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Article(id=1194344009238802543, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1194344006382486063, articleNumber=1001-2494(2025)10-1019-07, orderNo=null, doi=10.11669/cpj.2025.10.003, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1738944000000, receivedDateStr=2025-02-08, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1762683401640, onlineDateStr=2025-11-09, pubDate=1746028800000, pubDateStr=2025-05-01, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1762683401640, onlineIssueDateStr=2025-11-09, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1762683401640, creator=13701087609, updateTime=1762683401640, updator=13701087609, issue=Issue{id=1194344006382486063, tenantId=1146029695717560320, journalId=1190317699101192196, year='2025', volume='60', issue='10', pageStart='1005', pageEnd='1102', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=1, specialIssue=0, createTime=1762683400960, creator=13701087609, updateTime=1762844794786, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1195020941253128793, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1194344006382486063, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1195020941253128794, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1194344006382486063, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=1019, endPage=1025, ext={EN=ArticleExt(id=1194344009469489266, articleId=1194344009238802543, tenantId=1146029695717560320, journalId=1190317699101192196, language=EN, title=Expansion of Tuberculosis Pleural Effusion Mononuclear Cells in Vitro for Quality Control of Diagnostic Tests, columnId=null, journalTitle=Chinese Pharmaceutical Journal, columnName=null, runingTitle=null, highlight=null, articleAbstract=

OBJECTIVE To explore the feasibility of preparing samples for the quality control of tuberculosis interferon-gamma (IFN-γ) release assays (TB-IGRA) using in vitro expanded tuberculosis pleural fluid mononuclear cells (TB-PFMC). METHODS The cryopreserved TB-PFMC were stimulated and expanded in vitro using three expansion protocols based on CD3/CD28 antibody combined with IL-2, and the status of TB-PFMC was observed and the expansion fold was calculated. The amplification results of three different expansion protocols were compared to determine the optimal protocol. The enzyme-linked immunospot assay (ELISPOT) and enzyme-linked immunosorbent assay (ELISA)TB-IGRA kits were used to detect TB-PFMC before and after the expansion, and the changes of the results were analyzed. RESULTS Protocol 3 was determined to be optimal and used to amplify TB-PFMC from three different patients, and their expansion folds reached 3 535.00 %, 2 800.00 %, and 3 959.00%, respectively, on the 9th day. The results of two TB-IGRA kits after the expansion of TB-PFMC remained positive and no obvious non-specific reactions were observed. CONCLUSION This study proves that TB-PFMC can be successfully amplified by CD3/CD28 antibody combined with IL-2 and the optimal protocol was determined, and the expanded TB-PFMC is suitable for the preparation of reference materials and applied for the quality control of TB-IGRA products.

, correspAuthors=Tingmei CHEN, Dawei SHI, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, authorCompany=null, fund=null, authors=null, authorsList=Deyun JIANG, Haiping DONG, Jianrong LOU, Sihong XU, Tingmei CHEN, Dawei SHI), CN=ArticleExt(id=1194344195814027594, articleId=1194344009238802543, tenantId=1146029695717560320, journalId=1190317699101192196, language=CN, title=体外扩增结核性胸腔积液单个核细胞用于诊断试剂质量控制的研究, columnId=1190352405612040510, journalTitle=中国药学杂志, columnName=论著, runingTitle=null, highlight=null, articleAbstract=

目的 探索采用体外扩增的结核性胸腔积液(胸水)单个核细胞(tuberculosis pleural fluid mononuclear cells, TB-PFMC)制备结核γ干扰素释放试验[tuberculosis interferon-gamma(IFN-γ)release assays,TB-IGRA]质量控制用样本的可行性。方法 使用3种CD3/CD28抗体联合重组人白细胞介素2(interleukin 2,IL-2)的扩增方案对TB-PFMC进行体外扩增,观察TB-PFMC的生长情况并计算扩增倍数。通过比较3种不同方案的扩增效果,确定最优的方案。使用酶联免疫斑点(ELISPOT)法和酶联免疫吸附(ELISA)法TB-IGRA试剂检测扩增前后TB-PFMC的细胞免疫反应,分析检测结果的变化。结果 方案三被确定为最优扩增方案并用于对3例结核患者的TB-PFMC进行体外扩增,其第9 天的扩增倍数分别达到3 535.00%、2 800.00%和 3 959.00%。扩增后细胞在2种TB-IGRA试剂盒的检测结果中仍保持阳性且未观察到明显的非特异性反应。结论 本研究成功使用CD3/CD28抗体联合IL-2扩增TB-PFMC并确定了最优扩增方案,扩增后的TB-PFMC对于不同的TB-IGRA试剂盒具有良好的适用性,可制备参考品物质,用于TB-IGRA试剂的质量控制。

