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Article(id=1153986787883082278, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153986777279877909, articleNumber=null, orderNo=null, doi=10.19812/j.cnki.jfsq11-5956/ts.20240830002, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1724947200000, receivedDateStr=2024-08-30, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1753061490268, onlineDateStr=2025-07-21, pubDate=1736870400000, pubDateStr=2025-01-15, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1753061490268, onlineIssueDateStr=2025-07-21, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1753061490268, creator=13701087609, updateTime=1753061490268, updator=13701087609, issue=Issue{id=1153986777279877909, tenantId=1146029695717560320, journalId=1149652044408987649, year='2025', volume='16', issue='1', pageStart='1', pageEnd='320', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1753061487741, creator=13701087609, updateTime=1757901302572, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1174286432060453412, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153986777279877909, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1174286432060453413, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153986777279877909, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=258, endPage=265, ext={EN=ArticleExt(id=1153986788772274730, articleId=1153986787883082278, tenantId=1146029695717560320, journalId=1149652044408987649, language=EN, title=Spectral characteristics of bacterial endotoxin from different sources and their effects on the immune response of monocytes, columnId=1151895321388347923, journalTitle=Journal of Food Safety & Quality, columnName=Food Analysis and Detection, runingTitle=null, highlight=null, articleAbstract=

Objective To compare the differences in spectral characteristics and monocyte immune effects for bacterial endotoxin from different sources. Methods Ultraviolet absorption spectrum and three-dimensional fluorescence spectrum were used to identify excipients (polyethylene glycol 8000 and α-lactose) and biological impurities (protein and nucleic acid) in 5 kinds of control standard endotoxin. Biological contamination was further confirmed by the detection of Toll-like receptors (TLRs) in HL-60 cells. The monocyte activation test was used to compare the release of the inflammatory cytokines interleukin 6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) caused by 5 kinds of endotoxins on HL-60 cells. Results As for control standard endotoxin, absorption peaks related to excipients were observed in ultraviolet absorption spectrum, while fluorescence peaks related to excipients and proteins were shown in three-dimensional fluorescence spectrum. The expression levels of TLRs of HL-60 cells indicated that protein contamination was more significant than nucleic acid contamination. All endotoxins induced improvement in cellular IL-6 and IL-1β gene relative expression levels and their protein mass concentrations in HL-60 cells. However, the TNF-α gene relative expression levels and its protein concentrations were not elevated. Endotoxins from the same or different bacterial strains had significant differences in their ability to induce inflammation under the same endotoxin activities. Conclusion Excipients and protein contamination of endotoxins are identified by spectral detection, while proteins and nucleic acids in biological impurity are confirmed by the TLRs assay of HL-60 cells. Molecular structure of endotoxins, as well as protein contamination influence the ability of endotoxins to induce the release of the inflammatory cytokines.

, correspAuthors=Miao BAI, Ming-Lu ZHANG, 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=Can ZHANG, Chuan-Fu ZHANG, Yu-Chao BAI, Ping GUI, Chao-Hong TAN, Miao BAI, Ming-Lu ZHANG), CN=ArticleExt(id=1153986815456436571, articleId=1153986787883082278, tenantId=1146029695717560320, journalId=1149652044408987649, language=CN, title=不同来源细菌内毒素光谱特征及其对单核细胞免疫效应的影响, columnId=1151895321958773274, journalTitle=食品安全质量检测学报, columnName=食品分析与检测, runingTitle=null, highlight=null, articleAbstract=目的 比较不同来源细菌内毒素的光谱特征以及对单核细胞的免疫效应差异。方法 采用紫外吸收光谱和三维荧光光谱识别5种内毒素工作标准品中非内毒素成分如赋形剂(聚乙二醇8000和α-乳糖)和生物杂质(蛋白质和核酸), 结合HL-60细胞Toll样受体(Toll-like receptors, TLRs)测试进一步对生物杂质成分进行识别, 通过单核细胞活化反应测定法比较5种内毒素诱导HL-60细胞释放炎症因子白细胞介素-6 (interleukin 6, IL-6)、白细胞介素-1β (interleukin-1β, IL-1β)和肿瘤坏死因子-α (tumor necrosis factor-α, TNF-α)情况。结果 内毒素工作标准品紫外吸收光谱中出现与赋形剂相关的吸收峰, 三维荧光光谱中出现与赋形剂和蛋白质相关的荧光峰。HL-60细胞TLRs测试结果表明内毒素中蛋白质污染相较于核酸污染更明显。5种内毒素均引起HL-60细胞IL-6和IL-1β基因相对表达水平以及蛋白质量浓度升高, TNF-α基因相对表达水平及蛋白质量浓度未升高, 结果表明相同或不同菌种来源的内毒素在同一效价条件下的炎症效应差异明显。结论 光谱检测识别内毒素的赋形剂和蛋白质污染, HL-60细胞TLRs测试对生物污染成分进一步确认。内毒素分子结构差异以及非内毒素成分的蛋白质污染, 导致相同或不同菌种来源内毒素诱导炎症因子释放能力产生差异。, correspAuthors=白淼, 张明露, authorNote=null, correspAuthorsNote=
*白淼(1990—), 女, 硕士, 工程师, 主要研究方向为环境科学与工程。E-mail:
张明露(1980—), 女, 博士, 教授, 主要研究方向为环境微生物。E-mail:
, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=7TyNzQjgh5iyxOF+FljN3g==, magXml=q5BX3oBzVJjU+uQiVhPupg==, pdfUrl=null, pdf=s/1Cyy2d3LvdkYnfuuNkYQ==, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=yEBpUjIXzHRJgjJBgH34+Q==, mapNumber=null, authorCompany=null, fund=null, authors=

