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中华微生物学和免疫学杂志
北京生物制品研究所
中华微生物学和免疫学杂志

北京生物制品研究所

沈心亮

月刊

0254-5101

cjmia@163.com

010-52245168

100024

北京市朝阳区三间房南里4号

中华微生物学和免疫学杂志/Journal Chinese Journal of Microbiology and ImmunologyCSCD北大核心CSTPCD
查看更多>>中华医学会主办。本刊以医学微生物学和免疫学的基础和应用研究及其新理论、新技术以及国内外研究进展为报道内容。设有细菌学、病毒学、分子微生物学、临床微生物学、感染免疫、疫苗学、基础免疫学、临床免疫学、分子免疫学、免疫遗传、肿瘤免疫、中药免疫、免疫治疗、免疫学技术、检测技术、述评、综述、学术会议评述、人物述林、医药信息、新书介绍等栏目。读者为本专业科研人员、教师和高、中级卫生防疫、检验工作者及大学以上学生。本刊是中国生物科学和基础医学两学科核心期刊；被CA、BA、EM、Medline等国内外多种著名检索系统收录。杂志曾获国家优秀科技期刊奖并多次获省部级优秀科技期刊奖。2001年进入国家新闻出版署建设的“中国期刊方阵”。2008年获得中国科学技术信息研究所“中国精品科技期刊”称号。
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    潜心笃志　行稳致远

    李启明
    1页
    查看更多>>摘要：岁序更迭，华章日新。2023年，我们砥砺奋进，笃定前行，经受了磨砺，也取得了收获。
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    肺炎克雷伯菌耐药靶标KPC-2的免疫原性和保护效果评价及保护机制初步研究

    王晓琼明光阳陈致富苟强...
    2-10页
    查看更多>>摘要：目的 采用肺炎克雷伯菌耐药靶标KPC-2为抗原制备重组蛋白疫苗，在小鼠肺炎模型中评价疫苗的免疫原性、保护效果和保护机制。 方法 利用大肠埃希菌原核表达系统表达肺炎克雷伯菌KPC-2蛋白，经GST亲和层析对目的蛋白进行纯化；采用KPC-2蛋白制备疫苗，经皮下注射免疫新西兰兔，心脏采血分离血清，用Protein G亲和层析纯化多克隆抗体，同时运用调理吞噬杀菌试验检测抗体的体外杀菌活性。采用KPC-2疫苗免疫雌性BALB/c小鼠3次，间接ELISA检测血清中特异性IgG抗体滴度。末次免疫后7 d，通过气管插管构建小鼠肺炎克雷伯菌肺部感染模型，感染1 h后尾静脉给予0.1 mg美罗培南治疗，通过比较免疫组与佐剂组的生存率、细菌定植量和组织病理学差异，以及给药组与生理盐水组生存率的差异评价疫苗的保护效果。采用KPC-2多克隆抗体被动免疫评价抗体的保护效果。 结果 KPC-2免疫组小鼠血清特异性IgG水平显著高于对照组(t＝4.325, P<0.05)，免疫组生存率显著高于对照组[70% (7/10)vs 10% (1/10)，P<0.05]；免疫组小鼠肺组织炎性程度及细菌定植量显著低于对照组(t＝3.127, P<0.05)；疫苗主动免疫与被动免疫均具有良好的保护效果，且对抗生素治疗有明显的增效性；KPC-2多克隆抗体在体外表现出明显的吞噬杀菌活性(t＝5.427, P<0.05)。 结论 KPC-2疫苗具有良好的免疫原性和保护效果，体液免疫应答在其保护中发挥了重要作用，证明采用耐药靶标作为抗原制备疫苗具有可行性。 Objective To develop a recombinant protein vaccine based on KPC-2, a drug resistance target in Klebsiella pneumoniae, and evaluate its immunogenicity, protective efficacy and mechanism in a mouse model of pneumonia. Methods KPC-2 was expressed in Escherichia coli and purified using GST affinity chromatography. A recombinant protein vaccine was prepared with KPC-2 and used to immunize New Zealand rabbits through subcutaneous injection. Serum samples were isolated from cardiac blood and Protein G chromatography was used to purify polyclonal antibodies against KPC-2. Opsonophagocytic killing assay was used to assess the bactericidal activity of the polyclonal antibodies in vitro. Female BALB/c mice were immunized three times with the recombinant protein vaccine, and the titers of specific IgG antibodies in serum were measured by indirect ELISA. One week after the last vaccination, the mice were infected with Klebsiella pneumoniae strain SRT through tracheal intubation, and received a single intravenous dose of meropenem (0.1 mg) 1 h later. The protective efficacy of the KPC-2 recombinant protein vaccine was evaluated by comparing the survival rates, bacterial colonization and histopathological changes between vaccine group and adjuvant group as well as the survival rates between meropenem group and normal saline group. Moreover, the protective efficacy of polyclonal antibodies against KPC-2 was evaluated through passive immunization. Results The level of specific IgG antibodies in serum was significantly higher in the vaccine group than in the adjuvant group (t=4.325, P<0.05). The survival rate in the vaccine group was also higher than that of the adjuvant group [70% (7/10)vs 10% (1/10), P<0.05]. Furthermore, lung inflammation was less severe and bacterial burden was reduced in the vaccine group as compared with those of the control group (t=3.127, P<0.05). Both active and passive vaccination strategies demonstrated strong protective efficacy againstKlebsiella pneumoniae infection, and had a synergistic effect when used in combination with antibiotic therapy. The polyclonal antibodies against KPC-2 had bactericidal activity in vitro (t=5.427, P<0.05). Conclusions The prepared KPC-2 vaccine has better immunogenicity and protective efficacy. It can induce strong humoral immune responses. This study suggest that drug resistance target may be used as a candidate antigen for future vaccine development.