, correspAuthors=陈婷梅, 石大伟, authorNote=null, correspAuthorsNote=
*陈婷梅,女,博士,教授 研究方向:人工智能辅助检验新技术、精准检验新技术研发 Tel:(023)68485184;
石大伟,男,博士,研究员 研究方向:传染病诊断试剂质量控制和评价 Tel:(010)67095450
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蒋德云,女,学士 研究方向:临床检验技术,传染病诊断试剂质量控制和评价;

董海平,女,博士,主任医师 研究方向:结核病的诊断和治疗。蒋德云和董海平为共同第一作者

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蒋德云,女,学士 研究方向:临床检验技术,传染病诊断试剂质量控制和评价;

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蒋德云,女,学士 研究方向:临床检验技术,传染病诊断试剂质量控制和评价;

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董海平,女,博士,主任医师 研究方向:结核病的诊断和治疗。蒋德云和董海平为共同第一作者

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All three amplification protocols had the same “initial activation” procedures, so the microscope photographs of 0 d and 5 d were identical.

, figureFileSmall=73V35XHnJNq6+68BBPOJ5A==, figureFileBig=Rl4nFBATsVHIEE24oDuJJw==, tableContent=null), ArticleFig(id=1194372278738710631, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1194344009238802543, language=CN, label=图1, caption=结核性胸腔积液(胸水)单个核细胞(TB-PFMC)(1号)在不同体外扩增方案中的细胞形态(40×)

3个扩增方案的0 d和5 d为相同的“初始激活”结果,因此图片相同。

, figureFileSmall=73V35XHnJNq6+68BBPOJ5A==, figureFileBig=Rl4nFBATsVHIEE24oDuJJw==, tableContent=null), ArticleFig(id=1194372278830985320, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1194344009238802543, language=EN, label=Fig.2, caption=ELISPOT results of TB-PFMC(No.1) amplified by different protocols/spots forming unit

N-blank control well (AIM-V medium); A-ESAT-6 antigen testing well; B-CFP 10 antigen test well; P-positive control well (phytohemagglutinin, PHA); All three amplification protocols had the same “initial activation” procedures, so the ELISPOT results of 0 d were identical.

, figureFileSmall=QtCX9gCn/2chieITfg3Xsw==, figureFileBig=gH9XWU7H0HzOSiy9LZZ6Nw==, tableContent=null), ArticleFig(id=1194372278885511273, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1194344009238802543, language=CN, label=图2, caption=采用不同方案扩增的TB-PFMC(1号)的酶联免疫斑点(ELISPOT)法检测结果/斑点数

N-空白对照孔(AIM-V培养基);A-ESAT-6抗原测试孔;B-CFP 10抗原测试孔;P-阳性对照孔(植物血凝素PHA);3个扩增方案的0 d为相同的“初始激活”结果,因此图片相同。

, figureFileSmall=QtCX9gCn/2chieITfg3Xsw==, figureFileBig=gH9XWU7H0HzOSiy9LZZ6Nw==, tableContent=null), ArticleFig(id=1194372278952620138, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1194344009238802543, language=EN, label=Tab.1, caption=

Cell viability and expansion fold of TB-PFMC(No.1) in different in vitro expansion protocols

, figureFileSmall=null, figureFileBig=null, tableContent=
Expansion
days/d		 Protocol 1 Protocol 2 Protocol 3
Cell viability/% Expansion fold Cell viability/% Expansion fold Cell viability/% Expansion fold
0 93.65 1.00 93.65 1.00 93.65 1.00
5 77.12 1.58 79.45 4.06 86.89 3.71
7 79.87 5.95 80.36 9.45 92.57 13.09
9 81.66 14.60 88.89 23.04 95.28 35.35
), ArticleFig(id=1194372279019729003, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1194344009238802543, language=CN, label=表1, caption=

TB-PFMC(1号)在不同体外扩增方案中的细胞活率和扩增倍数

, figureFileSmall=null, figureFileBig=null, tableContent=
Expansion
days/d		 Protocol 1 Protocol 2 Protocol 3
Cell viability/% Expansion fold Cell viability/% Expansion fold Cell viability/% Expansion fold
0 93.65 1.00 93.65 1.00 93.65 1.00
5 77.12 1.58 79.45 4.06 86.89 3.71
7 79.87 5.95 80.36 9.45 92.57 13.09
9 81.66 14.60 88.89 23.04 95.28 35.35
), ArticleFig(id=1194372279095226476, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1194344009238802543, language=EN, label=Tab.2, caption=

Cell viability and expansion fold of TB-PFMC from different patients on day 0 and day 9 of the optimal in vitro expansion

, figureFileSmall=null, figureFileBig=null, tableContent=
TB-PFMC
No.		 Day 0 Day 9
Cell viability/% Expansion fold Cell viability/% Expansion fold
1 93.65 1.00 95.28 35.35
2 93.15 1.00 97.22 28.00
3 89.33 1.00 98.17 39.59
), ArticleFig(id=1194372279162335341, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1194344009238802543, language=CN, label=表2, caption=