张灿(1975—), 女, 博士, 研究员, 主要研究方向为环境科学与工程。E-mail:

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张灿(1975—), 女, 博士, 研究员, 主要研究方向为环境科学与工程。E-mail:

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张灿(1975—), 女, 博士, 研究员, 主要研究方向为环境科学与工程。E-mail:

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5. Department of Environmental Science and Engineering, Beijing Technology and Business University, Beijing 100048, China, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null), CN=AuthorExt(id=1174369665250902774, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986787883082278, authorId=1174369665141850866, language=CN, stringName=白淼, firstName=null, middleName=null, lastName=null, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=2, 5, *, address=2.中国人民解放军疾病预防控制中心, 北京 100071
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注: A. 1#样品(500 EU/mL); B. 2#样品(45 EU/mL); C. 聚乙二醇8000; D. α-乳糖。

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注: A. 基因相对表达水平; B. 蛋白相对表达水平。*. 与对照组相比具有显著性差异, P<0.05。下同。

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Sequence information of primers for real-time fluorescence quantitative PCR[9-13]

, figureFileSmall=null, figureFileBig=null, tableContent=
基因 序列5’-3’
TLR2-F GGCCAGCAAATTACCTGTGTG
TLR2-R AGGCGGACATCCTGAACCT
TLR9-F GCTGCGTCTCCGTGACAATTA
TLR9-R AGCTGACATCCAGCCTCCG
IL-6-F ACAGCCACTCACCTCTTCAG
IL-6-R TGGAAGCATCCATCTTTTTC
IL-1β-F CTGTACCTGTCCTCGGTGTTG
IL-1β-R GCAGACTCAAATTCCAGCTTGTT
TNF-α-F TGGAGAAGGGTGACCGACTCAG
TNF-α-R GTTTGGGAAGGTTGGATGTTCG
GAPDH-F CTCCTCCTGTTCGACAGTCA
GAPDH-R CGACCAAATCCGTTGACTCC
), ArticleFig(id=1174369667771679503, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986787883082278, language=CN, label=表1, caption=

荧光定量PCR引物序列信息[9-13]

, figureFileSmall=null, figureFileBig=null, tableContent=
基因 序列5’-3’
TLR2-F GGCCAGCAAATTACCTGTGTG
TLR2-R AGGCGGACATCCTGAACCT
TLR9-F GCTGCGTCTCCGTGACAATTA
TLR9-R AGCTGACATCCAGCCTCCG
IL-6-F ACAGCCACTCACCTCTTCAG
IL-6-R TGGAAGCATCCATCTTTTTC
IL-1β-F CTGTACCTGTCCTCGGTGTTG
IL-1β-R GCAGACTCAAATTCCAGCTTGTT
TNF-α-F TGGAGAAGGGTGACCGACTCAG
TNF-α-R GTTTGGGAAGGTTGGATGTTCG
GAPDH-F CTCCTCCTGTTCGACAGTCA
GAPDH-R CGACCAAATCCGTTGACTCC
), ArticleFig(id=1174369667842982672, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986787883082278, language=EN, label=Table 2, caption=

Reaction system for real-time fluorescence quantitative PCR

, figureFileSmall=null, figureFileBig=null, tableContent=
试剂 使用量/μL
2×SuperReal PreMix Plus 12.5
正向引物(10 μmol/L) 0.75
反向引物(10 μmol/L) 0.75
cDNA模板 1
RNase-free ddH2O 10
), ArticleFig(id=1174369667981394705, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986787883082278, language=CN, label=表2, caption=

