中华皮肤科杂志 ›› 2018, Vol. 51 ›› Issue (12): 879-884.doi: 10.3760/cma.j.issn.0412-4030.2018.12.006

• 论著 • 上一篇    下一篇

载芬维A铵脂质体体外对恶性黑素瘤细胞增殖、凋亡和迁移的影响

崔艾丽    金哲虎   

  1. 133000 吉林省延吉市，延边大学附属医院皮肤科
  • 收稿日期:2018-10-09 修回日期:2018-11-02 出版日期:2018-12-15 发布日期:2018-11-30
  • 通讯作者: 金哲虎 E-mail:Jinzh_621@163.com

In vitro effect of fenretinide-loaded liposomes on proliferation, apoptosis and migration of ma-lignant melanoma cells

Cui Aili, Jin Zhehu   

  1. Department of Dermatology, Yanbian University Hospital, Yanji 133000, Jilin, China 
  • Received:2018-10-09 Revised:2018-11-02 Online:2018-12-15 Published:2018-11-30
  • Contact: Jin Zhehu E-mail:Jinzh_621@163.com

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摘要： 目的 探讨载芬维A铵脂质体（4-HPR-L）对A375及B16F10黑素瘤细胞增殖、凋亡和迁移的影响。方法 采用薄膜-超声分散法制备载4-HPR-L。体外分别培养A375及B16F10黑素瘤细胞，分为3组，空白对照组仅加入细胞和新鲜培养基，4-HPR组和4-HPR-L组分别加入同浓度4-HPR和4-HPR-L。细胞增殖抑制实验（CCK-8法）检测各组细胞增殖情况；Hoechst33258染色法和流式细胞仪检测细胞凋亡情况；细胞划痕实验检测细胞迁移能力；通过激光共聚焦显微镜观察4-HPR-L入胞情况。采用SPSS22.0软件进行统计分析，多组间数据比较采用单因素方差分析，两组间比较采用t检验。结果 4-HPR与4-HPR-L对A375和B16F10的增殖抑制作用均表现出浓度依赖，0.1、1、15、30、50、70 mg/L 4-HPR和4-HPR-L处理A375和B16F10细胞48 h后，4-HPR组A375细胞存活率分别为（94.3 ± 1.4）%、（91.7 ± 2.5）%、（84.4 ± 2.5%）、（78.8 ± 2.1）%、（59.0 ± 1.1）%、（42.8 ± 2.0）%，4-HPR-L组分别为（86.0 ± 0.2）%、（76.5 ± 0.6）%、（60.9 ± 1.5）%、（49.0 ± 0.5）%、（32.9 ± 0.2）%、（18.9 ± 0.5）%，同浓度两组间比较，t值分别为8.019、8.298、11.455、19.978、33.672、16.314，均P < 0.01；4-HPR组B16F10细胞存活率分别是（95.4 ± 1.9）%、（90.5 ± 2.6）%、（77.0 ± 0.8%）、（64.4 ± 3.5）%、（59.1 ± 2.9）%、（49.9 ± 1.9）%，4-HPR-L组分别是（88.4 ± 2.0）%、（80.9 ± 3.4）%、（60.9 ± 2.2）%、（51.5 ± 2.9）%、（41.1 ± 1.2）%、（33.5 ± 2.4）%，同浓度两组间比较，均P < 0.05。相同浓度下，4-HPR同浓度组A375和B16F10细胞存活率均高于4-HPR-L组。Hoechst33258染色显示，对照组、4-HPR组细胞无明显变化，而4-HPR-L组细胞体积变小，细胞质浓缩，细胞核裂解为碎块，产生凋亡小体。流式细胞仪检测显示，4-HPR-L组A375和B16F10细胞凋亡率均显著高于4-HPR组，均P < 0.01。细胞划痕实验显示，4-HPR-L较4-HPR能更好地抑制细胞移行，显著降低划痕的愈合程度。激光共聚焦显微镜观察显示，C6脂质体入胞迅速。结论 4-HPR-L能更好地进入A375细胞、B16F10细胞，且能有效抑制A375、B16F10细胞的增殖和迁移，并诱导其凋亡。

