新城疫病毒对人前列腺癌细胞LNCaP免疫性细胞死亡标志物表达的影响
Effect of Newcastle disease virus on expression of immunogenic cell death markers expression in LNCaP human prostate cancer cells
投稿时间:2019-01-17  
DOI:10.11724/jdmu.2019.04.02
中文关键词:  新城疫病毒  人前列腺癌细胞  免疫性细胞死亡标志物
英文关键词:Newcastle disease virus  human prostate cancer  immunogenic cell death markers
基金项目:基金项目:国家自然科学基金项目(81803090,81702311);上海市科委科研项目(18DZ1910102,17411967300);申康临床三年行动计划项目(16CR3072B);崇明区科学技术委员会科技项目(CKY2018-34)
作者单位
王雪珂 同济大学医学院附属第十人民医院 肿瘤科上海 200072
同济大学医学院附属上海皮肤病医院 肿瘤科上海 200443 
邵晓雁 同济大学医学院附属第十人民医院 肿瘤科上海 200072
同济大学医学院附属上海皮肤病医院 肿瘤科上海 200443 
龚卫华 同济大学医学院附属第十人民医院崇明分院 肿瘤科上海 202150 
陈健华 同济大学医学院附属第十人民医院 肿瘤科上海 200072
同济大学医学院附属上海皮肤病医院 肿瘤科上海 200443 
孟松树 大连医科大学 肿瘤干细胞研究院辽宁 大连 116044 
许 青 同济大学医学院附属第十人民医院 肿瘤科上海 200072
同济大学医学院附属上海皮肤病医院 肿瘤科上海 200443 
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中文摘要:
      目的 探讨新城疫病毒(NDV)能否诱导人前列腺癌细胞LNCaP免疫性细胞死亡(ICD)标志物的表达。方法 病毒滴度和蛋白质免疫印迹(Western Blot,WB)检测NDV在细胞内的复制情况;CCK-8法检测细胞增殖情况;蛋白质免疫印迹(Western Blot,WB)检测细胞上清中的热休克蛋白70/90(HSP70/90)及高迁移率族蛋白1(HMGB1);流式细胞术和免疫荧光检测细胞膜上钙网蛋白(calreticulin,CRT)表达量;荧光素酶法检测细胞外ATP的释放。结果 NDV可以在LNCaP细胞内发生复制,并随着时间的增加而增加,P<0.05。NDV感染细胞的增殖能力较未感染组随时间逐渐降低,P<0.001。NDV感染细胞较未感染细胞,细胞上清中HSP90的表达明显增加,并呈时间依懒性,在细胞上清中可以检测到HSP70和HMGB1的表达。两组细胞的裂解液中HSP70/90和HMGB1的表达未有统计学差异,P>0.05。NDV感染后,细胞表面CRT表达阳性率较未感染组显著增加,差异有统计学意义,P<0.001。另外,病毒感染细胞较未感染组向外释放ATP增加,差异有统计学意义,P<0.05。结论 NDV可诱导人前列腺癌细胞发生免疫性细胞死亡,为溶瘤病毒NDV结合当前免疫疗法和化疗治疗前列腺癌提供了理论依据。
英文摘要:
      Objective To investigate the effect of Newcastle disease virus (NDV) on the expression of immunogenic cell death markers in castration-resistant prostate cancer cell line LNCaP. Methods Viral titer assay and Western Blot (WB) were used to detect NDV replication in cells. CCK-8 assay was used to examine the growth inhibiting effects of NDV on LNCaP cells. Western Blot(WB) was used to detect heat shock protein 70/90 (HSP70/90) and high mobility group protein 1(HMGB1). Flow cytometry and confocal microscopy were used to detect the expression of calreticulin(CRT)on the cell membrane. Fluorescein enzymatic method was used to detect ATP release. Results NDV can replicate in LNCaP cells and increase with time (P<0.05). Compared with the non-infected group, the proliferation ability of NDV infected cells decreased gradually with time (P<0.001). Compared with non-infected cells, HSP90 expression in the cell supernatant of NDV infected cells was significantly increased, showing time-dependence. HSP70 and HMGB1 may also be detected in the cell supernatant. In cell lysates, HSP70/90 and HMGB1 expression were not significantly different between two groups (P>0.05). After NDV infection, the positive rate of CRT expression on cell surface was significantly increased compared with that in the non-infected group (P<0.001). In addition, compared with the non-infected group, ATP release from cells increased (P<0.05).Conclusions NDV could induce immunogenic cell death in human prostate cancer cells. This study could provide a theoretical basis for the treatment of prostate cancer by oncolytic virus NDV combined with current immunotherapy and chemotherapy.
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