荧光定量PCR反应体系

, figureFileSmall=null, figureFileBig=null, tableContent=
试剂 使用量/μL
2×SuperReal PreMix Plus 12.5
正向引物(10 μmol/L) 0.75
反向引物(10 μmol/L) 0.75
cDNA模板 1
RNase-free ddH2O 10
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不同来源细菌内毒素光谱特征及其对单核细胞免疫效应的影响
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张灿 1 , 张传福 2 , 白宇超 2 , 桂萍 3 , 谭朝洪 4 , 白淼 2, 5, * , 张明露 5, *
食品安全质量检测学报 | 食品分析与检测 2025,16(1): 258-265
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食品安全质量检测学报 | 食品分析与检测 2025, 16(1): 258-265
不同来源细菌内毒素光谱特征及其对单核细胞免疫效应的影响
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张灿1 , 张传福2, 白宇超2, 桂萍3, 谭朝洪4, 白淼2, 5, * , 张明露5, *
作者信息
  • 1.中国检验检疫科学研究院卫生检验与检疫研究所, 北京 100176
  • 2.中国人民解放军疾病预防控制中心, 北京 100071
  • 3.中国城市规划设计研究院, 北京 100037
  • 4.北京建筑大学环境与能源工程学院, 北京 102616
  • 5.北京工商大学环境科学与工程系, 北京 100048

    • 张灿(1975—), 女, 博士, 研究员, 主要研究方向为环境科学与工程。E-mail:

通讯作者:

*白淼(1990—), 女, 硕士, 工程师, 主要研究方向为环境科学与工程。E-mail:
张明露(1980—), 女, 博士, 教授, 主要研究方向为环境微生物。E-mail:
Spectral characteristics of bacterial endotoxin from different sources and their effects on the immune response of monocytes
Can ZHANG1 , Chuan-Fu ZHANG2, Yu-Chao BAI2, Ping GUI3, Chao-Hong TAN4, Miao BAI2, 5, * , Ming-Lu ZHANG5, *
Affiliations
  • 1. Institute of Health Inspection and Quarantine, Chinese Academy of Inspection and Quarantine, Beijing 100176, China
  • 2. Center for Disease Control and Prevention of Chinese PLA, Beijing 100071, China
  • 3. China Academy of Urban Planning and Design, Beijing 100037, China
  • 4. School of Environmental and Energy Engineering, Beijing University of Architecture and Technology, Beijing 102616, China
  • 5. Department of Environmental Science and Engineering, Beijing Technology and Business University, Beijing 100048, China
出版时间: 2025-01-15 doi: 10.19812/j.cnki.jfsq11-5956/ts.20240830002
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摘要
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目的 比较不同来源细菌内毒素的光谱特征以及对单核细胞的免疫效应差异。方法 采用紫外吸收光谱和三维荧光光谱识别5种内毒素工作标准品中非内毒素成分如赋形剂(聚乙二醇8000和α-乳糖)和生物杂质(蛋白质和核酸), 结合HL-60细胞Toll样受体(Toll-like receptors, TLRs)测试进一步对生物杂质成分进行识别, 通过单核细胞活化反应测定法比较5种内毒素诱导HL-60细胞释放炎症因子白细胞介素-6 (interleukin 6, IL-6)、白细胞介素-1β (interleukin-1β, IL-1β)和肿瘤坏死因子-α (tumor necrosis factor-α, TNF-α)情况。结果 内毒素工作标准品紫外吸收光谱中出现与赋形剂相关的吸收峰, 三维荧光光谱中出现与赋形剂和蛋白质相关的荧光峰。HL-60细胞TLRs测试结果表明内毒素中蛋白质污染相较于核酸污染更明显。5种内毒素均引起HL-60细胞IL-6和IL-1β基因相对表达水平以及蛋白质量浓度升高, TNF-α基因相对表达水平及蛋白质量浓度未升高, 结果表明相同或不同菌种来源的内毒素在同一效价条件下的炎症效应差异明显。结论 光谱检测识别内毒素的赋形剂和蛋白质污染, HL-60细胞TLRs测试对生物污染成分进一步确认。内毒素分子结构差异以及非内毒素成分的蛋白质污染, 导致相同或不同菌种来源内毒素诱导炎症因子释放能力产生差异。
内毒素  /  鲎试验  /  单核细胞活化反应测定法  /  免疫效应  /  Toll样受体

