[1]姜 轩,付 超,赵瑞利*,等.新RGD嵌合体肽对黑色素瘤细胞B16增殖及其黑色素合成的影响[J].中国预防兽医学报,2019,(09):951-957.[doi:0.3969/j.issn.1008-0589.201903012]
 JIANG Xuan,FU Chao,ZHAO Rui-li*,et al.Effects of new chimeric peptide RGD-T-La (FS) on proliferation and melanin synthesis of B16 cells[J].Chinese journal of preventive veterinary medicine,2019,(09):951-957.[doi:0.3969/j.issn.1008-0589.201903012]
点击复制

新RGD嵌合体肽对黑色素瘤细胞B16增殖及其黑色素合成的影响()
分享到:

《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2019年09
页码:
951-957
栏目:
免疫学
出版日期:
2019-10-25

文章信息/Info

Title:
Effects of new chimeric peptide RGD-T-La (FS) on proliferation and melanin synthesis of B16 cells
文章编号:
1008-0589(2019)09-0951-07
作者:
 

姜 轩付 超赵瑞利*金天明马吉飞孙英峰于晓雪张 欣刘梦月潘晨浩

 (天津农学院 动物科学与动物医学学院,天津 300384)
Author(s):
 

JIANG Xuan FU Chao ZHAO Rui-li* JIN Tian-ming MA Ji-fei SUN Ying-feng YU Xiao-xueZHANG Xin LIU Meng-yue PAN Chen-hao

 (College of Animal Science and Animal Medicine, Tianjin Agricultural University, Tianjin 300384, China)
关键词:
RGD嵌合体肽B16细胞透射电镜细胞周期黑色素酪氨酸酶
Keywords:
  RGD chimera peptide  B16 cell  transmission electron microscope  cell cycle  melanin  tyrosinase
分类号:
S859.79
DOI:
0.3969/j.issn.1008-0589.201903012
文献标志码:
A
摘要:
为研究新合成的靶向抗肿瘤肽RGD-T-La(S)、RGD-T-La(FS)对小鼠黑色素瘤B16细胞增殖及其黑色素合成的影响,本研究将T-La (S)、T-La (FS)与RGD (精氨酸-谷氨酸-天冬氨酸)小分子蛋白质偶联,合成新的靶向抗肿瘤肽RGD-T-La (S)、RGD-T-La (FS)。通过CCK8法检测多肽对B16细胞增殖的影响;微量酶标法检测多肽作用于B16细胞后乳酸脱氢酶(LDH)含量变化;透射电镜观察多肽作用B16细胞后的超微结构;流式细胞仪检测多肽对B16细胞周期的影响;NaOH裂解法和L-Dopa氧化法分析多肽对黑色素合成含量和酪氨酸酶活性的影响;荧光定量PCR (q-PCR)法分析多肽对黑色素合成关键因子酪氨酸酶(TYR)和小眼相关转录因子(MITF)基因转录水平的影响。结果显示,RGD-T-La(S)、RGD-T-La (FS)在10 μg/mL时能够显著抑制B16细胞的增殖,且呈现时间浓度依赖性;RGD嵌合体肽在20 μg/mL时能够显著增加B16细胞中LDH含量,且呈现浓度依赖性;透射电镜结果显示RGD嵌合体肽作用B16后,细胞出现自噬体及线粒体自噬现象;同时RGD嵌合体肽对酪氨酸酶活性和细胞黑色素蛋白的生成具有明显的抑制作用,显著降低TYR和MITF基因的转录水平。上述结果表明,RGD嵌合体肽在体外具有诱导细胞凋亡,抑制B16细胞增殖的作用,能够促进抗肿瘤肽T-La (FS)对肿瘤细胞的靶向杀伤作用。本实验为研究RGD嵌合体肽RGD-T-La (S)和RGD-T-La (FS)靶向抗肿瘤的作用机制以及为临床相关抗肿瘤药物的研发提供科学依据。
  
Abstract:
In order to study the effects of newly synthesized targeted antitumor peptides RGD-T-La (S) and RGD-T-La (FS) on the proliferation and melanin synthesis of melanoma B16 cells, this study synthesized new targeted antitumor peptides RGD-T-La (S), RGD-T-La (FS) by coupling T-La (S) or T-La (FS) with RGD (arginine-glutamic acid-aspartate) peptide. CCK8 assay was used for testing the effect of peptides on the cell proliferation; Lactate dehydrogenase (LDH) content was detected by microtitration; Peptides acting on the ultrastructure of B16 cells was detected by transmission electron microscope; Flow cytometer(FCM) was used for investigating the cell cycle of B16 cells; NaOH lysis-assay and L-Dopa oxidation-assay were employed to investigate the melanin synthesis and tyrosinase(TYR) activity of B16 cells treated by peptides at different concentrations,respectively; and q-PCR to analyze the relative mRNA expression of TYR and microphthalmia associated transcription factor (MITF) in B16 cells. The results showed that RGD-T-La (S) and RGD-T-La (FS) significantly inhibited the proliferation of B16 cells at 10 μg/mL in a time-concentration-dependent manner; RGD chimeric peptide significantly increased LDH content in B16 cells at 20 μg/mL in a concentration-dependent manner; meanwhile, RGD chimeric peptide significantly inhibited tyrosinase activity and melanin synthesis in B16 cells, relative mRNA expressions of TYR and MITF genes in B16 cells were depressed remarkably. Conclusively, RGD chimeric peptide can induce apoptosis and inhibit the proliferation of B16 cells in vitro, and promote the targeting killing effect of anti-tumor peptide T-La (FS) on cancer cells. This study will help understand the mechanism of targeting anti-tumor effects of RGD chimeric peptides RGD-T-La (S) and RGD-T-La (FS) and provide scientific basis for the development of clinical related anti-tumor drugs.

