北京大学学报（医学版） ›› 2020, Vol. 52 ›› Issue (2): 214-220. doi: 10.19723/j.issn.1671-167X.2020.02.004
角蛋白18与剪切因子PSF相互作用介导PSF的膜易位并维持髓系白血病细胞的化疗敏感性
任思楣1,2,△(
),龙璐瑶1,2,许成山1
- 1. 北京医院 国家老年医学中心 国家卫生健康委员会临床检验中心 中国医学科学院老年医学研究院,北京 100730
2. 中国医学科学院北京协和医学院研究生院,北京 100730
Interaction between PSF and cytokeratin 18 mediates PSF relocation to cell membrane and maintains chemosensitivity of myeloid leukemia
Si-mei REN1,2,△(),Lu-yao LONG1,2,Cheng-shan XU1
- 1. National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology; Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing 100730, China
2. Graduate School of Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China
摘要：
目的 鉴定多嘧啶结合蛋白相关的剪切因子(polypyrimidine tract-binding protein-associated splicing factor, PSF)在髓系白血病细胞内的伴侣蛋白,探讨PSF在敏感HL60细胞和耐药的HL60/DOX细胞中易位细胞膜的模式和机制.方法: 通过脂质体瞬时转染PSF真核表达载体过表达PSF,进一步结合流式细胞术在转染后24 h,48 h,72 h检测PSF在细胞膜上的表达水平.构建链亲和素结合肽(streptavidin binding peptide,SBP)和PSF的融合表达载体,转染载体,过表达SBP-PSF融合蛋白.通过链亲和素磁珠共沉淀法和质谱分析,鉴定PSF在细胞内的伴侣蛋白.在慢病毒载体中插入角蛋白18 (cytokeratin 18,K18)干扰序列,转染293T细胞制备病毒液.用慢病毒感染HL60和HL60/DOX细胞,获得稳定干扰K18的细胞株.结合流式细胞术检测干扰K18的HL60和HL60/DOX细胞中细胞膜上PSF的表达水平,由此证实CK18在HL60和HL60/DOX细胞中协同转运PSF易位细胞膜机制的异同.结果: 瞬时过表达PSF后的24 h,48 h,72 h连续3 d检测细胞膜PSF表达水平,HL60敏感株细胞膜上PSF表达率分别为22.4%±3.5%, 37.9%±6.0%, 58.3%±8.8%;耐药株HL60/DOX细胞膜上PSF的表达率分别为4.7%±0.5%, 3.9%±0.6%, 2.9%±0.6%;各时间点下敏感株和耐药株的差异有统计学意义,P<0.01.免疫共沉淀和质谱证实K18为PSF在细胞内的伴侣蛋白.干扰K18的表达后,再次分析细胞膜PSF表达,发现PSF在敏感株细胞膜上的表达水平由原来的48.9%±5.4% 降至6.2%±1.0%;在耐药株细胞膜上的表达水平由9.11%±1.2%降至2.21%±0.51%.结论: K18是PSF在细胞内的伴侣蛋白,在敏感细胞中,K18与PSF相互作用可帮助PSF向细胞膜转运;而在耐药株中,由于该功能障碍导致PSF在胞内发生积累从而介导耐药性.
中图分类号:
- R557.1
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