Objective To compare the differences in spectral characteristics and monocyte immune effects for bacterial endotoxin from different sources. Methods Ultraviolet absorption spectrum and three-dimensional fluorescence spectrum were used to identify excipients (polyethylene glycol 8000 and α-lactose) and biological impurities (protein and nucleic acid) in 5 kinds of control standard endotoxin. Biological contamination was further confirmed by the detection of Toll-like receptors (TLRs) in HL-60 cells. The monocyte activation test was used to compare the release of the inflammatory cytokines interleukin 6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) caused by 5 kinds of endotoxins on HL-60 cells. Results As for control standard endotoxin, absorption peaks related to excipients were observed in ultraviolet absorption spectrum, while fluorescence peaks related to excipients and proteins were shown in three-dimensional fluorescence spectrum. The expression levels of TLRs of HL-60 cells indicated that protein contamination was more significant than nucleic acid contamination. All endotoxins induced improvement in cellular IL-6 and IL-1β gene relative expression levels and their protein mass concentrations in HL-60 cells. However, the TNF-α gene relative expression levels and its protein concentrations were not elevated. Endotoxins from the same or different bacterial strains had significant differences in their ability to induce inflammation under the same endotoxin activities. Conclusion Excipients and protein contamination of endotoxins are identified by spectral detection, while proteins and nucleic acids in biological impurity are confirmed by the TLRs assay of HL-60 cells. Molecular structure of endotoxins, as well as protein contamination influence the ability of endotoxins to induce the release of the inflammatory cytokines.