不同患者TB-PFMC在最优体外扩增方案下第0天和第9天的细胞活率及扩增倍数

, figureFileSmall=null, figureFileBig=null, tableContent=
TB-PFMC
No.		 Day 0 Day 9
Cell viability/% Expansion fold Cell viability/% Expansion fold
1 93.65 1.00 95.28 35.35
2 93.15 1.00 97.22 28.00
3 89.33 1.00 98.17 39.59
), ArticleFig(id=1194372279216861294, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1194344009238802543, language=EN, label=Tab.3, caption=

T-SPOT.TB kit results of TB-PFMC from different patients on day 0 and day 9 of the optimal in vitro expansion

, figureFileSmall=null, figureFileBig=null, tableContent=
TB-PFMC
No.		 Day 0(spots forming unit/50 000 cells) Day 9(spots forming unit/50 000 cells)
N A B P A/B ratio Result N A B P A/B ratio Result
1 0 6 32 34 0.2 positive 3 246 259 1212 0.9 positive
2 0 24 8 55 3.0 positive 1 183 36 1197 5.1 positive
3 0 24 59 37 0.4 positive 7 211 251 783 0.8 positive
), ArticleFig(id=1194372279288164463, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1194344009238802543, language=CN, label=表3, caption=

不同患者TB-PFMC在最优体外扩增方案下第0天和第9天的在结核γ干扰素(IFN-γ)释放试验T-SPOT.TB试剂盒检测结果

, figureFileSmall=null, figureFileBig=null, tableContent=
TB-PFMC
No.		 Day 0(spots forming unit/50 000 cells) Day 9(spots forming unit/50 000 cells)
N A B P A/B ratio Result N A B P A/B ratio Result
1 0 6 32 34 0.2 positive 3 246 259 1212 0.9 positive
2 0 24 8 55 3.0 positive 1 183 36 1197 5.1 positive
3 0 24 59 37 0.4 positive 7 211 251 783 0.8 positive
), ArticleFig(id=1194372279359467632, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1194344009238802543, language=EN, label=Tab.4, caption=

The QFT plus kit results of TB-PFMC from different patiens days 0 and days 9 of the optimal in vitro expansion

, figureFileSmall=null, figureFileBig=null, tableContent=
TB-PFMC
No.		 Day 0 Result Day 9 Result
IFN-γ concentration/IU·mL-1 IFN-γ concentration/IU·mL-1
Nil TB1 TB2 Mitogen Nil TB1 TB2 Mitogen
1 0.03 0.76 0.55 0.87 positive 0.36 12.56 13.68 16.07 positive
2 0.06 1.67 1.01 1.08 positive 0.28 11.59 10.51 15.45 positive
3 0.07 3.97 4.42 4.46 positive 0.62 14.31 16.02 17.50 positive
), ArticleFig(id=1194372279439159409, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1194344009238802543, language=CN, label=表4, caption=

不同患者的TB-PFMC在最优体外扩增方案下第0天和第9天的结核IFN-γ释放试验QFT-plus试剂盒试剂检测结果

, figureFileSmall=null, figureFileBig=null, tableContent=
TB-PFMC
No.		 Day 0 Result Day 9 Result
IFN-γ concentration/IU·mL-1 IFN-γ concentration/IU·mL-1
Nil TB1 TB2 Mitogen Nil TB1 TB2 Mitogen
1 0.03 0.76 0.55 0.87 positive 0.36 12.56 13.68 16.07 positive
2 0.06 1.67 1.01 1.08 positive 0.28 11.59 10.51 15.45 positive
3 0.07 3.97 4.42 4.46 positive 0.62 14.31 16.02 17.50 positive
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体外扩增结核性胸腔积液单个核细胞用于诊断试剂质量控制的研究
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蒋德云 1 , 董海平 2 , 楼建荣 3 , 许四宏 4 , 陈婷梅 1, * , 石大伟 1, 4, *
中国药学杂志 | 论著 2025,60(10): 1019-1025
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中国药学杂志 | 论著 2025, 60(10): 1019-1025
体外扩增结核性胸腔积液单个核细胞用于诊断试剂质量控制的研究
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蒋德云1, 董海平2, 楼建荣3, 许四宏4, 陈婷梅1, *, 石大伟1, 4, *
作者信息
  • 1 重庆医科大学检验医学院, 重庆 400016
  • 2 广州市胸科医院重症结核科, 广州市结核病研究重点实验室, 呼吸疾病全国重点实验室, 广州 510095
  • 3 广州市雷德医学检验实验室有限公司, 广州 510663
  • 4 中国食品药品检定研究院传染病诊断试剂二室, 国家药品监督管理局体外诊断试剂质量研究与评价重点实验室,国家卫生健康委员会生物技术检定方法及其标准化重点实验室, 北京 100050