    肺炎克雷伯菌KPC-2细菌耐药疫苗

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    鹦鹉热衣原体SINC蛋白激活宿主细胞MAPK/ERK信号通路抑制细胞凋亡

    李昀霏曾心靛罗宇辰肖翠...
    11-16页
    查看更多>>摘要：目的 探讨鹦鹉热衣原体SINC蛋白对宿主细胞凋亡的影响，及丝裂原活化蛋白激酶/细胞外信号调节激酶(mitogen-activated protein kinase/extracellular signal-regulated kinase，MAPK/ERK)信号通路在其中的调控作用。 方法 用重组SINC蛋白刺激HeLa细胞，Western blot检测凋亡相关蛋白Bax和Bcl-2的蛋白质表达水平及ERK1/2的磷酸化程度，Hoechst 33258染色检测SINC蛋白刺激对HeLa细胞凋亡的影响。用50 μmol/L MEK1/2抑制剂U0126预处理HeLa细胞，流式细胞术检测不同浓度SINC蛋白刺激后细胞凋亡率的变化，Western blot检测Bax和Bcl-2的蛋白质表达水平。 结果 Western blot检测结果显示，用10 μg/ml SINC蛋白刺激HeLa细胞18 h后，Bax表达下调，Bcl-2表达上调，Bax/Bcl-2比值最低；p-ERK1/2与ERK1/2的比值明显上调；Hoechst 33258染色检测发现5、10、15 μg/ml SINC蛋白刺激后，HeLa细胞内凋亡小体数量明显减少。加入抑制剂U0126后，Bcl-2表达下调，cleaved PARP表达量显著增加；流式细胞术显示细胞凋亡率明显增高。 结论 SINC蛋白通过激活MAPK/ERK信号通路抑制HeLa细胞发生凋亡。 Objective To investigate the effects of SINC, a novel secreted protein of Chlamydia psittaci, on the apoptosis of host cells and the regulatory role of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway in it. Methods HeLa cells were treated with recombinant SINC. The expression of Bax and Bcl-2 at protein level and the phosphorylation of ERK1/2 were analyzed by Western blot. Hoechst 33258 staining was used to detect the apoptosis of HeLa cells after SINC stimulation. Moreover, HeLa cells were pretreated with MEK1/2 inhibitor U0126 (50 μmol/L), and then stimulated with different concentrations of SINC for different time. Flow cytometry was used to detect the changes in cell apoptosis rates and Western blot was performed to detect the expression of Bax and Bcl-2 at protein level. Results Treating HeLa cells with 10 μg/ml of SINC for 18 h resulted in down-regulated Bax and up-regulated Bcl-2 at protein level. Besides, the ratio of Bax/Bcl-2 was the lowest and a significant increase in the ratio of phosphorylated ERK1/2 (p-ERK1/2) to ERK1/2 was observed. Hoechst 33258 staining showed that the number of apoptotic bodies decreased significantly after stimulating HeLa cells with 5, 10 and 15 μg/ml of SINC. In the presence of MEK1/2 inhibitor U0126, the expression of Bcl-2 at protein level was down-regulated, while the expression of cleaved PARP was significantly up-regulated. Flow cytometry showed a significantly enhanced apoptosis of HeLa cells. Conclusions SINC can inhibit the apoptosis of HeLa cells through activating the MAPK/ERK signaling pathway.