endotoxin  /  Limulus amebocyte lysate assay  /  monocyte activation test  /  immune response  /  Toll-like receptors
张灿, 张传福, 白宇超, 桂萍, 谭朝洪, 白淼, 张明露. 不同来源细菌内毒素光谱特征及其对单核细胞免疫效应的影响. 食品安全质量检测学报, 2025 , 16 (1) : 258 -265 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20240830002
Can ZHANG, Chuan-Fu ZHANG, Yu-Chao BAI, Ping GUI, Chao-Hong TAN, Miao BAI, Ming-Lu ZHANG. Spectral characteristics of bacterial endotoxin from different sources and their effects on the immune response of monocytes[J]. Journal of Food Safety & Quality, 2025 , 16 (1) : 258 -265 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20240830002
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0 引言
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内毒素, 又称脂多糖, 是革兰氏阴性菌和部分蓝藻的细胞壁成分, 主要通过细胞死亡裂解释放[1]。内毒素具有强免疫刺激性, 能够诱发强烈的免疫应答反应, 导致多种细胞因子释放, 引起发热、哮喘、慢性炎症、低血压等疾病, 严重可能出现休克、多器官衰竭甚至死亡[2]。内毒素在人体内超过1 ng/(kg·h)即可引起炎症反应, 美国食品药品监督管理局(Food and Drug Administration, FDA)规定内毒素的最大允许接触限值为5 EU/(kg·h)[3-4]。由于内毒素通过血液暴露引发严重健康风险, 国内外生物医药行业早已对其重点管控。近年来饮用水[5]、乳品[6]、果汁[7]等食品内毒素胃肠暴露风险也逐渐受到关注。食品在原料采集、生产加工等多个环节易发生微生物污染, 食品生产流程的杀菌处理工艺虽然能够杀灭微生物, 但是微生物死亡解体时会释放大量内毒素。由于内毒素具有热稳定性, 常用食品杀菌工艺如巴氏消毒等难以将其完全去除。目前国内外食品领域尚未将内毒素纳入管控, 食品内毒素污染风险亟需得到重视。
食品内毒素通常采用国内外药典规定的鲎试验进行检测[8]; 近年来国内外药典又收录了单核细胞活化反应测定法。鲎试验和单核细胞活化反应测定法均要求使用内毒素标准物质。内毒素标准物质由于选取的细菌来源不同, 导致分子结构不同, 影响生物活性。此外, 内毒素制备纯化过程会残留一些菌体自身组分和添加的试剂, 如蛋白质、核酸和赋形剂等, 也会影响生物活性。目前国内外对内毒素中非内毒素成分的理化性质、毒理学特征等均未明确, 使用过程中具有未知性和不确定性, 影响内毒素相关检测方法的协同统一性。本研究测定5种内毒素工作标准品的紫外吸收光谱与三维荧光光谱特征, 结合细胞Toll样受体(Toll-like receptors, TLRs)对非内毒素成分进行识别, 采用单核细胞活化反应测定法分析免疫效应差异, 旨在通过结合光谱特征和免疫效应指标识别内毒素中的非内毒素成分, 为内毒素相关检测方法的质量控制提供参考。
1 材料与方法
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1.1 材料与试剂
动态浊度法鲎试剂、细菌内毒素检查用水(小于0.003 EU/mL)(湛江安度斯生物有限公司); Iscove改良杜氏培养基(Iscove’s modified Dulbecco’s medium, IMDM)(武汉普诺赛生命科技有限公司); 优级胎牛血清(北京索莱宝科技有限公司); 人白细胞介素-6 (interleukin 6, IL-6)酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)试剂盒、人白细胞介素-1β (interleukin-1β, IL-1β) ELISA试剂盒和人肿瘤坏死因子-α (tumor necrosis factor-α, ‌TNF-α) ELISA试剂盒(北京欣博盛生物科技有限公司); 二喹啉甲酸法(bicinchoninic acid assay, BCA)蛋白定量试剂盒(美国Thermo Fisher Scientific公司); Toll样受体2兔单克隆抗体(Toll-like receptors 2 rabbit monoclonal antibody, TLR2 Rabbit mAb)(美国Cell Signaling Technology公司); 12~230 kDa分离试剂盒、抗兔二抗检测试剂盒(美国Protein Simple公司); 聚乙二醇8000(分析纯, 上海阿拉丁生化科技股份有限公司); α-乳糖(分析纯, 上海麦克林生化科技股份有限公司); 5种内毒素工作标准品分别购自国内外试剂公司, 其中1#内毒素菌种来源为大肠杆菌(Escherichia coli, E.coli) O113:H10, 2#、3#、4#和5#菌种来源为E.coli O111:B4。