    • 蒋德云,女,学士 研究方向:临床检验技术,传染病诊断试剂质量控制和评价;

    董海平,女,博士,主任医师 研究方向:结核病的诊断和治疗。蒋德云和董海平为共同第一作者

通讯作者:

*陈婷梅,女,博士,教授 研究方向:人工智能辅助检验新技术、精准检验新技术研发 Tel:(023)68485184;
石大伟,男,博士,研究员 研究方向:传染病诊断试剂质量控制和评价 Tel:(010)67095450
Expansion of Tuberculosis Pleural Effusion Mononuclear Cells in Vitro for Quality Control of Diagnostic Tests
Deyun JIANG1, Haiping DONG2, Jianrong LOU3, Sihong XU4, Tingmei CHEN1, *, Dawei SHI1, 4, *
Affiliations
  • 1 College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China
  • 2 Guangzhou Key Laboratory of Tuberculosis Research, State Key Laboratory of Respiratory Disease, Department of Tuberculosis, Guangzhou Chest Hospital, Guangzhou 510095, China
  • 3 Guangzhou Leide Medical Laboratory Co., Ltd., Guangzhou 510063, China
  • 4 Key Laboratory of National Health Commission for Research on Quality and Standardization of Biotech Products, NMPA Key Laboratory for Quality Research and Evaluation of in Vitro Diagnostics, Division Ⅱ of Diagnostic for Infectious Diseases, National Institutes for Food and Drug Control, Beijing 100050, China
出版时间: 2025-05-01 doi: 10.11669/cpj.2025.10.003
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摘要
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目的 探索采用体外扩增的结核性胸腔积液(胸水)单个核细胞(tuberculosis pleural fluid mononuclear cells, TB-PFMC)制备结核γ干扰素释放试验[tuberculosis interferon-gamma(IFN-γ)release assays,TB-IGRA]质量控制用样本的可行性。方法 使用3种CD3/CD28抗体联合重组人白细胞介素2(interleukin 2,IL-2)的扩增方案对TB-PFMC进行体外扩增,观察TB-PFMC的生长情况并计算扩增倍数。通过比较3种不同方案的扩增效果,确定最优的方案。使用酶联免疫斑点(ELISPOT)法和酶联免疫吸附(ELISA)法TB-IGRA试剂检测扩增前后TB-PFMC的细胞免疫反应,分析检测结果的变化。结果 方案三被确定为最优扩增方案并用于对3例结核患者的TB-PFMC进行体外扩增,其第9 天的扩增倍数分别达到3 535.00%、2 800.00%和 3 959.00%。扩增后细胞在2种TB-IGRA试剂盒的检测结果中仍保持阳性且未观察到明显的非特异性反应。结论 本研究成功使用CD3/CD28抗体联合IL-2扩增TB-PFMC并确定了最优扩增方案,扩增后的TB-PFMC对于不同的TB-IGRA试剂盒具有良好的适用性,可制备参考品物质,用于TB-IGRA试剂的质量控制。

结核病  /  结核γ干扰素释放试验  /  质量控制  /  T细胞扩增  /  胸腔积液

OBJECTIVE To explore the feasibility of preparing samples for the quality control of tuberculosis interferon-gamma (IFN-γ) release assays (TB-IGRA) using in vitro expanded tuberculosis pleural fluid mononuclear cells (TB-PFMC). METHODS The cryopreserved TB-PFMC were stimulated and expanded in vitro using three expansion protocols based on CD3/CD28 antibody combined with IL-2, and the status of TB-PFMC was observed and the expansion fold was calculated. The amplification results of three different expansion protocols were compared to determine the optimal protocol. The enzyme-linked immunospot assay (ELISPOT) and enzyme-linked immunosorbent assay (ELISA)TB-IGRA kits were used to detect TB-PFMC before and after the expansion, and the changes of the results were analyzed. RESULTS Protocol 3 was determined to be optimal and used to amplify TB-PFMC from three different patients, and their expansion folds reached 3 535.00 %, 2 800.00 %, and 3 959.00%, respectively, on the 9th day. The results of two TB-IGRA kits after the expansion of TB-PFMC remained positive and no obvious non-specific reactions were observed. CONCLUSION This study proves that TB-PFMC can be successfully amplified by CD3/CD28 antibody combined with IL-2 and the optimal protocol was determined, and the expanded TB-PFMC is suitable for the preparation of reference materials and applied for the quality control of TB-IGRA products.