    鹦鹉热衣原体SINC蛋白MAPK/ERK通路凋亡

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    粉防己碱通过cGAMP介导的cGAS-STING信号通路增强宿主的抗病毒反应

    王志文张亚明解智慧
    17-26页
    查看更多>>摘要：目的 研究粉防己碱是否可以联合cGAMP作为cGAMP的激动剂增强cGAS-STING信号通路，从而探究粉防己碱的抗病毒功能。 方法 采用不同浓度的粉防己碱联合cGAMP分别处理THP1-Lucia-ISRE及RAW-Lucia-ISRE细胞，荧光素酶报告试验探究IRF3免疫应答水平；Western blot检测cGAS-STING信号通路激活情况；实时荧光定量PCR检测IFN-β、CXCL10及CCL5的mRNA表达水平；ELISA检测IFN-β表达情况。构建稳定表达STING-GFP基因的HeLa细胞(HeLa-STNG-GFP)，粉防己碱联合cGAMP处理该细胞后，观察STING-GFP点聚集现象。Ⅰ型疱疹病毒(herpes simplex virus type 1，HSV-1)感染THP1-Lucia-ISG细胞，粉防己碱联合cGAMP处理细胞，Western blot及荧光强度探究粉防己碱的抗病毒能力。 结果 粉防己碱能够增强cGAMP介导的IRF3免疫应答，与cGAMP联用可激活cGAS-STING信号通路。粉防己碱联合cGAMP处理HeLa-STNG-GFP细胞后，观察到明显的STING-GFP点聚集现象。对感染HSV-1的THP1-Lucia-ISG细胞进行粉防己碱联合cGAMP孵育，HSV-1病毒滴度、HSV-糖蛋白D/UL30的表达及HSV-GFP荧光强度均显著下降。 结论 粉防己碱联合cGAMP可激活cGAS-STING信号通路从而增强宿主的抗病毒反应。 Objective To investigate whether tetrandrine could be used as an agonist of cGAMP to enhance the activation of cGAS-STING signaling pathway and analyze the antiviral function of tetrandrine. Methods THP1-Lucia-ISRE and RAW-Lucia-ISRE cells were incubated with different doses of tetrandrine in combination with cGAMP, respectively. IRF3 reporter activity was analyzed by luciferase reporter assay. Western blot was used to detect the activation of cGAS-STING signaling pathway. The expression of IFN-β, CXCL10 and CCL5 at mRNA level was quantified by real-time quantitative PCR. The expression of IFN-β at protein level was assessed by ELISA. HeLa cells stably expressing STING-GFP gene (HeLa-STNG-GFP cells) were constructed and stimulated with tetrandrine and cGAMP, then puncta-like structures were imaged by ZEISS LSM780. THP1-Lucia-ISRE cells were infected with herpes simplex virus type 1 (HSV-1) in the presence or absence of tetrandrine or cGAMP. The antiviral function of tetrandrine was analyzed by Western blot and fluorescence intensity assay. Results Tetrandrine enhanced cGAMP-mediated IRF3 responses and activated cGAS-STING signaling pathway in combination with cGAMP. Tetrandrine combined with cGAMP triggered STING translocation and the formation of puncta-like structures in HeLa-STNG-GFP cells. The titer of HSV-1, the expression of HSV-glycoprotein D/UL30 and the fluorescence intensity of HSV-GFP were all decline after treating HSV-1-infected THP1-Lucia-ISRE cells with tetrandrine and cGAMP. Conclusions Tetrandrine combined with cGAMP activates cGAS-STING signaling pathway, thus enhancing the host antiviral response.

