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细菌外膜囊泡肿瘤疫苗对胰腺癌小鼠肿瘤细胞增殖和CD8+ T细胞浸润的影响

陈志强, 马娜娜, 赵潇, 高松, 郝继辉

陈志强, 马娜娜, 赵潇, 等. 细菌外膜囊泡肿瘤疫苗对胰腺癌小鼠肿瘤细胞增殖和CD8+ T细胞浸润的影响[J]. 中华消化外科杂志, 2022, 21(4): 530-536. DOI: 10.3760/cma.j.cn115610-20220215-00087
引用本文: 陈志强, 马娜娜, 赵潇, 等. 细菌外膜囊泡肿瘤疫苗对胰腺癌小鼠肿瘤细胞增殖和CD8+ T细胞浸润的影响[J]. 中华消化外科杂志, 2022, 21(4): 530-536. DOI: 10.3760/cma.j.cn115610-20220215-00087
Chen Zhiqiang, Ma Nana, Zhao Xiao, et al. The influence of bacterial outer membrane vesicles tumor vaccine on tumor cell proliferation and CD8+ T cell infiltration of mouse with pancreatic cancer[J]. Chinese Journal of Digestive Surgery, 2022, 21(4): 530-536. DOI: 10.3760/cma.j.cn115610-20220215-00087
Citation: Chen Zhiqiang, Ma Nana, Zhao Xiao, et al. The influence of bacterial outer membrane vesicles tumor vaccine on tumor cell proliferation and CD8+ T cell infiltration of mouse with pancreatic cancer[J]. Chinese Journal of Digestive Surgery, 2022, 21(4): 530-536. DOI: 10.3760/cma.j.cn115610-20220215-00087

细菌外膜囊泡肿瘤疫苗对胰腺癌小鼠肿瘤细胞增殖和CD8+ T细胞浸润的影响

基金项目: 

国家自然科学基金 82072657

详细信息
    通讯作者:

    高松,Email:foxgao2004@163.com

The influence of bacterial outer membrane vesicles tumor vaccine on tumor cell proliferation and CD8+ T cell infiltration of mouse with pancreatic cancer

Funds: 

National Natural Science Foundation of China 82072657

More Information
  • 摘要:
    目的 

    探讨细菌外膜囊泡(OMVs)肿瘤疫苗对胰腺癌小鼠肿瘤细胞增殖和CD8+ T细胞浸润的影响。

    方法 

    采用实验研究方法。采用卵清蛋白(OVA)慢病毒载体质粒pLV‑EF1a‑hluc⁃P2A⁃mNeongreen‑CMV‑OVA‑3Xflag‑P2A‑puro构建小鼠胰腺癌Pan02‑OVA细胞;采用大肠杆菌来源ClyA⁃Catchers⁃OMVs(CC‑OMVs)与标签化抗原肽SpyTag‑OVA制备OMVs肿瘤疫苗;应用OMVs肿瘤疫苗刺激小鼠CD8+ T细胞生成,采用体外细胞杀伤实验(OMVs肿瘤疫苗刺激T细胞组和对照T细胞组)、小鼠皮下胰腺癌成瘤模型(OMVs肿瘤疫苗组和对照组)和免疫组织化学染色检测分析OMVs肿瘤疫苗抑制胰腺癌细胞增殖,刺激CD8+ T细胞浸润的效果。观察指标:(1)小鼠胰腺癌Pan02‑OVA细胞鉴定情况。(2)CC‑OMVs形态观察情况。(3)OMVs肿瘤疫苗特异性T细胞对小鼠胰腺癌Pan02‑OVA细胞增殖抑制情况。(4)OMVs肿瘤疫苗对小鼠胰腺癌的抑制情况。(5)OMVs肿瘤疫苗刺激小鼠胰腺癌组织CD8+ T细胞浸润情况。正态分布的计量资料以x±s表示,组间比较采用t检验。计数资料以绝对数或百分率表示。