tuberculosis  /  TB-IGRA  /  quality control  /  T cell expansion  /  pleural fluid
蒋德云, 董海平, 楼建荣, 许四宏, 陈婷梅, 石大伟. 体外扩增结核性胸腔积液单个核细胞用于诊断试剂质量控制的研究. 中国药学杂志, 2025 , 60 (10) : 1019 -1025 . DOI: 10.11669/cpj.2025.10.003
Deyun JIANG, Haiping DONG, Jianrong LOU, Sihong XU, Tingmei CHEN, Dawei SHI. Expansion of Tuberculosis Pleural Effusion Mononuclear Cells in Vitro for Quality Control of Diagnostic Tests[J]. Chinese Pharmaceutical Journal, 2025 , 60 (10) : 1019 -1025 . DOI: 10.11669/cpj.2025.10.003
正文
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结核病目前仍然是一个全球性的公共卫生问题。结核γ干扰素释放试验[tuberculosis interferon-gamma(IFN-γ)release assays,TB-IGRA]是一种以结核分枝杆菌特异抗原刺激T细胞产生的IFN-γ为检测靶标,对人体体液样本中的结核分枝杆菌特异性细胞免疫反应进行体外检测的方法,其检测过程大致分为抗原刺激和IFN-γ检测两部分[1]。该类试剂已被广泛应用于肺结核的临床诊断[2]和潜伏性结核感染的筛查[3],在推动我国结核病防治工作和实施世界卫生组织(WHO)终结结核病战略中具有重要作用[4-5]。目前常使用新鲜的结核病患者全血作为参考物质进行此类试剂盒的产品研发、质量控制和性能评价等工作[6-7],然而新鲜全血样本存在来源不稳定、难以长时间保存、单次采集量有限等问题,因此难以使用相同样本进行大量重复测试,从而限制了该类方法的质量控制以及对不同检测试剂质量的统一评价。T细胞体外扩增是过继性T细胞治疗中常用的实验方法,能有效增殖T细胞并使细胞保持较好的活性[8]。本研究拟建立结核性胸腔积液(胸水)单个核细胞(tuberculosis pleural fluid mononuclear cells, TB-PFMC)体外扩增方案并在不同患者来源的TB-PFMC中验证其适用性,然后使用2种常用的商品化TB-IGRA试剂验证扩增后TB-PFMC用于TB-IGRA试剂质量控制的适用性,为解决相关参考物质原料供应可持续差和相关试剂质量评价困难的问题提供新的思路。
1 材料和设备
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1.1 材料
结核性胸腔积液为广州市胸科医院临床胸腔穿刺3名结核病患者所得,患者经病原学证实为结核性胸膜炎,临床检测胸腔积液TB-IGRA结果为阳性。伦理和知情同意均按规定完成,伦理批件号:胸医伦理[2020]61号。RPMI-1640培养基、胎牛血清(fetal bovine serum, FBS)、AIM-V无血清培养基(美国Thermo Fisher Scientific公司);重组人白细胞介素2(interleukin 2,IL-2)、抗人T细胞CD3(OKT3)和CD28(15E8)鼠单克隆抗体(上海爱必信生物科技有限公司);淋巴细胞分离液(深圳市达科为生物技术股份有限公司);AO/PI细胞染色液(上海睿钰生物科技有限公司);T-SPOT.TB试剂(英国Oxford lmmunotec公司);QuantiFERON-TB Gold Plus试剂(商品名:QFT-plus,德国Qiagen公司)。
1.2 仪器设备
Countstar Mira FL细胞计数仪(上海睿钰生物科技有限公司);DM IL LED倒置显微镜(德国Leica公司);Echo Revovle荧光显微镜(美国Echo公司);ImmunoSpot S6 Entry M2酶联免疫斑点分析仪(美国CTL公司);Phomo 酶标仪(郑州安图生物工程股份有限公司)。
2 方法
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2.1 TB-PFMC分离与冻存
将胸腔积液缓慢加至淋巴细胞分离液上层(胸腔积液与淋巴细胞分离液体积比为4∶1),离心30 min。弃掉上层胸腔积液,收取中间白膜层单个核细胞,用RPMI-1640培养基稀释,离心10 min移除上清。用含有体积分数90% FBS的冻存液稀释TB-PFMC,调整细胞浓度为1×107·mL-1,以0.5 mL每管分装于冻存管中,采用程序降温的方法储存于液氮罐中。3例结核患者的TB-PFMC分别编号为1号、2号和3号。
2.2 TB-PFMC体外扩增方案
TB-PFMC的体外扩增主要包括初始激活、扩大培养和静息培养3个步骤。使用光学显微镜观察细胞形态,使用AO/PI细胞染色液和细胞计数仪对细胞染色后计数,并采用ELISPOT试剂检测细胞的结核特异性细胞免疫反应性。
