5种核碱丙氨酸的合成及其抑制大鼠肝癌细胞RH35增殖的作用机制  

Synthesis of five types of nucleobase-alanines and their mechanisms of inhibiting proliferation of rat hepatoma cells RH35

作　　者：徐存拴 宋亚萍 许凯 杨晖 丁一[1] 毕晶晶[2] 赵茜怡[2] 徐霆[2] XU Cun-shuan;SONG Ya-ping;XU Kai;YANG Hui;DING Yi;BI Jing-jing;ZHAO Qian-yi;XU Ting(State Key Laboratory Cultivation Base for Cell Differentiation Regulation,School of Life Science,He'nan Normal University,He'nan Xinxiang 453007,China;School of Chemistry and Chemical Engineering,He'nan Normal University,He'nan Xinxiang 453007,China)

机构地区：[1]河南师范大学生命科学学院,河南省-科技部共建细胞分化调控国家重点实验室培育基地,河南新乡453007 [2]河南师范大学化学化工学院,河南新乡453007

出　　处：《解剖学报》2020年第2期195-205,共11页Acta Anatomica Sinica

基　　金：细胞分化调控国家重点实验室培育基地研究项目(2013CKLB005,2015CKLB003)。

摘　　要：目的合成5种核碱丙氨酸,探讨其细胞毒性及机制。方法用丝氨酸-内酯亲核开环方法合成5种核碱丙氨酸,用MTT法检测它们对大鼠正常肝细胞BRL-3A和大鼠肝癌细胞RH35活性的影响,流式细胞术检测它们对上述BLR-3A和RH35细胞周期进程和凋亡的影响,用Real-time PCR检测它们对上述两种细胞增殖/凋亡相关基因表达的影响。结果合成了1-尿嘧啶-L-丙氨酸、1-胸腺嘧啶-L-丙氨酸、1-胞嘧啶-L-丙氨酸、9-腺嘌呤-L-丙氨酸和9-鸟嘌呤-L-丙氨酸等5种核碱丙氨酸。其中,1-胞嘧啶-L-丙氨酸和9-鸟嘌呤-L-丙氨酸对RH35的半数抑制浓度(IC50)值显著低于对BRL-3A的IC50值,它们促进RH35凋亡的作用显著强于对BRL-3A作用,它们促进RH35凋亡相关基因CASP3、CASP9和Bax的表达上调幅度,抑制细胞增殖相关基因CCNA2和PCAN的表达下调幅度和抑制抗凋亡相关基因Bcl-2的表达下调幅度均显著高于对BRL-3A作用。结论1-胞嘧啶-L-丙氨酸和9-鸟嘌呤-L-丙氨酸通过显著抑制RH35增殖相关基因表达,促进凋亡相关基因表达而抑制其活性和促进其凋亡。Objective Five types of nucleobase-alanines were synthesized to study their cytotoxicity and mechanism.Methods Five types of nucleobase-alanines were synthesized by the method of nucleophilic ring-opening of serine-lactone.MTT assay was used to detect their effects on the viability of rat normal hepatocytes BRL-3A and rat hepatoma cells RH35.Flow cytometry was used to detect their effects of cell cycle and apoptosis on BLR-3A and RH35.Real-time PCR was performed to detect their effects of proliferation/apoptosis-related gene expression on these two cells.Results Five types of nucleobase-alanines were synthesized,including 1-uracil-L-alanine,1-thymine-L-alanine,1-cytosine-L-alanine,9-adenine-L-alanine and 9-guanine-L-alanine.Among them,1-cytosine-L-alanine and 9-guanine-Lalanine had significant lower half inhibition concentration(IC50)values for RH35 than for BRL-3A,and their effects of promoting RH35 apoptosis were significantly stronger than that of BRL-3A,the amplitude of their promoting RH35 upregulation apoptosis-related genes CASP3,CASP9 and Bax,the down-regulation of cell proliferation-related genes CCNA2 and PCAN,as well as the down-regulation of anti-apoptosis-related gene Bcl-2 were significantly higher than those of BRL-3 A.Conclusion 1-cytosine-L-alanine and 9-guanine-L-alanine showed significant effects of inhibiting the viability and promoting apoptosis by up-regulating the expression of proliferation-related genes and down-regulating the expression of apoptosis-related genes more potently.

关 键 词：核碱丙氨酸 细胞毒性 细胞增殖动力学 流式细胞术 大鼠 

分 类 号：Q291[生物学—细胞生物学]