    结果 

    (1)小鼠胰腺癌Pan02‑OVA细胞鉴定情况。激光共聚焦检测结果显示:感染OVA慢病毒载体质粒pLV‑EF1a‑hluc‑P2A‑mNeongreen‑CMV‑OVA‑3Xflag‑P2A‑puro的小鼠胰腺癌Pan02⁃OVA细胞表达mNeongreen绿色荧光。流式细胞检测结果显示:以小鼠胰腺癌Pan02细胞为参照,Pan02‑OVA细胞Flag蛋白表达率为90.7%。(2)CC‑OMVs形态观察情况。透射电子显微镜检测结果显示:CC‑OMVs呈均匀球形,直径<50 nm。(3)OMVs肿瘤疫苗特异性T细胞对小鼠胰腺癌Pan02⁃OVA细胞增殖抑制情况。细胞增殖毒性检测结果显示:OMVs肿瘤疫苗刺激T细胞组小鼠胰腺癌Pan02‑OVA细胞450 nm吸光度为0.41±0.12,对照T细胞组为1.05±0.15,两组比较,差异有统计学意义(t=9.54,P<0.05)。(4)OMVs肿瘤疫苗对小鼠胰腺癌的抑制情况。OMVs肿瘤疫苗组小鼠背部皮下肿瘤组织质量为(81±10)g,对照组为(153±17)g,两组比较,差异有统计学意义(t=8.26,P<0.05)。(5)OMVs肿瘤疫苗刺激小鼠胰腺癌组织CD8+ T细胞浸润情况。免疫组织化学染色检测结果显示:OMVs肿瘤疫苗组小鼠背部皮下肿瘤组织中CD8+ T细胞染色数目为(28.7±3.5)个,对照组为(9.3±1.5)个,两组比较,差异有统计学意义(t=8.74,P<0.05)。

    结论 

    细菌OMVs肿瘤疫苗可抑制胰腺癌小鼠肿瘤细胞增殖并提高肿瘤组织中CD8+ T细胞浸润数目。

    Abstract:
    Objective 

    To investigate the influence of bacterial outer membrane vesicles (OMVs) tumor vaccine on tumor cell proliferation and CD8+ T cell infiltration of mouse with pancreatic cancer.

    Methods 

    The experimental study was conducted. The ovalbumin (OVA) lentivirus vector plasmid pLV‑EF1a‑hluc‑P2A‑mNeongreen‑CMV‑OVA‑3Xflag‑P2A‑puro was used to construct the mouse pancreatic cancer Pan02‑OVA cells. The ClyA‑Catchers‑OMVs (CC‑OMVs) originated from Escherichia coli and labeled antigenic peptide SpyTag‑OVA were used to construct the OMVs tumor vaccine. Mouse CD8+ T cells were stimulated by OMVs tumor vaccine, and the effects of OMVs tumor vaccine on inhibiting pancreatic cancer cells proliferation and stimulating CD8+ T cell infiltration were analy-zed by in vitro cell killing assay, including the OMVs tumor vaccine stimulated T cell group and the control T cell group, subcutaneous pancreatic cancer model, including the OMVs tumor vaccine group and the control group, and immunohistochemical staining. Observation indicators: (1) identification of mouse pancreatic cancer Pan02‑OVA cells; (2) morphological observation of CC-OMVs; (3) inhibi-tion of mouse pancreatic cancer Pan02‑OVA cells by OMVs tumor vaccine specific T cells; (4) inhibi-tion of mouse pancreatic cancer by OMVs tumor vaccine; (5) CD8+ T cell infiltration in pancreatic cancer tissue of mouse stimulated by OMVs tumor vaccine. Measurement data with normal distribu-tion were represented as Mean±SD, and comparison between groups was analyzed using the t test. Count data were described as absolute numbers or percentages.