    粉防己碱cGAMPcGAS-STING信号通路Ⅰ型疱疹病毒

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    单核/巨噬细胞在发热伴血小板减少综合征中的研究进展

    刘亚男郑昕
    27-33页
    查看更多>>摘要：发热伴血小板减少综合征(severe fever with thrombocytopenia syndrome, SFTS)是大别班达病毒(Dabie bandavirus, DBV)感染引起的一种以发热、白细胞和血小板减少、多器官损伤为临床特点的新发突发传染病。DBV导致的免疫系统紊乱是SFTS发病的重要机制之一。单核/巨噬细胞是固有免疫的重要成员，是DBV感染的靶细胞，其与病毒的相互作用在DBV致病过程中扮演重要角色。本文就DBV感染人体后单核/巨噬细胞介导的免疫效应特点及机制的相关研究进展进行综述。 Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by Dabie bandavirus (DBV), characterized by fever, leukopenia, thrombocytopenia and multiple organ damage. Immune dysfunction induced by DBV is closely associated with the pathogenesis of SFTS. Monocytes/macrophages that are essential in innate immunity are the target cells of DBV, and their interaction with DBV plays an important role in the pathogenesis of SFTS. The review summarizes the progress in the features and mechanisms of monocyte/macrophage-mediated immune responses to DBV infection.

    发热伴血小板减少综合征单核/巨噬细胞细胞因子风暴

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    微流控技术在抗感染免疫研究中的应用

    柳博洋朱琳静方芳齐妍...
    34-38页
    查看更多>>摘要：抗感染免疫是机体免疫系统抵抗病原体感染的第一道免疫防线，涉及多种免疫细胞活化、迁移及病原体清除过程。因此，免疫细胞行为及病原体检测就成为疾病诊断和预测的重要指标。近年来，基于微流控芯片开发的多种免疫细胞行为检测技术，以及细菌生长和药物筛查方法，因具有微型化、高通量、高敏感度、快速分析及低消耗等优势，已经在生物学、药理学及临床疾病研究和诊断中广泛使用。因此，本文对微流控技术在固有免疫细胞迁移、细胞核变形、致病菌及病毒快速检测等抗感染免疫研究中的应用进行综述，希望能进一步推动微流控技术在抗感染免疫研究和临床诊断中的应用发展。 The immune response against infection is a multifaceted process encompassing the activation and migration of diverse immune cells, as well as the clearance of pathogens. The behaviors of immune cells and the identification of pathogens play pivotal roles as indicators for disease diagnosis and prediction. In recent years, the utilization of microfluidic chip technology has gained substantial attention within the areas of biology, pharmacology, and clinical research and diagnosis. This is primarily attributed to the numerous advantages it offers, including miniaturization, enhanced throughput, heightened sensitivity, expedited analysis, and reduced sample consumption. As a result, microfluidic technology has facilitated the development and utilization of immune cell behavioral assays, bacterial growth studies, and drug-screening assays. This paper is to review the application of microfluidic technology in the field of anti-infection immunity research, focusing on the analysis of migratory behavior of innate immune cells, deformation of their nuclei, and rapid identification of pathogenic bacteria and viruses. The primary objective of this review is to advance the application of microfluidic technology in research on both anti-infection immunity and clinical diagnosis.

    微流控芯片感染免疫细胞迁移细菌快速检测

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    革兰阴性菌外膜囊泡在感染和免疫调节中的研究进展

    李一帆刘心伟李永伟
    39-43页
    查看更多>>摘要：革兰阴性菌释放的细胞外囊泡源于细胞最外层膜，被称为外膜囊泡(outer membrane vesicle，OMV)，含有磷脂、脂多糖、外膜蛋白和细菌特异性抗原等细菌外膜成分。OMV在细菌生理学和致病机制中发挥重要作用，可参与生物膜形成、基因水平转移、应激和炎症反应、毒素和其他生物分子的传递等，且在免疫调节以及肠道微生物群的建立和平衡中也起着重要作用。本文对OMV在细菌感染及免疫调节中的作用进行综述，为细菌导致的感染性疾病预防和治疗提供新的思路，并对OMV在肿瘤靶向治疗和新型疫苗制备中的潜在应用价值进行展望。 Outer membrane vesicle (OMV), originating from the outermost membrane of cells, is the extracellular vesicles released by gram-negative bacteria, containing bacterial outer membrane components such as phospholipids, lipopolysaccharide (LPS), outer membrane protein, and bacterial-specific antigens. OMV plays an important role in bacterial physiology and pathogenesis, involving in biofilm formation, horizontal gene transfer, stress and inflammatory responses, and delivery of toxins and other biomolecules. It also plays a vital role in immune regulation and the establishment and maintenance of balanced intestinal microflora. This article provides an overview of the roles of OMV in bacterial infections and immune regulation and the potential application value of OMV in tumor-targeted therapy and new vaccine preparation in the hope to provide new ideas for the prevention and treatment of bacterial infections.

