慢病毒载体介导Gag-Caspase-8诱导三阴性乳腺癌原代细胞凋亡及S期阻滞

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慢病毒载体介导Gag-Caspase-8诱导三阴性乳腺癌原代细胞凋亡及S期阻滞

Lentivirus vector-mediated Gag-Caspase-8 induces apoptosis and S-phase arrest in triple-negative breast cancer primary cells

慢病毒载体介导Gag-Caspase-8诱导三阴性乳腺癌原代细胞凋亡及S期阻滞

Lentivirus vector-mediated Gag-Caspase-8 induces apoptosis and S-phase arrest in triple-negative breast cancer primary cells

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doi:10.6040/j.issn.1671-7554.0.2024.1107

山东大学学报 (医学版) ›› 2025, Vol. 63 ›› Issue (1): 25-34.doi: 10.6040/j.issn.1671-7554.0.2024.1107

王敏¹,李习平²,檀军³,邱梅⁴,侯泽宇⁵,田莹³,罗鸿莹⁴,范超文¹,齐玲⁶,俞琦¹,谢薇⁴

1.贵州中医药大学基础医学院, 贵州贵阳 550025;2.贵州中医药大学第一附属医院急诊科, 贵州贵阳 550001;3.遵义医科大学组织胚胎学教研室, 贵州遵义 563006;4.贵州中医药大学护理学院, 贵州贵阳 550025;5.遵义医科大学附属医院甲乳外科, 贵州遵义 563099;6.贵州中医药大学第一附属医院重症医学科, 贵州贵阳 550001

出版日期:2025-01-10 发布日期:2025-02-20
通讯作者: 俞琦. E-mail:756128099@qq.com谢薇. E-mail:217180455@qq.com
基金资助:
贵州省科技厅基础研究计划(自然科学类)科技基金项目(黔科合基础-ZK［2024］一般403);贵州中医药大学研究生教育创新计划项目(YCXKYB2023011);贵州中医药大学研究生科研基金项目(YCXKYS2024008);贵州中医药大学学术新苗项目(贵科合学术新苗［2023］-04号);贵州省教育厅高等学校自然科学研究项目(青年科技人才成长项目)(黔教技［2024］120号);2023年贵州省中医药、民族医药重点学科项目［QZYYZDXK(JS)-2023-02］;遵义市科技与大数据局联合基金项目(遵市科合HZ字［2023］175号);贵州省大学生创新训练项目(S2024106621541);贵州中医药大学护理学院院级项目(HL2406003);贵州省卫生健康委科技基金项目(gzwkj2025-347)

WANG Min¹, LI Xiping², TAN Jun³, QIU Mei⁴, HOU Zeyu⁵, TIAN Ying³, LUO Hongying⁴, FAN Chaowen¹, QI Ling⁶, YU Qi¹, XIE Wei⁴

1. School of Basic Medical Sciences, Guizhou University of Traditional Chinese Medicine, Guiyang 550025, Guizhou, China;
2. Department of Emergency, The First Affiliated Hospital of Guizhou University of Traditional Chinese Medicine, Guiyang 550001, Guizhou, China;
3. Department of Histology and Embryology, Zunyi Medical University, Zunyi 563006, Guizhou, China;
4. School of Nursing, Guizhou University of Traditional Chinese Medicine, Guiyang 550025, Guizhou, China;
5. Department of Thyroid and Breast Surgery, Affiliated Hospital of Zunyi Medical University, Zunyi 563099, Guizhou, China;
6. Department of Critical Care Medicine, The First Affiliated Hospital of Guizhou University of Traditional Chinese Medicine, Guiyang 550001, Guizhou, China

Online:2025-01-10 Published:2025-02-20

摘要： 目的探讨携Gag-Caspase-8慢病毒样颗粒对三阴性乳腺癌原代细胞增殖、迁移、周期和凋亡的影响及作用机制。方法收集临床人乳腺癌标本3例,通过改良组织块培养法培养人乳腺癌原代细胞,通过HE染色、免疫组化及免疫荧光鉴定所培养的细胞是否为高纯度三阴性人乳腺癌原代细胞;慢病毒转染构建携Gag-Caspase-8慢病毒样颗粒,细胞分为PBS组、Gag-VLPs组和Gag-CASP8-VLPs组。采用MTT法评估细胞增殖,划痕实验检测细胞迁移能力,AO/EB染色检测细胞凋亡,流式细胞术分析细胞周期和凋亡,蛋白质免疫印迹考察凋亡相关蛋白表达。结果临床肿瘤标本经HE染色确定为乳腺癌病理组织,免疫组化鉴定乳腺癌原代细胞特异性分子标记物CA153阳性高表达,并通过计算阳性细胞比例评估细胞纯度约为98%,免疫荧光检测结果显示,HER2、ER、PR均阴性表达,证明所培养的细胞为高纯度三阴性人乳腺癌原代细胞;Western blotting检测发现构建的Gag-VLPs和Gag-CASP8-VLPs内有慢病毒载体特异性分子标志物P24表达,证明慢病毒样颗粒包装成功。Gag-CASP8-VLPs干预三阴性人乳腺癌原代细胞24、48 h后,与对照组PBS和Gag-VLPs相比,倒置显微镜下观察到细胞皱缩、体积减小、贴壁能力下降且漂浮细胞增多;MTT检测显示,Gag-CASP8-VLPs组显著抑制了人乳腺癌原代细胞的生长(P<0.01),并呈时间依赖性;细胞划痕实验观察到,Gag-CASP8-VLPs组可显著抑制人乳腺癌原代细胞的迁移能力(P<0.05);流式细胞术检测发现,Gag-CASP8-VLPs组S期细胞数量显著增高(P<0.01),细胞阻滞于S期;AO/EB染色及流式细胞术检测发现,Gag-CASP8-VLPs组可诱导细胞发生凋亡(P<0.01);Western blotting结果显示,Gag-CASP8-VLPs组三阴性乳腺癌原代细胞内Gag-Caspase-8、Pro caspase-8、Active caspase-8和Caspase-3凋亡相关蛋白表达显著增高(P<0. 01)。结论慢病毒介导Gag-Caspase-8将活化的Caspase-8导入三阴性乳腺癌原代细胞,通过激活下游Caspase-3,诱导细胞凋亡,并使细胞停滞在S期,从而抑制细胞增殖和迁移。

