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

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

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。

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.