基础医学与临床 ›› 2021, Vol. 41 ›› Issue (6): 786-791.

• 研究论文 • 上一篇    下一篇

精胺-普鲁兰联合地氯雷他定介导的NOTCH1沉默对急性T淋巴细胞白血病细胞系Jurkat的影响

王田, 许仕琳, 王涛, 温涛, 孟洁, 刘健*, 许海燕*   

  1. 中国医学科学院基础医学研究所 北京协和医学院基础学院 生物医学工程学系,北京 100005
  • 收稿日期:2021-03-24 修回日期:2021-04-16 出版日期:2021-06-05 发布日期:2021-05-31
  • 通讯作者: *liujian@ibms.pumc.edu.cn; xuhy@pumc.edu.cn
  • 基金资助:
    国家自然科学基金(81771969);中国医学科学院医学与健康科技创新工程项目(CIFMS 2016-I2M-3-004);国家重大科学研究计划(2017YFA0205500)

Effect of NOTCH1 silencing mediated by spermine-pullulan combined with desloratadine on acute T lymphocyte leukemia cell line Jurkat

WANG Tian, XU Shi-lin, WANG Tao, WEN Tao, MENG Jie, LIU Jian*, XU Hai-yan*   

  1. Department of Biomedical Engineering, Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing 100005, China
  • Received:2021-03-24 Revised:2021-04-16 Online:2021-06-05 Published:2021-05-31
  • Contact: *liujian@ibms.pumc.edu.cn; xuhy@pumc.edu.cn

摘要: 目的 以精胺-普鲁兰(Ps)作为NOTCH1 siRNA的载体,研究Ps联合溶酶体促渗剂地氯雷他定(DL)介导的NOTCH1沉默对急性T淋巴细胞白血病细胞系Jurkat的作用。方法 采用动态光散射仪分析不同N/P比siRNA/Ps复合物的粒径和Zeta电位。将Jurkat细胞与siNOTCH1/Ps共孵育6 h,换液后加入含10 μmol/L DL的RPMI 1640完全培养基继续孵育18 h;实时荧光定量PCR检测NOTCH1的沉默效率;流式细胞术检测细胞周期和细胞凋亡;Western blot检测NOTCH1下游蛋白C-MYC和凋亡相关蛋白caspase 3和cleaved caspase 3的表达。结果 N/P比为5的siRNA/Ps复合物粒径为(203.97±1.07)nm,Zeta电位为(46.13±0.07)mV;复合物在10% FBS培养基中稳定。在Jurkat细胞中,与无DL组相比,siNOTCH1/Ps联合10 μmol/L DL可高效沉默NOTCH1,诱导细胞周期阻滞在G1期,促进细胞凋亡(P<0.05);同时,Jurkat细胞中cleaved caspase 3蛋白的表达水平升高, C-MYC及caspase 3蛋白的表达水平降低(P<0.05)。结论 Ps联合溶酶体促渗剂DL有效介导Jurkat细胞中的NOTCH1沉默,从而诱导急性T淋巴细胞白血病细胞G1期阻滞,并促进细胞凋亡。

关键词: NOTCH1, 精胺-普鲁兰, 地氯雷他定, 急性T淋巴细胞白血病细胞系Jurkat, 凋亡

Abstract: Objective Using pullulan-spermine (Ps) as a carrier of NOTCH1 siRNA to find the effect of NOTCH1 silencing mediated by Ps combined with lysosomal penetration enhancer desloratadine (DL) on acute T lymphocytic leukemia cell line Jurkat. Methods A dynamic light scattering instrument was used to analyze the particle size and Zeta potential of siRNA/Ps complexes with different N/P ratios. Jurkat cells were incubated with siNOTCH1/Ps for 6 hours, then the medium was refreshed with RPMI 1640 complete medium containing 10 μmol/L DL and incubated for another 18 hours, real-time fluorescent quantitative PCR was used to detect the silencing efficiency of NOTCH1; flow cytometry was used to detect the cell cycle and apoptosis; Western blot was used to detect the expres- sion of C-MYC protein, which was the downstream of NOTCH1, and the expression of apoptosis-related proteins caspase 3 and cleaved caspase 3. Results The particle size of siRNA/Ps with N/P ratio of 5 was (203.97±1.07)nm, the zeta potential was (46.13±0.07)mV, and the particles were stable in 10% FBS medium. In Jurkat cells, compared with the no DL group, the siNOTCH1/Ps combined with 10 μmol/L DL induced cell cycle arrest in G1 phase and promoted cell apoptosis (P<0.05). At the same time, the expression level of cleaved caspase 3 protein in Jurkat cells increased, the expression level of C-MYC and caspase 3 protein decreased (P<0.05). Conclusions Ps combined with lysosomal penetration enhancer DL effectively mediates the silencing of NOTCH1 in Jurkat cell, thereby inducing the G1 phase arrest of acute T lymphocyte leukemia cells and promoting cell apoptosis.

Key words: NOTCH1, spermine-pullulan, desloratadine, acute T lymphocyte leukemia cell line Jurkat, apoptosis

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