2.2.1 初始激活
培养当天(第0天)将抗人T细胞CD3鼠单克隆抗体以1 μg·mL-1的浓度包被至6孔板中,37 ℃水浴复苏1号TB-PFMC细胞,用RPMI-1640培养基清洗TB-PFMC细胞,离心10 min后移除上清。使用含体积分数10% FBS、1 μg·mL-1抗人T细胞CD28鼠单克隆抗体、10 ng·mL-1IL-2的T细胞扩增培养基重悬细胞,以每毫升1×106个的细胞密度在包被好抗体的6孔板中培养至第3天。
2.2.2 扩大培养
将步骤1中的细胞悬液分为3份,分别按3个实验方案进行扩大培养。方案一(持续激活方案),继续采用步骤1的条件进行培养,每隔2或3 d扩大培养至第9天。方案二(单IL-2+半换液方案),改用含体积分数10% FBS、10 ng·mL-1IL-2的RPMI-1640的培养基通过半换液的方式扩大培养,每隔2或3 d 用前述培养基培养至第9天。方案三(单IL-2+离心换液方案),离心换液去除刺激因子,改用含体积分数10% FBS、10 ng·mL-1IL-2的RPMI-1640的培养基扩大培养,每隔2或3 d用前述培养基培养至第9天。
2.2.3 静息培养
细胞培养至第9天,对上述3个方案培养的细胞进行离心换液,用含体积分数10% FBS的RPMI-1640培养基培养1 d。
2.3 不同患者TB-PFMC的体外扩增
采用“2.2”项下确定的最优的体外扩增方案对来源于另2例结核患者的TB-PFMC(2号和3号)进行体外扩增,以验证该方案在不同来源细胞上的适用性。在培养当天(第0天)和第9天,将细胞吹打分散后,取部分细胞使用AO/PI细胞染色液对细胞染色并用细胞计数仪计数。同时采用酶联免疫斑点(ELISPOT)法和酶联免疫吸附(ELISA)法试剂检测3例TB-PFMC细胞的结核特异性细胞免疫反应性。
2.4 ELISPOT检测
将细胞吹打分散后,取适量TB-PFMC细胞,用AIM-V无血清培养基调整细胞浓度,以每个测试孔50 000个细胞用T-SPOT.TB试剂进行ELISPOT检测。ELISPOT具体实验操和结果判读标准详见试剂盒说明书。主要操作步骤包括50μL抗原、对照试剂以及100μL细胞悬液的加入,微孔培养板的孵育、清洗、二抗孵育和底物显色。采用酶联免疫斑点分析仪对微孔培养板各孔拍照并计数斑点。
2.5 ELISA检测
将细胞吹打分散后,取适量TB-PFMC细胞,用AIM-V无血清培养基调整细胞浓度,以5×105·mL-1的细胞浓度用QFT-plus试剂进行ELISA检测。QFT-plus的具体实验操作和结果判读标准详见试剂盒说明书。主要操作步骤包括1 mL细胞悬液的加入,空白对照管、结核抗原测试管和阳性对照管的孵育,收集上清,以及ELISA检测。采用酶标仪对微孔板中各孔中的OD值进行检测并计算IFN-γ浓度。
2.6 统计分析
使用Excel(office 2010,Microsoft)软件进行数据整理、统计分析。扩增倍数为扩增后某天的细胞总数除以扩增第0天(扩增前)细胞总数的百分比。
3 结果
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3.1 TB-PFMC在3种方案中的细胞生长情况和结核特异性细胞免疫反应的变化
显微镜观察结果(图1)可见,第0天,TB-PFMC单个散落在培养板中,细胞呈圆形,透亮有光泽,状态良好。第3天少量细胞聚集成团,提示发生增殖,同时有部分细胞发生皱缩,提示有细胞死亡。第5天,细胞团开始增大,细胞快速扩增。第7天, 3种扩增方案下的TB-PFMC呈现不同的扩增状态。方案一培养至静息后,细胞仍持续扩增。而在方案二和方案三中,培养至第7天,TB-PFMC中的细胞团减少,第9天,细胞团少见,静息后几乎看不到细胞团,表明细胞扩增减慢。在3种扩增方案中,TB-PFMC的细胞活率均呈现先降低后升高的趋势(表1),其中,方案一中的细胞活率在第3天下降至最低,后期升高最慢,并且升高后的细胞活率低于扩增前,而方案三中的细胞活率在第3天下降的幅度最小并且升高后的细胞活率高于扩增前,提示方案三中的细胞状态最好。此外,培养第9天,TB-PFMC在3种方案中的扩增倍数分别为1 460.00%、2 304.00%和3 535.00%,方案三的扩增效率最高。
ELISPOT结果显示(图2),1号TB-PFMC细胞的N、A、B和P孔分别为0、6、32和34个斑点,在扩增前呈现出“阳性”的检测结果。在方案一(Protocol 1,持续激活方案)中,随着扩增培养时间的增加,TB-PFMC细胞高度激活并释放出大量非特异性IFN-γ,静息后N孔中形成的斑点数由262减少到12,但仍大于10。按T-SPOT.TB试剂盒说明书,结果判读为“不确定”。在方案二(Protocol 2,单IL-2+半换液方案)中,培养第5天的TB-PFMC在N孔中生成19个斑点,相较方案一中的非特异性IFN-γ释放明显减少。在培养第9天,N孔斑点减少为8个,同时A孔和B孔中产生的结核抗原特异性斑点分别为200和347,结果判读为阳性。静息后,N孔中的非特异性斑点减少到0,但A孔和B孔斑点数相比静息前有减少。在方案三(Protocol 3,单IL-2+离心换液方案)中,培养第5天、9天,N孔中分别只有0和3个斑点,较方案二更少,结果判读和扩增前相同,为“阳性”,且结核抗原特异性斑点的扩增效果和方案二基本相同,结核ESAT-6(A孔)和CFP 10(B孔)特异性细胞免疫反应水平(spots forming unit/50000 cells)在扩增后分别增加了41倍和8倍。此外,方案二和方案三的静息后A孔和B孔斑点数相比静息前均有一定减少,而方案一则大量减少,提示静息会使结核特异性T细胞的活性降低。
