Quantitative Analysis of Mitochondrial Damage in T Lymphocytes from Patients with Autoimmune Diseases and Evaluation of Its Clinical Value
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摘要:目的 评估自身免疫性疾病(autoimmune disease,AID)患者外周血T淋巴细胞线粒体损伤情况,以期为AID病因学研究提供思路。方法 回顾性收集2023年3—4月北京协和医院诊治的AID患者及同期体检健康人群的临床资料,并依据外周血辅助性T淋巴细胞(T helper cell, Th)百分比/杀伤性T淋巴细胞(T cytotoxic cell, Tc)百分比比值,将AID患者分为免疫功能低下亚组和免疫功能正常亚组。采用流式细胞术评估AID患者T淋巴细胞线粒体损伤情况,以线粒体膜电位偏低细胞百分比(mitochondrial membrane potential low percentage,MMP-low%)表示线粒体功能障碍程度并分析其与AID的相关性。结果 共入选符合纳入与排除标准的AID患者70例,健康人群20名。AID患者中,免疫功能低下者20例(Th/Tc比值<0.70),免疫功能正常者50例(Th/Tc比值≥0.70);系统性红斑狼疮(systemic lupus erythematosus,SLE)患者33例,类风湿关节炎(rheumatoid arthritis,RA)患者19例,干燥综合征(Sjögren syndrome,SS)患者18例。SLE、RA、SS患者CD3+T淋巴细胞线粒体膜电位偏低细胞百分比(T MMP-low%)、CD3+CD4+T淋巴细胞线粒体膜电位偏低细胞百分比(Th MMP-low%)、CD3+CD8+T淋巴细胞线粒体膜电位偏低细胞百分比(Tc MMP-low%)均低于健康人群(P均<0.05)。AID患者中,免疫功能低下亚组和免疫功能正常亚组T MMP-low%、Th MMP-low%、Tc MMP-low%均低于健康人群(P均<0.05);相较于免疫功能正常亚组,免疫功能低下亚组T MMP-low%、Th MMP-low%、Tc MMP-low%均呈降低趋势,但差异无统计学意义(P均>0.05)。Spearman相关性分析显示,线粒体损伤指标中,仅发现Th MMP-low%/Tc MMP-low%比值与AID患者免疫功能(Th/Tc比值)具有相关性(r=-0.39,P=0.001)。受试者工作特征曲线显示,T MMP-low%、Tc MMP-low%、Th MMP-low%在AID的识别中均具有良好效能,曲线下面积分别为0.83(95% CI: 0.74~0.92)、0.82(95% CI: 0.73~0.92)、0.77(95% CI: 0.67~0.88)。结论 AID患者存在一定程度的外周血T淋巴细胞线粒体损伤, 尤其在免疫功能低下者中此现象更显著。T淋巴细胞线粒体损伤相关指标或可作为AID辅助诊断的分子标志物。Abstract:Objective To evaluate the mitochondrial damage of peripheral blood T lymphocytes in patients with autoimmune diseases (AID) and provide insights for etiological research.Methods Clinical data were retrospectively collected from the AID patients treated at the Peking Union Medical College Hospital from March 2023 to April 2023 and from a population that was physically healthy during the same period. Based on the ratio of peripheral blood helper T cells (Th) to cytotoxic T cells (Tc), the AID patients were divided into an immunodeficiency subgroup and an immunocompetent subgroup. Flow cytometry was used to assess the mitochondrial damage of T lymphocytes in the AID patients, with the percentage of cells showing low mitochondrial membrane potential (MMP-low%) as an indicator of mitochondrial dysfunction, and its correlation with AID was analyzed.Results A total of 70 AID patients and 20 healthy individuals who met the inclusion and exclusion criteria were included. Among the AID patients, there were 20 immunodeficient cases (Th/Tc ratio < 0.70) and 50 immunocompetent cases (Th/Tc ratio ≥0.70); 33 patients had systemic lupus erythematosus (SLE), 19 had rheumatoid arthritis (RA), and 18 had Sjögren syndrome (SS). The percentage of CD3+ T lymphocytes showing low mitochondrial membrane potential (T MMP-low%), CD3+CD4+ T lymphocytes showing low mitochondrial membrane potential (Th MMP-low%), and CD3+CD8+ T lymphocytes showing low mitochondrial membrane potential (Tc MMP-low%) in SLE, RA, and SS patients were all lower than those in healthy individuals (all P < 0.05). In the AID patients, the percentages of T MMP-low%, Th MMP-low%, and Tc MMP-low% in both the immunodeficient subgroup and immunocompetent subgroup were lower than those in healthy individuals (P < 0.05). Compared to the immunocompetent subgroup, the immunodeficient subgroup showed a decreasing trend in the percentages of T MMP-low%, Th MMP-low%, and Tc MMP-low%, but the differences were not statistically significant (all P > 0.05). Spearman correlation analysis showed that among the mitochondrial damage indicators, only the Th MMP-low%/Tc MMP-low% ratio was correlated with the immune function (Th/Tc ratio) of the AID patients (r=-0.39, P=0.001). The receiver operating characteristic curve showed that Tc MMP-low%, Tc MMP-low%, and Th MMP-low% all had good performance in identifying AID, with respective areas under the curve of 0.83(95% CI: 0.74-0.92), 0.82(95% CI: 0.73-0.92), and 0.77(95% CI: 0.67-0.88), respectively.Conclusions Peripheral blood T lymphocytes in AID patients have varying degrees of mitochondrial damage, especially in immunodeficient individuals. Mitochondrial damage-related indicators of T lymphocytes may serve as molecular markers for auxiliary diagnosis of AID.