    革兰阴性菌外膜囊泡细菌感染免疫调节

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    一株降低了抗体依赖性增强效应的西尼罗病毒中和抗体的表达与体外活性研究

    郝相君陈楠朱婉露王晶...
    44-49页
    查看更多>>摘要：目的 建立降低抗体依赖性增强(antibody-dependent enhancement，ADE)效应的抗体表达系统，以期降低目标抗体的ADE效应。 方法 对哺乳细胞抗体表达载体Fc区进行L234A和L235A点突变，建立抗体表达载体pFRT-IgG1κ-FcM。选取前期工作中获得的1株具有显著ADE效应的抗体Wt-WNV利用pFRT-IgG1κ-FcM系统进行表达，获得突变后抗体FcM-WNV。ELISA检测FcM-WNV与靶抗原西尼罗病毒包膜蛋白DⅢ(West Nile virus envelope protein-DⅢ，WNV E-DⅢ)的结合，流式细胞术分析FcM-WNV与人高亲和力IgG Fc受体hFcγRⅠ(hCD64)的结合。假病毒感染宿主细胞(BHK21和K562)试验检测FcM-WNV的体外中和活性。 结果 FcM-WNV与Wt-WNV表达水平相当，能识别结合WNV E-DⅢ，且呈浓度依赖效应；与Wt-WNV相比，FcM-WNV与hCD64的结合力明显减弱，表现为荧光强度明显降低；与前期实验结果一致，Wt-WNV在5 μg/ml的浓度下具有明显增强WNV假病毒感染K562细胞的作用，而FcM-WNV在5 μg/ml浓度下，能有效阻断假病毒感染K562和BHK21细胞。 结论 本研究建立的抗体表达系统可有效降低目标抗体的ADE效应。 Objective To establish an antibody expression system to reduce the antibody-dependent enhancement (ADE) effect of target antibody. Methods Site-directed mutagenesis was used to mutate the 234 and 235 sites of the Fc region of the mammalian cell antibody expression vector-L234A and L235A to establish the antibody expression vector pFRT-IgG1κ-FcM. An antibody Wt-WNV with significant ADE effect obtained in previous work was selected and expressed by the pFRT-IgG1κ-FcM system to obtain mutant antibody FcM-WNV. The binding ability of FcM-WNV to target antigen West Nile virus envelope protein-DⅢ (WNV E-DⅢ) was detected by ELISA, and the its binding ability to human high-affinity IgG Fc receptor hFcγRⅠ (hCD64 ) was analyzed by flow cytometry. The neutralizing activity of FcM-WNVin vitro was detected by pseudovirus infection of host cells (BHK21 and K562). Results The expression levels of FcM-WNV and Wt-WNV were comparable, and FcM-WNV could recognize and bind to WNVE-DIII in a concentration-dependent manner. Compared with Wt-WNV, the binding ability of FcM-WNV to hCD64 was significantly weakened, showing a significant decrease in fluorescence intensity. Consistent with the previous experimental results, Wt-WNV at a concentration of 5 μg/ml significantly enhanced the infection of K562 by WNV pseudovirus, while FcM-WNV at a concentration of 5 μg/ml could effectively block pseudovirus infection in both K562 and BHK21 cells. Conclusions The established antibody expression system can effectively reduce the ADE effect of the target antibody.

    中和抗体西尼罗病毒抗体依赖性增强效应

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    过敏原对过敏性鼻炎患者外周血CD4 ＋Th17细胞IL-18、IL-18结合蛋白a和IL-18受体α表达水平的影响