ATI320-06动态试管仪(英国Lab Kinetic公司); U-3010型紫外分光光度计、F-7000荧光分光光度计(日本HITACHI公司); Jess全自动蛋白免疫印迹定量分析系统(美国Protein Simple公司); CFX96荧光定量PCR仪(美国Bio-rad公司); SpectraMax iD3多功能酶标仪(美国Molecular Devices公司)。
1.2 动态浊度法鲎试验
将0.1 mL动态浊度法鲎试剂加入反应试管, 加入0.1 mL样品混合均匀, 立即放入ATI320-06动态试管仪进行检测。样品和鲎试剂在37 ℃温育条件下发生反应, ATI320-06动态试管仪及软件生成每个样品的反应动态曲线, 记录反应试管在波长405 nm处达到95%透光率的达限时间。样品达限时间和内毒素活性呈负相关, 根据标准曲线来确定样品内毒素活性。将内毒素标准品溶液分别稀释为2.00、0.50、0.20和0.04 EU/mL, 以细菌内毒素检查用水作为阴性对照。每个样品在测试过程中采用2个平行样, 并以0.25 EU/mL内毒素标准品溶液作为阳性对照, 记录加标回收率、稀释倍数、达限时间等参数。样品测试3次取平均值。
1.3 内毒素光谱测定
1.3.1 紫外吸收光谱
采用U-3010型紫外分光光度计对内毒素工作标准品溶液的紫外吸收光谱进行测定, 扫描范围为190~400 nm, 采样间隔为0.5 nm。以细菌内毒素检查用水作为参比, 对紫外分光光度计进行基线校准。
1.3.2 三维荧光光谱
采用F-7000荧光分光光度计对内毒素工作标准品溶液的三维荧光光谱进行测定, 氙灯激发源, 激发和发射光谱步长均为5 nm, 激发光谱波长(excitation wavelength, Ex)范围200~400 nm, 发射光谱波长(emission wavelength, Em)范围250~500 nm, 狭缝宽度5 nm, 响应时间为自动方式。光电倍增管电压设定为600 V, 扫描速度设定为1200 nm/min。以细菌内毒素检查用水作为参比, 扣除空白水样光谱数据, 以消除拉曼散射影响。
1.4 单核细胞活化反应测定
选取生长状态良好、处于对数生长期的HL-60细胞, 在96孔细胞培养板中加入不含胎牛血清的IMDM培养液制备成细胞密度约为4.0×106 cells/mL的细胞悬液(160 μL/孔)。将配制好的内毒素工作标准品溶液加入含有HL-60细胞的96孔细胞培养板中(40 μL/孔), 每孔培养体系内毒素最终活性均为18 EU/mL。以细菌内毒素检查用水代替内毒素工作标准品溶液作为对照组。将96孔细胞培养板置于37 ℃, 5% CO2培养箱中孵育24 h。
1.4.1 细胞TLRs蛋白检测
提取经过内毒素工作标准品溶液处理后的HL-60细胞总蛋白, 使用BCA法对蛋白浓度进行测定。采用样品稀释液(0.1×Sample Buffer)和上样缓冲液(5×Master Mix)配制样品, 样品终质量浓度为2 mg/mL。分别加入TLR2抗体(稀释比例1:50, V:V)、兔抗GAPDH抗体(稀释比例1:50, V:V)、发光液(Lumino-S和Peroxide)等, 2500 r/min离心 5 min, 确认孔底无气泡后放入全自动蛋白免疫印迹定量分析系统进行检测。
1.4.2 细胞炎症因子蛋白检测
收集经过内毒素工作标准品溶液处理后的HL-60细胞上清液, 分别用人IL-6 ELISA试剂盒、人IL-1β ELISA试剂盒和人TNF-α ELISA试剂盒检测IL-6、IL-1β和TNF-α的含量。
1.4.3 细胞TLRs和炎症因子相关基因表达检测
收集经过内毒素工作标准品溶液处理后的HL-60细胞沉淀, 使用动物组织/细胞总RNA快速提取试剂盒提取RNA后逆转录为互补DNA (complementary DNA, cDNA)。荧光定量聚合酶链反应(polymerase chain reaction, PCR)检测3种炎症因子(IL-6、IL-1β和TNF-α)和两种TLRs (TLR2和TLR9)基因相对表达水平, 引物序列如表1所示, 反应体系见表2。反应条件: 95 ℃ 15 min; 95 ℃ 10 s, 60 ℃ 32 s, 40个循环; 熔解曲线分析。以甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase, GAPDH)作为内参, 使用2-ΔΔCt法对结果进行分析。
1.5 数据处理
采用SPSS 22.0软件进行统计学分析, 组间比较使用单因素方差分析, P<0.05认为差异具有统计学意义。
2 结果与分析
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2.1 紫外吸收光谱特征
如图1所示, 不同内毒素的紫外吸收光谱具有明显差异。1#样品在190~400 nm范围内没有发现紫外吸收峰, 2#和3#样品在190 nm处具有明显的紫外吸收峰, 4#和5#样品在190 nm和277 nm处出现紫外吸收峰。由于不同细菌来源的内毒素分子结构差异较大, 并且国内外内毒素标准物质的组分信息也均未明确, 因此目前国内外研究报道中尚无内毒素的紫外吸收光谱可直接参考。国外研究发现100 mJ/cm2低压紫外线(254 nm)和100 mJ/cm2中压紫外线(200~400 nm)均不能显著降低水中内毒素活性[14-15], 这间接表明内毒素分子典型结构中可能不存在明显的共轭体系或发色官能团, 导致其没有紫外吸收峰。1#样品紫外吸收光谱也说明内毒素(180~2000 EU/mL)在该光谱范围内不具有紫外吸收特性, 和前期报道结果一致[14-15]。