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Keywords:
- autoimmune disease /
- T lymphocytes /
- mitochondrial damage /
- immunocompromised
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尿路上皮癌是最常见的恶性肿瘤之一,可发生于肾盂、输尿管、膀胱和尿道,其中以膀胱尿路上皮癌最为常见。膀胱尿路上皮癌常用的诊断方法包括尿液细胞学检查、膀胱镜检查,但尿液细胞学检查的灵敏度较低,膀胱镜检查为有创性操作,因此,亟需探寻灵敏度、特异度均较高的分子标志物用于膀胱尿路上皮癌的诊断。胰岛素样生长因子2(insulin-like growth factor 2, IGF2)是一种包含67个氨基酸的肽类激素,在多种恶性肿瘤组织中呈过表达[1]。外泌体是细胞主动分泌的一类穿梭于细胞间的细胞外囊泡,其中富含各种蛋白质、脂质、核酸等物质,在细胞间传递信号等方面发挥着重要作用[2]。本研究通过荧光定量反转录PCR法检测尿液外泌体中IGF2表达水平,探索其在膀胱尿路上皮癌诊断中的价值。
1. 对象与方法
1.1 研究对象
前瞻性选取2020年9月至12月北京协和医院诊治的膀胱尿路上皮癌患者和非尿路上皮癌患者为研究对象。
膀胱尿路上皮癌患者纳入标准:(1)具有完整的病历资料;(2)经膀胱镜检查或经尿道膀胱肿瘤电切术(transurethral resection of bladder tumor, TURBT)病理确诊为膀胱尿路上皮癌;(3)近期未行泌尿生殖系统操作或手术。排除标准:(1)有其他系统恶性肿瘤史;(2)近期曾行放化疗或者免疫抑制剂治疗者。
非尿路上皮癌患者纳入标准:(1)与膀胱尿路上皮癌患者同期入院;(2)通过CT/MRI等检查诊断为泌尿系结石、泌尿系感染、前列腺增生、肾上腺良性肿瘤等疾病;(3)临床资料完整。排除标准:(1)有其他系统恶性肿瘤史;(2)近期行泌尿生殖系统操作或手术者。
本研究已通过北京协和医院伦理委员会审批(审批号:JS-3178),患者均签署知情同意书。
1.2 方法
1.2.1 样本量估算
依据公式n=Uα2P(1-P)/δ2估算样本量,其中P分别为估计的IGF2诊断膀胱尿路上皮癌的灵敏度(用于膀胱尿路上皮癌组样本量估算,取0.90)和估计的IGF2诊断膀胱尿路上皮癌的特异度(用于非尿路上皮癌组样本量估算,取0.80);检验水准α=0.05,Uα为正态分布中累积概率为α/2时的U值(U0.05=1.96)。经计算膀胱尿路上皮癌患者样本量为35例,非尿路上皮癌患者为61例。
1.2.2 尿液采集和外泌体提取
患者入院后均留取新鲜晨起清洁中段尿。取尿液标本(约5 mL),置于无菌无酶离心管中,采用外泌体提取试剂盒(广州恒泰生物科技有限公司产品,粤穗械备20202151号)提取尿液中的外泌体,具体操作步骤依据试剂盒说明书。
1.2.3 核酸提取和反转录
取外泌体沉淀物,加入Trizol 1 mL,15~30 ℃放置5 min,4 ℃, 离心半径8 cm,2000 r/min离心10 min保留上清液。加入200 μL氯仿,剧烈震荡15 s,室温放置3 min,4 ℃, 离心半径8 cm,12 000 r/min离心10 min后样本分成3层:黄色的有机相、中间层和无色的水相。将水相转移至新离心管,加入等体积异丙醇,混匀,15~30 ℃静置10 min;4 ℃, 离心半径8 cm,12 000 r/min离心5~10 min;去上清液,缓慢沿离心管管壁加入75% 乙醇1 mL,轻轻上下颠倒洗涤离心管;4 ℃, 离心半径8 cm,12 000 r/min离心2 min后弃乙醇;重复洗涤一次,获取RNA沉淀,室温干燥2~5 min,加入适量DEPC水以溶解RNA沉淀,必要时可用移液器轻轻吹打沉淀,待沉淀完全溶解后立刻进行反转录。反转录体系:1 pg~1 μg规格的RNA 6 μL,gDNA去除液2 μL,移液器吹打混匀,42 ℃保存2 min;向PCR反应管中加入反转录反应液2 μL,移液器吹打混匀后进行反转录,反转录条件:50 ℃ 15 min,85 ℃ 5 s,4 ℃ 2 min。