    王君灵湛萌萌杜恩明王思勤...
    50-57页
    查看更多>>摘要：目的 分析过敏性鼻炎(allergic rhinitis，AR)患者外周血CD4＋Th17细胞中IL-18、IL-18结合蛋白a(IL-18-binding protein a, IL-18BPa)和IL-18受体α(IL-18 receptor α，IL-18Rα)的表达水平及过敏原对其表达的影响。 方法 选择2019年10月—2020年9月锦州医科大学附属第一医院过敏反应科门诊AR患者45例和该医院体检中心体检者23例(健康对照组)为研究对象。根据皮肤点刺试验结果，将患者分为阳性组(AR组，24例)和阴性组(nAR组，21例)。收集受试者血液样本，流式细胞术检测过敏原对CD4＋Th17细胞中IL-18、IL-18BPa和IL-18Rα蛋白质表达水平的影响。Bioplex检测血浆中IL-17A水平，并分析其与IL-18＋Th17细胞比例的相关性。 结果 与健康对照组比较，AR组患者CD4＋Th17细胞和IL-18＋Th17细胞比例均增加(P<0.01)，IL-18BPa＋Th17细胞比例降低(P<0.01)，IL-18BPa的平均荧光强度(mean fluorescence intensity，MFI)增加(P<0.01)，IL-18Rα的MFI降低(P<0.01)；nAR组患者CD4＋Th17细胞IL-18BPa的MFI增加(P<0.000 1)，IL-18Rα的MFI降低(P<0.000 1)。与nAR组患者比较，AR组患者IL-18＋Th17细胞比例(P<0.05)和IL-18Rα的MFI均增加(P<0.01)。户尘螨和梧桐花粉过敏原粗提液能分别诱导AR组患者IL-18＋Th17和IL-18BPa＋Th17细胞比例增加(P<0.05)。户尘螨过敏原粗提液可直接诱导分选后的健康对照者CD4＋Th17细胞表达IL-18和IL-18Rα(P<0.05)。AR组患者血浆IL－17A水平升高(P<0.001)，并与IL-18＋Th17细胞比例中度相关(P<0.05)。 结论 过敏原可能通过诱导外周血CD4＋Th17细胞表达IL-18和IL-18Rα参与AR发病。 Objective To investigate the expression of IL-18, IL-18-binding protein a(IL-18BPa) and IL-18 receptor α(IL-18Rα) by peripheral blood CD4 + Th17 cells of patients with allergic rhinitis (AR) and the effects of allergens on their expression. Methods This study enrolled 45 outpatients with AR and 23 healthy control subjects receiving physical examination in the First Affiliated Hospital of Jinzhou Medical University from October 2019 to September 2020. According to the results of skin prick test, the 45 patients were divided into two groups: AR group with positive results (24 cases) and nAR group with negative results (21 cases). Blood samples of them were collected. Flow cytometry was used to analyze the effects of allergens on the expression of IL-18, IL-18BPa and IL-18Rα at protein level by peripheral blood CD4+ Th17 cells. The level of IL-17A in plasma was measured by Bioplex system, and its correlation with the percentage of IL-18+ Th17 cells was analyzed. Results Compared with the healthy control group, the AR group showed increased ratios of CD4+ Th17 and IL-18+ Th17 cells (P<0.01), decreased ratio of IL-18BPa+ Th17 cells (P<0.01), enhanced mean fluorescence intensity (MFI) of IL-18BPa (P<0.01) and reduced MFI of IL-18Rα (P<0.01) the nAR group showed enhanced MFI of IL-18BPa (P<0.000 1) and reduced MFI of IL-18Rα (P<0.000 1). The ratio of IL-18+ Th17 cells and the MFI of IL-18Rα in the AR group were higher than those in the nAR group (P<0.05,P<0.01). House dust mite extract andPlatanus pollen extract induced the expression of IL-18 and IL-18BPa by CD4+ Th17 cells of AR patients (P<0.05). Moreover, house dust mite extract directly induced the CD4+ Th17 cells isolated from the healthy control subjects to express IL-18 and IL-18R (P<0.05). Compared with healthy control subjects, AR patients had higher level of IL-17A in plasma and it was moderately correlated with the ratio of IL-18+ Th17 cells (P<0.05). Conclusions Allergens may be involved in the pathogenesis of AR by inducing blood CD4+ Th17 cells to express IL-18 and IL-18Rα.