内毒素中的非内毒素成分主要涉及赋形剂和生物污染, 这些成分均会影响内毒素的紫外吸收光谱特征。常用赋形剂为聚乙二醇和乳糖, 不仅具有成型支架作用, 还能减少冻干过程影响, 降低支间差异; 常见生物污染成分主要包括蛋白质和核酸。如图1所示, 聚乙二醇8000和α-乳糖在190 nm出现紫外吸收峰。由于1#样品未添加赋形剂, 因此紫外吸收光谱中没有出现与赋形剂相关的紫外吸收峰。2#、3#、4#和5#样品均在190 nm处发现紫外吸收峰, 这可能与添加的赋形剂有关。此外, 4#和5#样品在277 nm处的紫外吸收峰与α-乳糖在相同波长附近的紫外吸收峰相似, 表明该紫外吸收峰可能与添加的α-乳糖有关。据报道核酸中的嘌呤环和嘧啶环的共轭双键使其具有紫外吸收, 最大紫外吸收峰在260 nm[16]。蛋白质的共轭双键使其在200~300 nm波长范围内具有紫外吸收特性, 最大紫外吸收峰通常在230 nm和280 nm[17-18]。紫外吸收光谱能显示内毒素中与赋形剂相关的吸收峰, 生物污染成分则需要进一步识别。
2.2 三维荧光光谱特征
图2展示具有不同菌种来源的1#样品和2#样品以及赋形剂的三维荧光光谱。1#样品检测出荧光峰A (Ex/Em为230/340)(图2A)。按照三维荧光的不同区域划分, 荧光峰A处于类蛋白物质荧光区域范围(Ex/Em为200~250 nm/ 320~380 nm)[19], 表明1#样品的荧光峰A与蛋白质污染有关。由于1#样品未添加赋形剂, 因此未发现与赋形剂相关的荧光峰。聚乙二醇8000检测出荧光峰B (Ex/Em为220/290)(图2C), α-乳糖检测出荧光峰B (Ex/Em为220/290)和荧光峰C (Ex/Em为275/335)(图2D)。因此, 2#样品的荧光峰均与非内毒素成分有关, 其中峰A (Ex/Em为230/340)为蛋白质污染引起, 峰B (Ex/Em为220/290)和峰C (Ex/Em为275/335)与赋形剂有关(图2B)。如图1所示, 通过紫外吸收光谱识别出与赋形剂相关的吸收峰, 但是未能识别内毒素中含量较低的生物污染。三维荧光法灵敏度通常比紫外吸收光谱法高2~3个数量级, 因此不仅检出赋形剂的荧光峰, 还能进一步识别蛋白质污染。
2.3 HL-60细胞TLR2和TLR9表达差异
TLRs是先天免疫系统的重要模式识别受体, 通过跨膜结构将相关刺激信号传导至细胞内, 引发复杂的级联信号反应, 激活NF-κB等核转录因子, 诱导IL-6、IL-1β以及TNF-α等细胞炎症因子的合成与释放, 启动机体免疫反应[20]。TLR2能够识别和结合蛋白质等多种微生物成分[21]; TLR9配体则为微生物的DNA、RNA等核酸类物质[22]。如图3A所示, 与对照组相比, 同为18 EU/mL的4种内毒素刺激HL-60细胞后, TLR2基因相对表达水平升高(P<0.05); TLR9基因相对表达水平未升高(P>0.05)。其中, 1#、2#、3#和5#样品TLR2基因相对表达水平升高, 分别为对照组的1.14、1.26、2.42和1.18倍, 3#样品TLR2基因相对表达水平最高; 4#样品与对照组相比, TLR2基因相对表达水平未升高(P>0.05)。相较于TLR2, 5种内毒素均未引起TLR9基因相对表达水平升高(图3A)。
如图3B所示, 3#样品TLR2蛋白表达水平高于对照组以及其他4种样品, 这与其基因相对表达水平变化情况基本一致(图3A)。相较于对照组, 1#、2#和5#样品TLR2基因相对表达水平虽然升高, 但是表达变化倍数均较低(图3A)。因此, 1#、2#和5#样品TLR2蛋白表达水平未检测到明显改变。由于2#、3#和5#样品具有相同的菌种来源, TLR2基因相对表达水平升高说明这些内毒素中可能含有被TLR2识别的蛋白质。5种内毒素的TLR9基因相对表达水平均未升高, 说明核酸类物质含量水平较低。据报道热酚水法提取内毒素的过程中会添加核酸酶去除核酸污染, 残留的核酸酶是引起蛋白质污染的重要原因之一[23]。图3结果表明5种内毒素的蛋白质污染相较于核酸污染更明显。因此, 内毒素提取纯化过程中应加强对蛋白质污染的控制, 尤其避免核酸酶过量添加。本研究结果表明通过单核细胞活化反应测定法对细胞TLR2和TLR9进行测试, 能够有效识别生物污染中的蛋白质和核酸成分。目前内毒素中的蛋白质和核酸分别采用Lowry-Folin法和紫外分光光度法进行检测[24]; 相较而言, TLRs测试对生物污染成分的检测灵敏度更高, 有利于识别内毒素中蛋白质与核酸污染。
2.4 HL-60细胞IL-6、IL-1β和TNF-α表达差异