参考文献/References:

 
[1]赵瑞利,韩俊友,韩文瑜,等. 新牛蛙活性肽Catesbeianin-1a的抗菌抗肿瘤作用[J]. 中国兽医学报,2013,33(09):1407-1411.
[2]王心怡,卫旭东. RGD肽对喉癌干细胞接种裸鼠的影响[J].世界最新医学信息文摘,2018,18(38):6-8.
[3]Miyano K, Cabral H, Miura Y, et al. cRGD peptide installation on cisplatin-loadednanomedicines enhances efficacy against locally advanced head and necksquamous cell carcinoma bearing cancer stem-like cells [J]. J Control Release, 2017, 261: 275- 286.
[4]Sun Cui-cui, Qu Xian-jun, Gao Zu-hua. Integrins: players in cancer progression andtargets in cancer therapy[J]. Anticancer Drugs, 2014, 25(10): 1107-1121.
[5]Hagen B, Trinh V A. Managing side effects of vemurafenib therapy for advanced melanoma [J]. J Adu Pract Oncol, 2014, 5(6): 400-410.
[6]Jaukovic L, Sijan G, Rajovié M, et al. Lymphoscintigraphy and sentinel lymph node biopsy, in cutaneous melanoma staging and treatment decisions [J]. Hell J Nucl Med, 2014, 18(2): 146-151.
[7]Speijers M J, Bastiaannet E, Sloot S, et al. Tumor mitotic rate added to the equation:melanoma prognostic factors changed?: a single-institution database study on the prognostic value of tumor mitotic rate for sentinel lymph node status and survival of cutaneous melanoma patients [J]. Ann Surg Oncol, 2015, 22(9): 2978-2987.
[8]Stanelle E J. Busam K J, Rich B S, et al. Early-stage non-Spitzoid cutaneous melanoma in patients younger than 22 years of age at diagnosis: long-term follow-up and survival analysis [J]. J Pediatr Surgery, 2015, 50(6): 1019-1023.
[9]程杏安,张淑明,周晓武,等. 两种天然产物对B16F10细胞增殖及黑色素合成抑制机理研究[J]. 生物技术通报,2017,33(08):199-205.
[10]Steingrimsson E, Copeland N G, Jenkins N A. Melanocytes and the microphthalmia- transcription factor network [J]. Ann Rev Genet, 2004, 38: 365-411.
[11]Levy C, Khaled M, Fisher D E. MITF: master regulator of melanocyte development and melanoma oncogene [J]. Trends Mol Med, 2006, 12: 406-414.
[12]刘梦月,刘珊珊,付超,等. 新牛蛙抗肿瘤肽RGD-T-La(FS)嵌合体的设计及其抗肿瘤作用[J]. 中国兽医学报,2018,38(01):39-50.
[13]刁昱文. 肿瘤细胞靶向抗菌肽嵌合体的设计及其抗肿瘤活性机制研究[D]. 长春:吉林大学,2012.
[14]苏雅. 银杏外种皮提取物抑制B16-F10黑色素瘤转移及其对PI3K/Akt相关信号通路的影响[D]. 扬州:扬州大学,2018.
[15]Dziudyk J M, Sui Mei-hua, Zhu Xue-ming, et al. Puclituxel - induced apoplosis may occur without a prior G2/M phase arrest[J]. Anticaneer Res, 2004, 24(1): 27-36.
[16]殷珊. 栀子黄色素对小鼠B16黑素瘤细胞增殖、酪氨酸酶活性及黑素生成影响的实验研究[D]. 长沙:湖南中医药大学,2016.
[17]Levy C, Khaled M, Fisher D E. MITF: master regulator of melanocyte development and melanoma oncogene [J]. Trends Mol Med, 2006, 12: 406-414.
[18]鄢文慧. TNF-α诱导B16细胞自体吞噬的机制研究[D]. 天津:天津医科大学,2012.
[19]Chiu H W, Lin Wei, Ho S Y, et al. Synergistic effects of arsenic trioxide andradiation in osteosarcoma cells through the induction of both autophagy andapoptosis [J]. Radiat Res, 2011, 175(5): 547-560.
(本文编辑:李 爽;英文编辑:张振宇)

更新日期/Last Update: 2019-10-29