    Results 

    (1) Identification of mouse pancreatic cancer Pan02‑OVA cells. Results of laser scanning confocal microscopy showed that the mNeongreen fluorescence was expressed in Pan02‑OVA cells infected with the OVA lentivirus vector plasmid of pLV‑EF1a‑hluc‑P2A‑mNeongreen‑CMV-OVA‑3Xflag-P2A-puro. Results of Flow cytometry showed that using the mouse pancreatic cancer Pan02 cells as references, the protein expression rate of Flag on the Pan02‑OVA cells was 90.7%. (2) Morphological observation of CC‑OMVs. Results of transmission electron microscopy analysis showed that the CC‑OMVs were in spherical shape, with a diameter <50 nm. (3) Inhibition of mouse pancreatic cancer Pan02‑OVA cells by OMVs tumor vaccine specific T cells. Results of cell proliferation toxicity test showed that the absorbance at 450 nm of mouse pancreatic cancer Pan02‑OVA cells was 0.41±0.12 and 1.05±0.15 in the OMVs tumor vaccine‑stimulated T cell group and the control T cell group, respectively, showing a significant difference between the two groups (t=9.54, P<0.05). (4) Inhibition of mouse pancreatic cancer by OMVs tumor vaccine. The weight of subcutaneous tumor tissue in the back of mouse was (81±10)g and (153±17)g in the OMVs tumor vaccine group and the control group, respectively, showing a significant difference between the two groups (t=8.26, P<0.05). (5) CD8+ T cell infiltration in pancreatic cancer tissue of mouse stimulated by OMVs tumor vaccine. Results of immuno-histochemical staining showed that the numbers of CD8+ T cells staining in the mouse back subcu-taneous tumor tissues was 28.7±3.5 and 9.3±1.5 in the OMVs tumor vaccine group and the control group, respectively, showing a significant difference between the two groups (t=8.74, P<0.05).

    Conclusion 

    Bacterial OMVs tumor vaccine can inhibit proliferation of pancreatic cancer cells and increase the numbers of CD8+ T cells infiltrated in pancreatic cancer tissue of mouse.

  • 胰腺癌是一种高度恶性的消化系统肿瘤,手术切除率低,化疗等治疗效果有限,患者预后差,5年生存率仅10%[14]。近年来,肿瘤免疫治疗,特别是免疫检查点抑制剂等发展迅速,在多种肿瘤治疗中取得较好效果[511]。但免疫检查点抑制剂单药治疗或联合化疗均未能有效延长胰腺癌患者生存时间[1213]。主要原因是胰腺癌为“冷肿瘤”,其肿瘤组织内浸润的肿瘤特异性T细胞数量不足[1213]。笔者团队前期工作构建基于细菌外膜囊泡(outer membrane vesicles,OMVs)的肿瘤疫苗制备平台,可高效激活抗原特异性免疫反应,刺激肿瘤特异性T细胞产生[14]。本研究通过制备小鼠胰腺癌Pan02⁃OVA细胞、构建小鼠皮下胰腺癌成瘤模型,探讨细菌OMVs肿瘤疫苗对胰腺癌小鼠肿瘤细胞增殖和CD8+ T细胞浸润的影响。

    采用实验研究方法。

    1.小鼠胰腺癌Pan02细胞株购自中国医学科学院基础医学研究所细胞资源中心;293T细胞获取自中国科学院动物研究所。

    2.可稳定表达ClyA‑Catchers标记OMVs(CC⁃OMVs)的大肠杆菌由笔者团队构建[14]

    3.卵清蛋白(ovalbumin,OVA)慢病毒载体质粒pLV‑EF1a‑hluc‑P2A‑mNeongreen‑CMV‑OVA‑3Xflag‑P2A‑puro由北京合生生物科技有限公司构建;包装质粒psPAX2、pMD2G获取自中国科学院生物物理研究所。

    4.主要实验试剂:胎牛血清、DMEM培养基、Lipofectamine 3000转染试剂购自赛默飞世尔科技(中国)有限公司;病毒转染试剂聚凝胺购自北京兰博利德商贸有限公司;嘌呤霉素、4′,6‑二脒基‑2‑苯基吲哚(4′,6‑diamidino‑2‑phenylindole,DAPI)购自北京索莱宝科技有限公司;4%多聚甲醛购自北京兰杰柯科技有限公司;PE标记大鼠抗小鼠Flag单克隆抗体购自美国加州BioLegend公司;Bicincho⁃ninic acid(BCA)蛋白定量试剂盒购自天根生化科技(北京)有限公司;醋酸双氧铀购自北京中镜科仪技术有限公司;标签化抗原肽SpyTag‑OVA购自上海强耀生物科技有限公司;RBC裂解液购自上海碧云天生物技术有限公司;细胞增殖毒性检测(cell counting kit‑8,CCK‑8)试剂盒购自日本九州岛同仁化学研究所;基质胶购自美国纽约州康宁公司;兔抗小鼠CD8单克隆抗体购自英国剑桥Abcam公司;辣根过氧化物酶标记山羊抗兔二抗购自美国马萨诸塞州SeraCare公司;二氨基联苯胺(diamnobenzi⁃dine,DAB)染色试剂盒购自江苏世泰实验器材有限公司。

