抗体阳性皮肌炎(DM)患者真菌感染的风险指,将抗MDA5抗体阳性皮肌炎(DM)患者区分为真菌感染高风险组和低风险组。
4.根据权利要求3所述的应用,其特征在于,当GPX4水平降低时,
(1)所述DM患者为抗MDA5抗体阳性DM患者;或
(2)抗MDA5抗体阳性DM患者真菌感染的风险高。
5.根据权利要求3所述的应用,其特征在于,
(1)当外周血CD3+T细胞中的GPX4蛋白量低于0.785时(数值为GPX4相对表达量,指GPX4相对于GAPDH的表达量的比值)时,该患者为抗MDA5抗体阳性DM患者;或
(2)当外周血CD3+T细胞中的GPX4蛋白量低于0.74时(数值为GPX4相对表达量,指GPX4相对于GAPDH的表达量的比值),该患者真菌感染风险高。
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