Liu Yajing, Zhu Pan, Huang Zhiwei, Zhou Li, Shi Ping
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China; Key Laboratory of Organofluorine Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China.
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2018 Feb 15;1076:1-7. doi: 10.1016/j.jchromb.2018.01.011. Epub 2018 Jan 7.
A well-known suicide gene therapy approach, cytosine deaminase (CD) in combination with prodrug 5-flurocytosine (5-FC), has become an effective strategy of tumor treatment. However, there are short of simple and convenient detection methods to evaluate the efficiency of 5-FC conversion to 5-fluorouracil (5-FU) in human cells carrying various CD/5-FC systems. In this study, we developed an effective capillary zone electrophoresis (CZE) method to simultaneously measure 5-FC and 5-FU in cells carrying CD/5-FC suicide gene system. Under the condition of 60 mM borate buffer (pH 9.5) and 25 kV separation voltage with 0.5 psi × 15 s injection in 210 nm, the separation of 5-FC and 5-FU could be completely achieved within 15 min. The linearity of the calibration curve of standard 5-FC and 5-FU was in the range from 1 to 1000 μM (r > 0.999) and their recoveries were 98.4% and 96.0%, respectively. Due to the simple sample preparation and easy detection, this method is suitable for the study of the conversion efficiency of CD/5-FC suicide gene system. It aims to intuitively evaluate CD/5-FC systems and helps to guide the improvement of more effective CD/5-FC suicide gene systems.
一种著名的自杀基因治疗方法，即胞嘧啶脱氨酶（CD）与前药5-氟胞嘧啶（5-FC）联合使用，已成为一种有效的肿瘤治疗策略。然而，缺乏简单便捷的检测方法来评估在携带各种CD/5-FC系统的人类细胞中5-FC转化为5-氟尿嘧啶（5-FU）的效率。在本研究中，我们开发了一种有效的毛细管区带电泳（CZE）方法，用于同时测量携带CD/5-FC自杀基因系统的细胞中的5-FC和5-FU。在60 mM硼酸盐缓冲液（pH 9.5）、25 kV分离电压、0.5 psi×15 s进样且检测波长为210 nm的条件下，15分钟内可完全实现5-FC和5-FU的分离。标准5-FC和5-FU校准曲线的线性范围为1至1000 μM（r>0.999），其回收率分别为98.4%和96.0%。由于样品制备简单且检测方便，该方法适用于研究CD/5-FC自杀基因系统的转化效率。其目的是直观地评估CD/5-FC系统，并有助于指导改进更有效的CD/5-FC自杀基因系统。