关键词: 慢病毒载体, Gag-Caspase-8, 三阴性人乳腺癌原代细胞, Caspase-8, S期阻滞

Abstract: Objective To investigate the effects and mechanisms of Gag-Caspase-8-containing lentivirus-like particles on the proliferation, migration, cell cycle, and apoptosis in primary triple-negative breast cancer(TNBC)cells. Methods Three clinical specimens of human breast cancer were collected, and primary cells were cultured using an improved tissue block culture method. The cultured cells were identified as high-purity triple-negative breast cancer primary cells through HE staining, immunohistochemistry, and immunofluorescence. Lentiviral transduction was employed to construct virus-like particles(VLPs)carrying Gag-Caspase-8. The cells were divided into the PBS group, Gag-VLPs group, and Gag-CASP8-VLPs group. Cell proliferation was assessed by MTT assay, cell migration was evaluated by scratch assay, apoptosis was detected by AO/EB staining and flow cytometry, and the expression of apoptosis-related proteins was examined by Western blotting. Results Clinical tumor specimens were confirmed as breast cancer tissues by HE staining. Immunohistochemistry revealed high expression of CA153 in the primary breast cancer cells, with a purity of approximately 98% based on the proportion of positive cells. Immunofluorescence analysis showed that HER2, ER, and PR were all negatively expressed, confirming the cultured cells as high-purity triple-negative human breast cancer primary cells. Western blotting detected the expression of the lentiviral vector-specific marker P24 in both Gag-VLPs and Gag-CASP8-VLPs, indicating successful packaging of lentivirus-like particles. Upon intervention with Gag-CASP8-VLPs for 24 and 48 hours, compared to PBS and Gag-VLPs controls, inverted microscopy observed cellular shrinkage, reduced size, decreased adherence, and increased floating cells. MTT assays demonstrated significant inhibition of cell growth in the Gag-CASP8-VLPs group(P<0.01), with time-dependent effects. Wound healing assays showed significant inhibition of cell migration(P<0.05). Flow cytometry revealed a significant increase in S-phase cells(P<0.01), indicating cell cycle arrest at the S phase. AO/EB staining and flow cytometry detected induced apoptosis in the Gag-CASP8-VLPs group(P<0.01). Western blotting results indicated significantly increased expressions of Gag-Caspase-8, Pro caspase-8, Active caspase-8, and Caspase-3(P<0.01), demonstrating that lentivirus-mediated Gag-Caspase-8 effectively enters and activates downstream apoptotic executioner Caspase-3 in triple-negative human breast cancer primary cells, leading to their apoptosis. Conclusion Lentivirus-mediated Gag-Caspase-8 can deliver activated Caspase-8 into primary TNBC cells, induce apoptosis through activation of caspase-3, arrest cells at the S phase, and inhibit cell proliferation and migration.

Key words: Lentiviral vectors, Gag-Caspase-8, Primary triple-negative breast cancer cells, Caspase-8, S phase arrest

中图分类号:

R736.3

王敏, 李习平, 檀军, 邱梅, 侯泽宇, 田莹, 罗鸿莹, 范超文, 齐玲, 俞琦, 谢薇. 慢病毒载体介导Gag-Caspase-8诱导三阴性乳腺癌原代细胞凋亡及S期阻滞[J]. 山东大学学报 (医学版), 2025, 63(1): 25-34.

WANG Min, LI Xiping, TAN Jun, QIU Mei, HOU Zeyu, TIAN Ying, LUO Hongying, FAN Chaowen, QI Ling, YU Qi, XIE Wei. Lentivirus vector-mediated Gag-Caspase-8 induces apoptosis and S-phase arrest in triple-negative breast cancer primary cells[J]. Journal of Shandong University (Health Sciences), 2025, 63(1): 25-34.

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