结合体外扩增倍数和ELISPOT结果,可以确定方案三的TB-PFMC体外扩增效果最优,即:使用CD3/CD28对TB-PFMC进行初始激活从而有效倍增具有结核特异性细胞免疫反应性的TB-PFMC的数量,并且通过离心换液更充分地去除刺激因子从而降低扩增后TB-PFMC在ELISPOT检测结果中的非特异反应,同时不需进行静息培养。
3.2 不同患者TB-PFMC的扩增效果和结核特异性细胞免疫反应的变化
采用方案三对另2例结核患者的TB-PFMC进行体外扩增。如表2所示,和1号相似,2号和3号TB-PFMC也发生大量扩增,培养第9天的扩增倍数分别达到28.00和39.59,细胞活率均大于90%,细胞状态良好。这表明,方案三在不同患者来源的TB-PFMC中均表现出良好且相似的扩增效果。
Note: the data of cell viability and amplification factor of TB-PFMC No.1 were derived from Tab.1.
ELISPOT结果显示(表3),在扩增前(培养第0天),2号和3号TB-PFMC的结果判读均为“阳性”,但结核特异性斑点数较少,均低于100。 在第9天,TB-PFMC在大量扩增后仍保持了“阳性”(positive)结果,且结核特异性斑点数明显增加:2号TB-PFMC的ESAT-6(A孔)和CFP 10(B孔)特异性细胞免疫反应水平(spots forming unit/50 000 cells)分别增加了约7.6倍和4.5倍,而3号则分别增加了约8.8倍和4.3倍。值得注意的是,扩增后的TB-PFMC中的ESTA-6特异性细胞免疫反应水平增加倍数均高于CFP 10,A和B孔斑点的比值(A/B ratio)分别从0.2、3.0和0.4增加到0.9、5.1和0.8,显示该扩增方案可能更利于结核ESAT-6特异性T细胞扩增。此外,培养第9天后的TB-PFMC在阳性质控孔中生成的斑点相较扩增前明显增多,表明此时细胞的细胞免疫反应基础水平升高。
Notes: N-blank control well (AIM-V medium); A-ESAT-6 antigen testing well; B-CFP 10 antigen test well; P-Positive control well (phytohemagglutinin, PHA); the data of cell viability and amplification factor of TB-PFMC No.1 were derived from Fig.2.
和ELISPOT结果一致,ELISA法TB-IGRA试剂的检测结果显示(表4),培养第9天,3例TB-PFMC在大量扩增后仍保持了“阳性”(positive)结果,且结核特异性细胞免疫反应水平明显提高,和扩增前比CD4 T细胞免疫反应(TB1管)分别增加了约16.5、6.9和3.6倍,而CD4和CD8 T细胞免疫反应(TB2管)分别增加了约24.9、10.4和3.6倍。
T-SPOT.TB和QFT-plus试剂的结果表明,扩增后的TB-PFMC仍保持很好的结核特异性细胞免疫反应性和细胞状态,适用于TB-IGRA该类试剂的质量控制。
4 讨论
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结核病患者的胸腔积液中含有大量结核致敏T细胞[9-10]是制备TB-IGRA试剂参考物质的良好原料,然而从每位患者中能收集到的TB-PFMC是有限的,无法满足对大量试剂进行质量控制和性能评价的需求。采用体外扩增的方法来增加具有特异性细胞免疫反应的T细胞的数量是解决该问题的一个策略。CD3/CD28抗体活化免疫细胞是常用的T细胞体外扩增的方法[11]。 Han等[12]利用CD3/CD28单抗、IFN-γ等因子对分离自肿瘤患者胸腔积液来源的T细胞进行体外培养,将肿瘤特异性杀伤效果的T细胞的数量扩增了5~6倍。本研究利用CD3/CD28单抗联合IL-2的方法成功实现TB-PFMC的大量扩增,扩增倍数可以达到约30倍,并保持较好的结核特异性细胞免疫反应性,为有效解决TB-IGRA试剂参考物质所用细胞原料不足的问题奠定了基础。
IFN-γ等细胞因子的释放是T细胞被激活后的重要特征[13],持续的CD3/CD28抗体刺激使得TB-PFMC一直扩增并且非特异性释放大量的IFN-γ导致“不确定”ELISPOT结果的产生(T-SPOT.TB试剂中规定N孔斑点数大于10个则为“不确定”结果)。培养ELISPOT(cultured ELISPOT)方法被认为可以用于中央记忆T细胞(central memory T cells)的表型检测[14],该方法在初始培养时使用特定抗原激活T细胞,后续培养通常采用半换液方式补充IL-2,不再持续刺激T细胞。因此,笔者借鉴了该方法,在CD3/CD28抗体初始激活后采用半换液补充IL-2,发现TB-PFMC非特异性释放的IFN-γ明显减少,并且结核特异性细胞免疫反应得到了保持甚至增强。进一步地,笔者采用离心的方式去除刺激因子从而更好地降低非特异性IFN-γ释放的影响,最终确定方案三为扩增TB-PFMC制备TB-IGRA参考物质的最优方法。
