裂亡前列腺癌细胞外囊泡对PC3细胞活性的影响  

The effect of mitotic death prostate cancer extracellular vesicles on PC3 cells

作　　者：史剑 邹一鸣 孙阳洋[1] 吴晓彤 沈宇炜 范敏[1] Shi Jian;Zou Yiming;Sun Yangyang;Wu Xiaotong;Shen Yuwei;Fan Min(Department of Urology,The First People's Hospital of Changzhou Affliated with Soochow University,Changzhou 213000,China;Department of Urology,Wuhan East and West Lake People's Hospital Affiliated with Wuhan University of Science and Technology,Wuhan 430000,China)

机构地区：[1]苏州大学附属常州市第一人民医院泌尿外科,常州213000 [2]武汉科技大学附属东西湖区人民医院泌尿外科,武汉430000

出　　处：《中华泌尿外科杂志》2024年第10期776-782,共7页Chinese Journal of Urology

摘　　要：目的探讨裂亡PC3细胞来源的细胞外囊泡(MEV)对前列腺癌细胞活性的影响。方法采用1μmol/L顺铂连续诱导PC3细胞5d,用免疫荧光法检测细胞核损伤,原子力显微镜检测细胞刚度,流式细胞术检测细胞的衰老标志物(SA-β-Gal)、增殖、周期、线粒体膜电位和线粒体数量,qRT-PCR检测衰老分泌表型相关因子CDKN1A、CDKN2A、IL-6、IL-8、TNF-α、IFN-α/β/mRNA表达量。提取MEV,用电镜和纳米颗粒跟踪分析技术检测MEV的形态和粒径,qRT-PCR检测MEV的DNA含量和成分(β-actin和mtDNA),原子力显微镜检测MEV刚度。检测MEV对PC3细胞的作用,免疫荧光法检测细胞对MEV吞噬情况,流式细胞术检测PC3细胞增殖、凋亡情况,qRT-PCR检测PC3细胞IFNα/β/表达情况。将5、15、50μmol/L阿霉素与PC3细胞共孵育24h后检测PC3细胞凋亡情况,选取可使PC3细胞明显凋亡浓度的阿霉素与50μgMEV混合后处理PC3细胞24h,流式细胞术检测PC3细胞凋亡情况。结果成功诱导PC3细胞裂亡并提取MEV。与正常培养PC3细胞分泌的胞外囊泡(NEV)相比,MEV的数量明显增多[(4530.9±353.6)×10^(6)粒子数/1×10^(6)个细胞与(33.7±5.4)×10^(6)粒子数/1×10^(6)个细胞,P<0.01],粒径显著变小[(122.0±2.6)nm与(163.6±2.6)nm,P<0.01],核DNA[(111.0±20.7)/1×10^(6)个细胞]与mtDNA[(26.2±3.8)/1×10^(6)个细胞]相对含量均明显上升(P均<0.01),硬度变大[(0.18±0.01)MPa与(0.11±0.01)MPa,P<0.01],且MEV更易被吸收。与NEV相比,MEV能显著诱导PC3细胞凋亡[(641.0±42.5)平均荧光强度(MFI)与(351.7±37.0)MFI,P<0.01],抑制其增殖[(1523.0±64.9)MFI与(1336.3±94.1)MFI,P<0.05],对PC3细胞的IFNβmRNA水平无影响,显著降低IFNα/表达量[(0.6±0.1)与(0.8±0.1)](P均<0.01)。与单独应用15μmol/L阿霉素相比,MEV和15μmol/L阿霉素联用后能显著促进PC3细胞凋亡[(14290.3±1315.9)MFI与(2669.3±241.5)MFI,P<0.01]。结论裂亡PC3细胞能高效分泌富含DNA的柔性细胞外囊泡,且MEV易被PC3细胞吸收并能Objective To investigate the effect of extracellular vesicles derived from mitotic cell death PC3 cells(MEV)on prostate cancer cells.Methods After the phenotype of mitotic death was continuously induced by 1μmol/L cisplatin for 5 days,nuclear damage was detected by immunofluorescence,cell stiffness was measured by Atomic Force Microscope,cell senescence marker(SA-β-Gal),proliferation,cycling,mitochondrial membrane potential,mitochondrial number by flow cytometry,and expression of senescence-secreting phenotype-associated factors including CDKNIA,CDKN2A,IL-6,IL-8,TNF-α,IFN-α/βγby qRT-PCR.Mitotic-death PC3 cells derived extracellular vesicles(MEV)were extracted,and the morphology and size of MEV were detected by electron microscopy and nanoparticle tracking analysis,and their stiffness and substance(β-actin and mtDNA)were separately detected by atomic force microscope and qRT-PCR.The effects of MEV on PC3 cells were further detected by immunofluorescence to detect phagocytosis,flow cytometry to detect proliferation and apoptosis,and qRT-PCR to detect the level of IFNα/β/γ.Finally,5Oμg MEV were treated to PC3 cells combined with 15μmol/L adriamycin(Dox),and apoptosis was detected by flow assay.Results Mitotic cell death PC3 cells and MEV were both obtained successfully.In comparison with normal PC3's,the number of MEV group was significantly increased[(4530.9±353.6)×10^(6)(particle)/1×10^(6)cell and(33.7±5.4)×10^(6)particle/1×10^(6)cell,P<0.01].Additionally,the particle size of the extracellular vesicles were significantly smaller[(122.0±2.6)nm and(163.6±2.6)nm,P<0.01],along with significantly increased content of nuclear DNA[(111.0±20.7)/1×10^(6)cell,P<0.01]and mtDNA[(26.2±3.8)/1×10^(6)cell,P<0.01].MEV were also easier to absorb by PC3 for their softness[(0.11±0.01)MPa and(0.18±0.01)MPa,P<0.01].MEV could significantly induce PC3 cell apoptosis[(641.0±42.5)MFI and(351.7±37.0)MFI,P<0.01]and inhibit their proliferation[(1523.0±64.9)MFI and(1336.3±94.1)MFI,P<0.05].Besides,they did not affe

关 键 词：PC3细胞 细胞裂亡 细胞外囊泡 联合阿霉素治疗 

分 类 号：R737.25[医药卫生—肿瘤]