1.2.4 PCR检测
从试剂盒中取出PCR反应液、ROX、引物探针混合物、阳性质控品、阴性质控品,于冰上或4 ℃融化后震荡混匀, 离心半径8 cm,8000 r/min瞬时离心后备用。计算待测样本数(n),取n+2个(包含1个阳性质控品和1个阴性质控品)PCR反应管,单份扩增体系总体积为26 μL,其中15 μL PCR反应液1,11 μL PCR反应液2。加入4 μL待测样本反转录产物,离心半径8 cm,8000 r/min瞬时离心后进行PCR扩增。参数设置:窗口设置:Reporter Dye1:FAM,Quencher Dye1:none,Reporter Dye2:CY5,Quencher Dye2:none,Passive Reference:ROX。循环条件:37 ℃ 2 min,1个循环;95 ℃ 8 min,1个循环;95 ℃ 10 s,60 ℃ 34 s,45个循环。分别随机选取4例膀胱尿路上皮癌患者和4例非尿路上皮癌患者的PCR产物,行琼脂糖凝胶电泳(图 1)。
1.2.5 结果分析
收集PCR产物,使用配套仪器自动调整基线和阈值,基线Start值为3~15,End值为5~20。在log图谱中调整阈值处于扩增曲线指数期,调整完毕后获取待测样本数据。若CY5通道Ct值≤35且FAM通道Ct值≤36,判定IGF2阳性;若CY5通道Ct值≤35且FAM通道Ct值>36,判断定IGF2阴性;若CY5通道Ct值>35,则样本的RNA浓度达不到最低检测限要求,需重新检测。
1.3 偏倚控制
本研究入选的膀胱尿路上皮癌患者均由膀胱镜检查或TURBT病理确诊,非尿路上皮癌患者均为非恶性肿瘤疾病,以避免其他类型恶性肿瘤(如肾癌、前列腺癌等)对研究结果的影响。
1.4 统计学处理
采用SPSS 26.0软件进行统计学分析。年龄符合正态分布,以均数±标准差表示。IGF2阳性表达率为计数资料,以频数(百分数)表示。以膀胱镜检查或TURBT病理结果为金标准,计算尿液外泌体中IGF2诊断膀胱尿路上皮癌的灵敏度、特异度、阳性似然比与阴性似然比。其中灵敏度=真阳性例数/(真阳性例数+假阴性例数)×100%;特异度=真阴性例数/(真阴性例数+假阳性例数)×100%;阳性似然比=灵敏度/(1-特异度);阴性似然比=(1-灵敏度)/特异度。以P<0.05为差异具有统计学意义。
2. 结果
2.1 一般临床资料
共入选符合纳入和排除标准的膀胱尿路上皮癌患者35例,非尿路上皮癌患者60例(肾上腺疾病患者24例,良性前列腺增生症患者22例,泌尿系结石患者14例)。膀胱尿路上皮癌患者中,男性26例,女性9例;平均年龄(65.43±12.22)岁(范围:45~86岁)。非尿路上皮癌患者中,男性35例,女性25例;平均年龄(61.68±13.07)岁(范围:12~82岁),两组患者年龄、性别比例无统计学差异(P均>0.05)。
2.2 尿液外泌体中IGF2阳性表达率比较
膀胱尿路上皮癌患者IGF2阳性表达25例(71.4%),阴性表达10例(28.6%);非尿路上皮癌患者IGF2阳性表达6例(10.0%),阴性表达54例(90.0%)。膀胱尿路上皮癌患者尿液外泌体中IGF2阳性表达率显著高于非尿路上皮癌患者(P=0.000)。
2.3 尿液外泌体中IGF2诊断膀胱尿路上皮癌的性能
尿液外泌体中IGF2诊断膀胱尿路上皮癌的灵敏度为71.43%(95% CI:53.48%~84.76%),特异度为90.00%(95% CI:78.83%~95.87%),阳性似然比为7.14(95% CI:3.25~15.70),阴性似然比为0.32(95% CI:0.19~0.54)。