    5.主要实验器材:共聚焦培养皿购自康宁(美国)公司;电镜铜网购自北京中镜科仪技术有限公司;超速离心管购自美国贝克曼库尔特有限公司;孔径分别为0.45 μm和0.22 μm的滤器购自美国密理博公司;孔径为70 μm细胞筛网购自韩国京畿道SPL Lifesciences公司。

    6.实验动物:6~8周龄雌性C57BL/6小鼠购自北京维通利华实验动物技术有限公司。本研究符合动物实验和研究的相关伦理规范,所有动物实验方案均经国家纳米科学中心实验动物伦理委员会审批,批号为NCNST21‑2101‑YC01。

    7.主要实验仪器:激光共聚焦扫描显微镜为德国蔡司公司LSM700产品;流式细胞仪为安捷伦科技中国有限公司NovoCyte产品;超速离心机为美国贝克曼库尔特有限公司Optima L‑100XP产品;透射电子显微镜为日本电子株式会社JEM‑1400产品;酶标仪为美国佛蒙特州博腾仪器有限公司SILFA产品。

    1.细胞培养:小鼠胰腺癌Pan02细胞、293T细胞采用含10%胎牛血清的DMEM培养基,添加100 U/mL青霉素和100 μg/L链霉素,置于37 ℃、5% CO2培养箱中培养。

    2.OVA慢病毒制备、收集:OVA慢病毒载体质粒pLV‑EF1a‑hluc‑P2A‑mNeongreen‑CMV‑OVA‑3Xflag‑P2A‑puro、包装质粒psPAX2、包装质粒pMD2G按4∶3∶1体积比例混合后,采用Lipofectamine 3000转染试剂,按说明书感染对数生长期293T细胞;36 h后收集培养上清液,经0.45 μm孔径滤器过滤获得OVA慢病毒。

    3.小鼠胰腺癌Pan02‑OVA细胞的制备:OVA慢病毒、聚凝胺按1 000∶1体积比例混合后,感染对数生长期小鼠胰腺癌Pan02细胞;36 h后采用含2 μg/mL嘌呤霉素、10%胎牛血清的DMEM培养基筛选小鼠胰腺癌Pan02‑OVA细胞,经连续3次传代培养获得小鼠胰腺癌Pan02‑OVA细胞。

    4.小鼠胰腺癌Pan02‑OVA细胞的鉴定:(1)激光共聚焦检测。取对数生长期小鼠胰腺癌Pan02⁃OVA细胞接种于共聚焦培养皿,24 h后去除培养基,采用4 ℃ PBS漂洗细胞3次,采用4%多聚甲醛固定细胞,采用DAPI染色细胞核,采用激光共聚焦扫描显微镜观察细胞荧光。(2)流式细胞检测。收集对数生长期小鼠胰腺癌Pan02细胞和Pan02⁃OVA细胞,采用4 ℃ PBS分别漂洗细胞3次,PE标记大鼠抗小鼠Flag单克隆抗体按1∶100稀释后于4 ℃下避光孵育细胞30 min,经流式细胞仪检测小鼠胰腺癌Pan02细胞和Pan02‑OVA细胞Flag表达。

    5.CC‑OMVs的制备:参照文献[14]。收集可稳定表达CC‑OMVs的大肠杆菌培养基,于4 ℃条件下,5 000 r/min(离心半径为14.275 cm),离心10 min;收集上清液分别经0.45 μm和0.22 μm孔径滤器过滤;收集滤液于4 ℃条件下,150 000 g离心3 h;收集沉淀物用无菌PBS充分溶解,并采用BCA蛋白定量试剂盒,按照说明书检测CC‑OMVs溶液的浓度。