内毒素和非内毒素致炎效应物质均可激活机体免疫反应, 诱导细胞释放促炎细胞因子, 但是鲎试验中的鲎试剂仅与内毒素发生特异性凝集反应。因此, 鲎试验测试结果相同的内毒素, 在机体炎症诱导能力上仍可能存在差异。如图4A所示, 与对照组相比, 同为18 EU/mL的5种内毒素刺激HL-60细胞后, 从同种炎症因子基因相对表达水平看, 5种内毒素对HL-60细胞的炎症诱导能力不同。例如, 1#、2#和3#样品IL-6基因相对表达水平升高, 分别为对照组的4.39、1.61和2.55倍, 其中1#样品IL-6基因相对表达水平最高; 4#和5#样品与对照组相比, IL-6基因相对表达水平未显著升高(P>0.05)。1#、3#、4#和5#样品IL-1β基因相对表达水平升高, 分别为对照组的1.86、1.71、2.49和2.05倍, 其中4#样品IL-1β基因相对表达水平最高; 2#样品与对照组相比, IL-1β基因相对表达水平未显著升高(P>0.05)。相较于IL-6和IL-1β, TNF-α基因相对表达水平和蛋白质量浓度均未显著升高(P>0.05)(图4)。据报道革兰氏阴性菌诱导细胞释放TNF-α能力低于革兰氏阳性菌, 并且TNF-α浓度在18 h达到峰值后开始下降[25]。本研究所用内毒素均提取自革兰氏阴性菌大肠杆菌, 单核细胞活化反应测定法孵育时间为24 h, 这导致未能检测到TNF-α浓度升高(图4)。
如图4B所示, 与对照组相比, 同为18 EU/mL的5种内毒素刺激HL-60细胞后, IL-6和IL-1β蛋白质量浓度升高(P<0.05), 与二者基因相对表达水平变化情况基本一致(图4A)。从同种炎症因子蛋白释放情况看, 5种内毒素对HL-60细胞的炎症诱导能力不同。1#和3#样品IL-6蛋白质量浓度较高, 分别为103.49 pg/mL和118.16 pg/mL; 5#样品IL-6蛋白浓度最低, 为36.22 pg/mL。4#样品IL-1β蛋白质量浓度最高, 为176.34 pg/mL; 2#和5#样品IL-1β蛋白质量浓度较低, 分别为67.70 pg/mL和80.25 pg/mL。
如图4所示, 相同或不同菌种来源的内毒素在同一效价条件下诱导HL-60细胞释放炎症因子的能力不同。裴宇盛等[26]报道我国现行内毒素国家标准物质的人源单核细胞活化反应测定结果与通过鲎试验标定得到的标示值存在较大差距, 这与本研究结果一致。内毒素生物活性与其结构密切相关, 不同菌种来源内毒素的结构形态差异较大, 如脂肪酸排列方式、数目及种类等[27-28], 影响内毒素生物活性, 导致其致炎效应具有差异。此外, 非内毒素成分中的核酸、蛋白质等具有免疫刺激能力, 能引发免疫反应, 促进炎症因子释放[29-30]。本研究中2#、3#、4#和5#样品来源于同一菌种, 但是相同内毒素活性条件下炎症因子释放具有显著差异, 推测蛋白质污染是导致相同菌种来源的内毒素炎症诱导能力具有差异的重要原因之一。
3 结论
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本研究探究5种内毒素工作标准品的紫外吸收光谱与三维荧光光谱特征, 并结合细胞TLRs测试对非内毒素成分中的生物污染成分进行识别。紫外吸收光谱显示赋形剂的吸收峰; 三维荧光光谱进一步识别蛋白质污染。采用单核细胞活化反应测定法对HL-60细胞TLR2和TLR9进行测试, 结果表明5种内毒素的蛋白质污染相较于核酸污染更明显, 内毒素提取纯化过程中应加强对蛋白质污染的控制。此外, 5种内毒素均引起HL-60细胞IL-6和IL-1β基因相对表达水平以及蛋白质量浓度升高, TNF-α基因相对表达水平及其蛋白质量浓度未升高。相同或不同菌种来源的内毒素在同一效价条件下诱导HL-60细胞释放炎症因子能力具有差异。内毒素分子结构差异以及非内毒素成分中蛋白质污染是导致相同或不同菌种来源内毒素致炎效应具有差异的重要原因。本研究为内毒素相关检测方法的质量控制提供参考, 旨在提升内毒素风险评估的准确性。
基金
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  • 国家重点研发计划项目(2022YFF0609103)
  • 国家自然科学基金项目(52070193)
  • 北京市自然科学基金项目(8192053)
文献
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2025年第16卷第1期
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doi: 10.19812/j.cnki.jfsq11-5956/ts.20240830002
  • 接收时间：2024-08-30
  • 首发时间：2025-07-21
  • 出版时间：2025-01-15
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  • 收稿日期：2024-08-30
基金
国家重点研发计划项目(2022YFF0609103)
国家自然科学基金项目(52070193)
北京市自然科学基金项目(8192053)
作者信息
    1.中国检验检疫科学研究院卫生检验与检疫研究所, 北京 100176
    2.中国人民解放军疾病预防控制中心, 北京 100071
    3.中国城市规划设计研究院, 北京 100037
    4.北京建筑大学环境与能源工程学院, 北京 102616
    5.北京工商大学环境科学与工程系, 北京 100048

通讯作者:

*白淼(1990—), 女, 硕士, 工程师, 主要研究方向为环境科学与工程。E-mail:
张明露(1980—), 女, 博士, 教授, 主要研究方向为环境微生物。E-mail:
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2种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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