3. 讨论
本研究对尿液外泌体中IGF2与膀胱尿路上皮癌的相关性进行了初步分析,结果表明IGF2在膀胱尿路上皮癌中呈明显高表达(阳性率:71.4%),其诊断膀胱尿路上皮癌的灵敏度为71.43%(95% CI:53.48%~84.76%),特异度为90.00%(95% CI:78.83%~ 95.87%)。
全球每年约550 000例新发尿路上皮癌病例[3],其中膀胱尿路上皮癌占比90%以上。80%的膀胱尿路上皮癌确诊时为非肌层浸润性膀胱癌(non-muscle invasive bladder cancer, NMIBC),TURBT术切除后复发率高达50%,其中30%进展为肌层浸润性膀胱癌(muscle invasive bladder cancer, MIBC)[4]。因此,早期诊断对改善膀胱尿路上皮癌患者预后至关重要。虽然常规尿液细胞学检查对膀胱尿路上皮癌具有较高的诊断特异度(88.1%),但总体灵敏度极低(32%),其中对高级别膀胱尿路上皮癌的诊断灵敏度为50.6%,对低级别膀胱尿路上皮癌的诊断灵敏度仅为10.3%[5]。
有研究分析了NMP22、CD44、CK20、CEACAM1和BTA 5种尿液肿瘤标志物诊断膀胱尿路上皮癌的临床价值,结果表明NMP22的灵敏度最高(85.1%),但特异度仅为54.7%;虽然CD44的灵敏度(83.2%)和特异度(84.4%)均可[6],但尚需临床进一步验证。本次研究旨在探索一种新的无创标志物作为膀胱尿路上皮癌筛查、诊断和随访的替代方案。
外泌体是由不同类型细胞(包括肿瘤细胞)主动分泌直径为50~150 nm的双分子层结构囊泡样小体[7],在细胞间发挥通讯作用,特别是在肿瘤发展过程中的作用,已得到临床认可。外泌体相关的RNA、微RNA、蛋白质、DNA,甚至代谢物均可通过自分泌和旁分泌方式作用于受体细胞。在血液、尿液、唾液和脑脊液等体液中均可检测到外泌体,是肿瘤辅助诊断理想的非侵袭性或侵袭性生物标志物[8]。
人类IGF2基因定位于染色体11p15,包括9个外显子和4个启动子,是一种有效的促分裂原和凋亡抑制剂,可抑制细胞凋亡,促进细胞周期进展、血管生成,对细胞生长和存活至关重要。IGF2在包括乳腺癌[9]、卵巢癌[10]、结直肠癌[11]和前列腺癌[12]等在内的不同恶性肿瘤组织均呈过表达。早期研究表明,膀胱尿路上皮癌患者尿液中IGF2水平明显升高,证实IGF2是膀胱尿路上皮癌诊断的一种有前景的标志物[13]。一项前瞻性多中心队列研究对4种尿液分子标志物诊断膀胱膀胱癌的性能进行了测试,结果显示IGF2是诊断膀胱尿路上皮癌的核心基因之一[14]。但目前尚缺乏其单独作为标志物诊断膀胱尿路上皮癌临床价值的相关研究。
本研究初步探索了尿液外泌体中IGF2诊断膀胱尿路上皮癌的临床效能,结果表明相较于非尿路上皮癌患者,膀胱尿路上皮癌患者尿液外泌体中IGF2阳性表达率显著升高(71.4%比10.0%),IGF2诊断膀胱尿路上皮癌的灵敏度为71.43%,特异度为90.00%,相比传统尿液细胞学检测,其灵敏度和特异度均有不同程度的提高[5];与肿瘤标志物CD44比较,特异度更高,但灵敏度稍低[6]。
本研究局限性:对照人群仅为非尿路上皮癌患者。后期可增加病例收集范围,比较膀胱尿路上皮癌与泌尿系统其他恶性肿瘤患者尿液外泌体中IGF2表达差异,以进一步分析IGF2在膀胱尿路上皮癌鉴别诊断中的作用。
综上,膀胱尿路上皮癌患者尿液外泌体中IGF2呈高表达,其对膀胱尿路上皮癌具有较高的诊断特异度,但灵敏度稍低。由于尿液样本收集简便、易行,尿液外泌体中IGF2有望作为分子标志物辅助膀胱尿路上皮癌的诊断。
作者贡献:吴子燕负责论文撰写;李昊隆负责实验研究;奉福泰、徐洪琳负责数据分析;张蜀澜、李永哲负责研究设计。利益冲突:所有作者均声明不存在利益冲突 -
图 1 AID患者与健康人群CD3+T淋巴细胞、CD3+CD4+T淋巴细胞、CD3+CD8+T淋巴细胞线粒体质量比较