    6.CC‑OMVs的形态观察:取CC‑OMVs溶液10 μL滴加在电镜铜网上,室温下静置15 min后轻甩电镜铜网弃去溶液,滴加10 μL醋酸双氧铀在电镜铜网上,室温下静置15 min后轻甩电镜铜网弃去溶液,于阴凉干燥处静置电镜铜网1~2 h,采用透射电子显微镜检测CC‑OMVs形态。

    7.OMVs肿瘤疫苗的制备:参照文献[14]。分别取50 μg CC‑OMVs和50 μg SpyTag‑OVA于室温下混合获得OMVs肿瘤疫苗。

    8.小鼠T细胞的制备:取6~8周龄雌性C57BL/6小鼠,分为OMVs肿瘤疫苗组和PBS对照组,每组3只;分别于第0、3天通过腹部皮下注射100 μL OMVs肿瘤疫苗或100 μL PBS;于第10天采用脊髓脱臼法处死小鼠,取脾脏研磨后采用70 μm孔径细胞筛网过滤;收集过滤液按1∶5体积比例加入RBC裂解液,混匀后室温下静置2 min;4 ℃条件下,500 g离心5 min;收集沉淀物用无菌PBS充分重悬,经细胞计数后备用。

    9.细胞杀伤实验:取对数生长期小鼠胰腺癌Pan02‑OVA细胞接种于96孔板,分为OMVs肿瘤疫苗刺激T细胞组和对照T细胞组,每组8孔,每孔接种5 000个细胞;接种12 h后,OMVs肿瘤疫苗刺激T细胞组、对照T细胞组每孔分别加入20 000个上述实验8中OMVs肿瘤疫苗组小鼠T细胞或PBS对照组小鼠T细胞,继续培养24 h后,按照CCK‑8试剂盒操作步骤,采用酶标仪检测450 nm吸光度。

    10.OMVs肿瘤疫苗小鼠胰腺癌抑制实验:取6~8周龄雌性C57BL/6小鼠,分为OMVs肿瘤疫苗组和对照组,每组5只;取对数生长期小鼠胰腺癌Pan02‑OVA细胞,与基质胶等体积混合制成细胞浓度为1×107个/mL细胞悬液;采用1 mL胰岛素注射针吸取100 μL细胞悬液接种于OMVs肿瘤疫苗组和对照组小鼠背部皮下;OMVs肿瘤疫苗组于细胞接种后第5、9、13天通过小鼠腹部皮下注射100 μL OMVs肿瘤疫苗。对照组于同一时间点同一位置注射100 μL PBS;于细胞接种后第20天采用脊髓脱臼法处死小鼠,收集小鼠背部皮下肿瘤组织并拍照、称重。

    11.免疫组织化学染色检测:采用DAB染色方法,操作步骤参照试剂盒说明书,兔抗小鼠单克隆CD8抗体按1∶2 000稀释,辣根过氧化物酶标记山羊抗兔二抗按1∶200稀释。由2位独立的病理科医师进行双盲阅片,评估染色结果。

    (1)小鼠胰腺癌Pan02‑OVA细胞鉴定情况:激光共聚焦检测结果和流式细胞检测结果。(2)CC‑OMVs形态观察情况:透射电子显微镜检测结果。(3)OMVs肿瘤免疫特异性T细胞对小鼠胰腺癌Pan02⁃OVA细胞增殖抑制情况:CCK‑8检测结果。(4)OMVs肿瘤疫苗对小鼠胰腺癌的抑制情况:小鼠背部皮下肿瘤组织质量。(5)OMVs肿瘤疫苗刺激小鼠胰腺癌组织CD8+ T细胞浸润情况:小鼠背部皮下肿瘤组织中CD8+ T细胞免疫组织化学染色结果。

    应用SPSS 20.0统计软件进行分析。正态分布的计量资料以x±s表示,组间比较采用t检验。计数资料以绝对数或百分率表示。P<0.05为差异有统计学意义。

    激光共聚焦检测结果显示:感染OVA慢病毒载体质粒pLV‑EF1a‑hluc⁃P2A‑mNeongreen⁃CMV⁃OVA⁃3Xflag‑P2A‑puro的小鼠胰腺癌Pan02‑OVA细胞表达mNeongreen绿色荧光。见图1