关键词: 黑色素瘤, 实验性； 芬维A铵； 脂质体； 细胞增殖； 细胞凋亡； 细胞运动； A375细胞； B16F10细胞

Abstract: Cui Aili, Jin Zhehu Department of Dermatology, Yanbian University Hospital, Yanji 133000, Jilin, China Corresponding author: Jin Zhehu, Email: Jinzh_621@163.com 【Abstract】 Objective To evaluate the effect of fenretinide （4-HPR）-loaded liposomes （4-HPR-L） on the proliferation, apoptosis and migration of A375 and B16F10 melanoma cells. Methods A film-ultrasonic dispersion method was used to prepare 4-HPR-L. In vitro cultured A375 cells, as well as B16F10 melanoma cells, were divided into the following groups: blank control group treated with fresh culture medium alone, 4-HPR groups treated with 4-HPR at concentrations of 0.1, 1, 15, 30, 50 and 70 mg/L separately, and 4-HPR-L groups treated with 4-HPR-L at concentrations of 0.1, 1, 15, 30, 50 and 70 mg/L separately. Cell counting kit-8 （CCK8） assay was conducted to detect cell proliferation, Hoechst33258 staining and flow cytometry to detect cell apoptosis, wound healing assay to evaluate cell migration ability, and laser scanning confocal microscopy to observe endocytic uptake of the liposomes. Statistical analysis was done by one-way analysis of variance （ANOVA） for intergroup comparison, and a two-sample t-test for comparisons between 2 groups with SPSS 20.0 software. Results Both 4-HPR and 4-HPR-L showed inhibitory effects on the proliferation of A375 and B16F10 melanoma cells. After 48-hour treatment, the survival rates of A375 cells in the 0.1-, 1-, 15-, 30-, 50- and 70-mg/L 4-HPR groups were （94.3 ± 1.4）%, （91.7 ± 2.5）%, （84.4 ± 2.5%）, （78.8 ± 2.1）%, （59.0 ± 1.1）% and （42.8 ± 2.0）% respectively, and those in the 0.1-, 1-, 15-, 30-, 50- and 70-mg/L 4-HPR-L groups were （86.0 ± 0.2）%, （76.5 ± 0.6）%, （60.9 ± 1.5）%, （49.0 ± 0.5）%, （32.9 ± 0.2）% and （18.9 ± 0.5）% respectively. After 48-hour treatment, the survival rates of B16F10 cells in 0.1-, 1-, 15-, 30-, 50- and 70-mg/L 4-HPR groups were （95.4 ± 1.9）%, （90.5 ± 2.6）%, （77.0 ± 0.8%）, （64.4 ± 3.5）%, （59.1 ± 2.9）% and （49.9 ± 1.9）% respectively, and those in the 0.1-, 1-, 15-, 30-, 50- and 70-mg/L 4-HPR-L groups were （88.4 ± 2.0）%, （80.9 ± 3.4）%, （60.9 ± 2.2）%, （51.5 ± 2.9）%, （41.1 ± 1.2）% and （33.5 ± 2.4）% respectively. The survival rates of A375 and B16F10 cells were significantly higher in the 0.1-, 1-, 15-, 30-, 50- and 70-mg/L 4-HPR groups than in the 4-HPR-L groups at the same concentrations（A375 cells: t = 8.019, 8.298, 11.455, 19.978, 33.672, 16.314 respectively, all P < 0.01; B16F10 cells: t = 3.573, 3.153, 9.953, 4.019, 8.097, 7.53 respectively, all P < 0.05）. Hoechst33258 staining showed that the morphology of the melanoma cells in the control group and 4-HPR groups did not change obviously, but the cells in the 4-HPR-L groups became smaller, with the cytoplasm concentrated, nuclei dissociated into fragments, and apoptotic bodies formed. Flow cytometry showed that the apoptosis rates of A375 and B16F10 cells were significantly higher in the 4-HPR-L groups than in the 4-HPR groups （all P < 0.01）. Cell wound healing assay showed that the inhibitory effect of 4-HPR-L on the migration of cells was stronger than that of 4-HPR, and 4-HPR-L could markedly decrease the degree of wound healing. Laser scanning confocal microscopy showed that C6 liposomes could be rapidly and successfully internalized into both A375 and B16F10 cells. Conclusion The 4-HPR-L can be internalized into A375 and B16F10 cells better than 4-HPR, effectively inhibit the proliferation and migration of A375 and B16F10 cells, and induce the apoptosis of these cells.

Key words: Melanoma, experimental, Fenretinide, Liposomes, Cell proliferation, Apoptosis, Cell movement, A375 cell, B16F10 cell