T MMP-low%、Th MMP-low%、Tc MMP-low%、AID、SLE、RA、SS:同表 1
图 2 AID患者Th MMP-low%/Tc MMP-low%比值与Th/Tc比值的相关性
Th MMP-low%、Tc MMP-low%、AID:同表 1;Th/Tc:Th百分比与Tc百分比的比值
表 1 AID患者与健康人群外周血T淋巴细胞及其亚群线粒体质量比较[M(P25, P75), %]
组别 T MMP-low% Th MMP-low% Tc MMP-low% SLE患者(n=33) 20.91(11.96,28.17)* 14.52(10.18,18.75)* 22.83(13.49,32.95)* RA患者(n=19) 25.51(21.59,36.07)*# 18.20(12.31,27.46)* 35.64(24.12,41.97)*# SS患者(n=18) 19.92(11.09,34.50)* 19.09(4.78,30.85)* 26.56(11.99,43.09)* 健康人群(n=20) 38.24(31.35,48.54) 25.05(23.47,33.32) 48.41(35.59,65.41) P值 <0.0001 0.0006 <0.0001 AID:自身免疫性疾病;SLE:系统性红斑狼疮;RA:类风湿关节炎;SS:干燥综合征;T MMP-low%:CD3+T淋巴细胞线粒体膜电位偏低细胞百分比;Th MMP-low%:CD3+CD4+T淋巴细胞线粒体膜电位偏低细胞百分比;Tc MMP-low%:CD3+CD8+T淋巴细胞线粒体膜电位偏低细胞百分比;与健康人群比较,* P<0.05;与SLE患者比较,# P<0.05 表 2 不同免疫状态的AID患者与健康人群T淋巴细胞及其亚群线粒体质量比较[M(P25, P75)]
组别 T MMP-low%(%) Th MMP-low%(%) Tc MMP-low%(%) Th MMP-low%/Tc MMP-low%比值 AID组(n=70) 免疫功能低下亚组(n=20) 17.15 (10.71,31.16)* 15.36 (10.62,25.46)* 18.07 (10.99,35.85)* 0.86(0.59,1.27) 免疫功能正常亚组(n=50) 23.88 (17.16,32.52)* 16.27 (10.30,22.67)* 30.61 (18.15,38.85)* 0.56(0.38,0.85) 健康人群(n=20) 38.24 (31.35,48.54) 25.05(23.47,33.32) 48.41(35.59,65.41) 0.60(0.43,0.72) P值 0.0002 0.005 0.0001 0.056 T MMP-low%、Th MMP-low%、Tc MMP-low%、AID:同表 1;与健康人群比较,* P<0.05 表 3 T MMP-low%、Tc MMP-low%、Th MMP-low% 识别AID的临床价值
诊断指标 T MMP-low% Th MMP-low% Tc MMP-low% AUC 0.83 0.77 0.82 最佳临界值(%) 24.07 21.95 42.57 灵敏度(%) 100 85.00 70.00 特异度(%) 57.14 72.86 82.86 阴性预测值(%) 100 94.44 90.63 阳性预测值(%) 40.00 47.22 53.85 假阳性率(%) 42.86 27.14 17.14 假阴性率(%) 0 15.00 30.00 准确度(%) 66.67 75.56 80.00 Youden指数 0.57 0.58 0.53 AUC:曲线下面积;T MMP-low%、Th MMP-low%、Tc MMP-low%、AID:同表 1 -
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