    图  1  激光共聚焦检测小鼠胰腺癌Pan02‑OVA细胞 1A:细胞核4′,6‑二脒基‑2‑苯基吲哚染色;1B:细胞质或细胞核mNeongreen绿色荧光显色;1C:细胞核、细胞质荧光重叠 免疫荧光染色 高倍放大
    Figure  1.  Analysis of mouse pancreatic cancer Pan02‑OVA cells by laser scanning confocal microscopy 1A: 4′,6‑diamidino‑2‑phenylindole staining in nucleus; 1B: mNeongreen green fluorescence in cytoplasm or nucleus; 1C: Overlapping the nucleus fluorescence with the cytoplasm fluorescence Immunofluorescence staining High magnification

    流式细胞检测结果显示:以小鼠胰腺癌Pan02细胞为参照,Pan02‑OVA细胞Flag蛋白表达率为90.7%。见图2

    图  2  流式细胞检测小鼠胰腺癌Pan02细胞和Pan02⁃OVA细胞Flag蛋白表达率
    Figure  2.  Flow cytometry analysis of Flag expression in pancreatic cancer Pan02 cells and Pan02‑OVA cells

    透射电子显微镜检测结果显示:CC‑OMVs呈均匀球形,直径<50 nm。见图3

    图  3  透射电子显微镜检测ClyA‑Catchers‑细菌外膜囊泡
    Figure  3.  Transmission electron microscope analysis of ClyA‑Catchers‑bacterial outer membrane vesicles

    CCK‑8检测结果显示:OMVs肿瘤疫苗刺激T细胞组小鼠胰腺癌Pan02‑OVA细胞450 nm吸光度为0.41±0.12,对照T细胞组为1.05±0.15,两组比较,差异有统计学意义(t=9.54,P<0.001)。

    OMVs肿瘤疫苗组小鼠背部皮下肿瘤组织质量为(81±10)g,对照组为(153±17)g,两组比较,差异有统计学意义(t=8.26,P<0.001)。

    免疫组织化学染色检测结果显示:OMVs肿瘤疫苗组小鼠背部皮下肿瘤组织中CD8+ T细胞染色数目为(28.7±3.5)个,对照组为(9.3±1.5)个,两组比较,差异有统计学意义(t=8.74,P<0.001)。免疫组织化学染色检测见图4

    图  4  免疫组织化学染色检测外膜囊泡肿瘤疫苗组和对照组小鼠胰腺癌组织CD8+ T细胞 高倍放大 4A:外膜囊泡肿瘤疫苗组小鼠胰腺癌组织CD8+ T细胞染色;4B:对照组小鼠胰腺癌组织CD8+ T细胞染色
    注:外膜囊泡肿瘤疫苗组小鼠于腹部皮下注射外膜囊泡肿瘤疫苗,对照组小鼠于腹部皮下注射磷酸盐缓冲液
    Figure  4.  Immunohistochemical staining of CD8+ T cell in the mouse pancreatic cancer tissue of outer membrane vesicles group and control group 4A: CD8+ T cell staining in the the mouse pancreatic cancer tissue of outer membrane vesicles group; 4B: CD8+ T cell staining in the the mouse pancreatic cancer tissue of control group High magnification

    已有的研究结果显示:肿瘤疫苗可以刺激机体免疫系统产生肿瘤特异性T细胞,发挥杀伤肿瘤细胞、抑制肿瘤生长的作用[1517]。目前,胰腺癌肿瘤疫苗包括细胞疫苗、蛋白质或多肽类疫苗、基因疫苗等[1824]。其中,蛋白质或多肽类疫苗可以被T细胞识别免疫显性抗原决定簇,进而引发抗肿瘤免疫反应,且具有易合成、成本低、性状稳定等优点[25]

    肿瘤免疫反应的强度取决于肿瘤的“抗原强度”,但肿瘤相关抗原的免疫反应强度可能由于对自身蛋白的中央免疫耐受从而受到限制[2627]。在肿瘤形成过程中,发生非同义基因突变可产生肿瘤细胞唯一表达的新抗原,其具备的肿瘤特异性等优点成为设计肿瘤疫苗的理想靶点[2830]。已有的研究结果显示:人胰腺癌表达中等程度的非同义抗原性突变[31]。有研究者发现可以预测免疫原性的新抗原质量指标,并与患者预后相关[3234]