大部分TB-IGRA试剂采用ESAT-6和CFP 10这两种结核特异性抗原作为刺激抗原[15],因此分别对单个结核特异性抗原的检测效果进行质量控制具有重要意义。本研究中,初始TB-PFMC中的ESAT-6和CFP 10特异性T细胞的数量经过扩增均大量增加,但二者呈现出不同的细胞免疫水平增强效果。其原因可能是,不同扩增方法对于不同的T细胞亚群具有偏好性[16-17]。另一个原因可能是T细胞的特异性细胞免疫水平在9天的体外扩增后已达到或接近“天花板”,从而压平了不同抗原反应之间差异。在后续研究中,笔者需要验证是否在其他患者来源的TB-PFMC在扩增后出现该现象,并研究导致这一现象的原因,以进一步优化扩增方案。
根据全球性非营利组织FIND的统计,目前约有十几个商品化的TB-IGRA试剂已用于或将用于结核病的防控,这些试剂的刺激抗原、孵育方法和检测原理各有不同[18]。因此,评估扩增后TB-PFMC对于这些试剂的适用性是确保其可以用于相关试剂质量控制的一项重要工作。典型的TB-IGRA试剂有两大类,为分别基于ELISPOT和刺激管ELISA的原理检测结核抗原特异性T细胞免疫反应[3,7]。T-SPOT.TB和QFT-plus分别是基于这两种原理的常用TB-IGRA试剂,并且已被美国食品药品监督管理局批准[3,19],因此使用这两种试剂进行适用性评估具有很好的代表性。本研究中不同患者来源的TB-PFMC在体外扩增后的均能在两种试剂的检测结果中保持阳性结果,表明体外扩增得到的TB-PFMC用于制备TB-IGRA质量控制和性能评价用参考物质具有良好的适用性。
本研究在如下方面还存在局限性,并在下一步研究中改进:①使用了3例患者来源的样本用于建立TB-PFMC体外扩增的方法,代表性有限;②扩增后TB-PFMC的均匀性和冻存稳定性需要进一步验证。
综上所述,本研究成功建立了TB-PFMC的体外扩增方法,扩增后的细胞可用于TB-IGRA试剂盒的质量控制,为研制TB-IGRA试剂参考物质提供了新的方案,从而有利于对TB-IGRA试剂进行质量控制及性能评价。
基金
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  • 科技部重大传染病防治专项资助(2018ZX10102001)
  • 广州市科技计划项目资助(2024A03J0587)
  • 广州市科技计划项目资助(202201010744)
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参考文献 引证文献
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doi: 10.11669/cpj.2025.10.003
  • 接收时间：2025-02-08
  • 首发时间：2025-11-09
  • 出版时间：2025-05-01
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  • 收稿日期：2025-02-08
基金
科技部重大传染病防治专项资助(2018ZX10102001)
广州市科技计划项目资助(2024A03J0587)
广州市科技计划项目资助(202201010744)
作者信息
    1 重庆医科大学检验医学院, 重庆 400016
    2 广州市胸科医院重症结核科, 广州市结核病研究重点实验室, 呼吸疾病全国重点实验室, 广州 510095
    3 广州市雷德医学检验实验室有限公司, 广州 510663
    4 中国食品药品检定研究院传染病诊断试剂二室, 国家药品监督管理局体外诊断试剂质量研究与评价重点实验室,国家卫生健康委员会生物技术检定方法及其标准化重点实验室, 北京 100050

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*陈婷梅,女,博士,教授 研究方向:人工智能辅助检验新技术、精准检验新技术研发 Tel:(023)68485184;
石大伟,男,博士,研究员 研究方向:传染病诊断试剂质量控制和评价 Tel:(010)67095450
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鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
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红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
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