    过敏性鼻炎CD4＋Th17细胞IL-18IL-18Rα过敏原

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    表达荧光素酶的胰腺癌细胞系的建立及其在免疫细胞杀伤效力检测中的应用

    梁谦许崇凤李国亚张丽霞...
    58-66页
    查看更多>>摘要：目的 构建表达报告基因荧光素酶和肿瘤相关抗原间皮素(mesothelin，MSLN)基因的胰腺癌细胞系，评估其作为靶细胞在评价免疫细胞的肿瘤杀伤效力中的应用。 方法 构建表达荧光素酶、MSLN基因的慢病毒载体，包装慢病毒，感染胰腺癌细胞系，抗生素筛选后，有限稀释法获得单细胞克隆，并验证目的基因的稳定表达。实时无标记细胞分析(real-time cell analysis，RTCA)和荧光素酶活性检测方法体外检测免疫细胞对肿瘤细胞的杀伤效力。将构建的细胞系接种B-NDG小鼠建立表达荧光素酶的胰腺癌肿瘤模型，利用活体成像技术检测荧光素酶表达水平，监测小鼠体内胰腺癌肿瘤生长情况。在胰腺癌小鼠模型中验证嵌合抗原受体T(chimeric antigen receptor T，CAR-T)细胞的肿瘤杀伤能力。 结果 本研究建立了稳定表达荧光素酶和MSLN基因的胰腺癌细胞系panc-1-luc、panc-1-luc-MSLN和capan-2-luc细胞。3种细胞的报告基因表达水平较高，与细胞数量呈正相关，panc-1-luc-MSLN细胞的MSLN阳性率达95.6%。体外肿瘤细胞杀伤试验结果表明MSLN-CAR-T细胞能特异性杀伤MSLN抗原阳性的panc-1-luc-MSLN细胞和capan-2-luc细胞，最小杀伤率分别为(70.00±18.19)%和(57.00±5.29)%，而对MSLN阴性的panc-1-luc细胞无杀伤作用。RTCA结果显示MSLN-CAR-T细胞对构建的3种胰腺癌细胞均有杀伤能力，最小杀伤率分别为(56.33±7.64)%、(93.00±2.65)%和(26.33±28.15)%；NK-92MI细胞对靶细胞的杀伤不受MSLN限制。免疫缺陷小鼠胰腺癌模型结果显示，体内杀伤效力与体外荧光素酶活性检测结果一致。体内外试验证明，检测靶细胞荧光素酶表达活性，可以反映免疫细胞对靶细胞的杀伤效力。 结论 本研究建立了稳定表达荧光素酶的多株胰腺癌单克隆细胞系，可用于体内外免疫治疗产品肿瘤杀伤效力的研究评价。 Objective To construct pancreatic cancer cell lines expressing luciferase and mesothelin (MSLN), and evaluate the feasibility of using them as target cells in analyzing the cytotoxicity activity of immune cells. Methods Lentiviral vectors expressing luciferase and MSLN genes were constructed, and pancreatic cancer cell lines were infected after lentivirus packaging. Single-cell clones were obtained by limited dilution following antibiotic screening, and the stable expression of the target genes were verified. These cells were used as target cells to detect the cytotoxicity of immune cells by real-time cell analysis (RTCA) and luciferase activity. Besides, these luciferase-expressing cells were transplanted into B-NDG mice to establish the animal models of pancreatic cancer, and in vivo optical imaging technology was used to detect the expression of luciferase and monitor the tumor growth in mice. The cytotoxicity of chimeric antigen receptor T (CAR-T) cells was verified in these animal models. Results Three pancreatic cancer cell lines, panc-1-luc, panc-1-luc-MSLN and capan-2-luc, that could stably express luciferase and MSLN genes were successfully constructed. The expression of the reporter gene in these cells were high, and positively correlated with the number of cells. There were 95.6% of panc-1-luc-MSLN cells expressing MSLN. MSLN-CAR-T cells had specific killing effect on MSLN-positive panc-1-luc-MSLN cells and capan-2-luc cells, with the minimum killing rates of (70.00±18.19)% and (57.00±5.29)%, respectively. But they had no cytotoxicity to MSLN-negative panc-1-luc cells. RTCA results showed that MSLN-CAR-T cells were able to lyse all three pancreatic cancer cell lines, and the minimum killing rates were (56.33±7.64)%, (93.00±2.65)% and (26.33±28.15)%, respectively. The killing of target cells by NK-92MI cells was not depended on MSLN expression. The cytotoxicity in the mice models of pancreatic cancer was consistent with the results in vitro. The in vivo and in vitro test results suggested that the expression of luciferase by target cells could reflect the cytotoxicity of immune cells. Conclusions This study establishes three pancreatic cancer cell lines stably expressing luciferase, which can be used to evaluate the cytotoxicity of immunotherapy products targeting tumor cells in vitro and in vivo.

    胰腺癌荧光素酶间皮素活体成像技术免疫治疗

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