    本研究中,笔者基于团队前期构建的OMVs肿瘤疫苗制备平台,选择模式抗原OVA模拟肿瘤细胞特异性抗原,通过与CC‑OMVs快速反应制备OMVs肿瘤疫苗。其结果显示:OMVs肿瘤疫苗可提高小鼠胰腺癌组织中CD8+ T细胞的浸润,体外实验中OMVs肿瘤疫苗刺激的T细胞可抑制小鼠胰腺癌细胞增殖。该结果为以OMVs肿瘤疫苗制备平台,选择胰腺癌新型特异性抗原快速制备OMVs特异性肿瘤疫苗奠定基础。笔者认为:高效、经济地针对不同胰腺癌患者筛选新型特异性抗原是未来研究的发展方向[35]。此外,由于胰腺癌存在复杂的免疫微环境,联合应用针对不同分子机制的肿瘤免疫治疗,可能为提高胰腺癌治疗效果提供新思路[3640]

    综上,细菌OMVs肿瘤疫苗可抑制胰腺癌小鼠肿瘤细胞增殖并提高肿瘤组织中CD8+ T细胞浸润数目。

    陈志强、马娜娜:实验设计与实施、数据收集与统计分析、论文撰写;赵潇、高松、郝继辉:研究指导、论文修改、经费支持
    所有作者均声明不存在利益冲突
    陈志强, 马娜娜, 赵潇, 等. 细菌外膜囊泡肿瘤疫苗对胰腺癌小鼠肿瘤细胞增殖和CD8+ T细胞浸润的影响[J]. 中华消化外科杂志, 2022, 21(4): 530-536. DOI: 10.3760/cma.j.cn115610-20220215-00087.

    http://journal.yiigle.com/LinkIn.do?linkin_type=cma&DOI=10.3760/cma.j.cn115610-20220215-22087

  • 图  1   激光共聚焦检测小鼠胰腺癌Pan02‑OVA细胞 1A:细胞核4′,6‑二脒基‑2‑苯基吲哚染色;1B:细胞质或细胞核mNeongreen绿色荧光显色;1C:细胞核、细胞质荧光重叠 免疫荧光染色 高倍放大

    Figure  1.   Analysis of mouse pancreatic cancer Pan02‑OVA cells by laser scanning confocal microscopy 1A: 4′,6‑diamidino‑2‑phenylindole staining in nucleus; 1B: mNeongreen green fluorescence in cytoplasm or nucleus; 1C: Overlapping the nucleus fluorescence with the cytoplasm fluorescence Immunofluorescence staining High magnification

    图  2   流式细胞检测小鼠胰腺癌Pan02细胞和Pan02⁃OVA细胞Flag蛋白表达率

    Figure  2.   Flow cytometry analysis of Flag expression in pancreatic cancer Pan02 cells and Pan02‑OVA cells

    图  3   透射电子显微镜检测ClyA‑Catchers‑细菌外膜囊泡

    Figure  3.   Transmission electron microscope analysis of ClyA‑Catchers‑bacterial outer membrane vesicles

    图  4   免疫组织化学染色检测外膜囊泡肿瘤疫苗组和对照组小鼠胰腺癌组织CD8+ T细胞 高倍放大 4A:外膜囊泡肿瘤疫苗组小鼠胰腺癌组织CD8+ T细胞染色;4B:对照组小鼠胰腺癌组织CD8+ T细胞染色

    注:外膜囊泡肿瘤疫苗组小鼠于腹部皮下注射外膜囊泡肿瘤疫苗,对照组小鼠于腹部皮下注射磷酸盐缓冲液

    Figure  4.   Immunohistochemical staining of CD8+ T cell in the mouse pancreatic cancer tissue of outer membrane vesicles group and control group 4A: CD8+ T cell staining in the the mouse pancreatic cancer tissue of outer membrane vesicles group; 4B: CD8+ T cell staining in the the mouse pancreatic cancer tissue of control group High magnification

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出版历程
  • 收稿日期:  2022-02-14
  • 网络出版日期:  2024-07-04
  • 刊出日期:  2022-04-19

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