Yu Tingyu, Lin Jiao, Zhao Jin, Huang Wei, Zeng Lingwen, Fang Zhiyuan, Xu Ning
Second Clinical Medical College, Guangzhou University of Chinese Medicine, Guangzhou, China.
Department of Clinical Laboratory, Guangdong Provincial Hospital of Chinese Medicine (The Second Affiliated Hospital of Guangzhou University of Chinese Medicine), Guangzhou, China.
PLoS One. 2017 Nov 10;12(11):e0186184. doi: 10.1371/journal.pone.0186184. eCollection 2017.
The analysis of intracellular ATP can reveal the response of cells to different treatments and is important for individualized medicine. In the present study, we developed a cell penetrating peptides (CPPs) tagged luciferase (TAT-LUC) for tumor chemosensitivity assay. The activity of recombinant TAT-LUC was evaluated using ATP standard solution and tumor cells. This recombinant TAT-LUC was then used for the analysis of sensitivity index (SI) of four strains of tumor cells. The results showed that TAT-LUC could detect less than 10 nM extracellular ATP with a strong correlation between the luminescence intensity and the ATP content (R2 = 0.994). Without cell lysis, the detection limit for intracellular ATP analysis was 40 tumor cells. Furthermore, chemosensitivity of four strains of tumor cells (Skov-3/DDP, A549/DDP, MDA-MB-231, Huh-7) was determined by this assay successfully. The cell penetration ability of TAT-LUC enables the assay not only to reflect drug resistance of tumor cells real-timely but also to minimize the test time, which can be a valuable aid for personalized cancer chemotherapy.
细胞内ATP的分析能够揭示细胞对不同处理的反应，对个体化医疗具有重要意义。在本研究中，我们开发了一种用于肿瘤化学敏感性测定的、带有荧光素酶的细胞穿透肽（TAT-LUC）。使用ATP标准溶液和肿瘤细胞对重组TAT-LUC的活性进行了评估。然后将这种重组TAT-LUC用于分析四株肿瘤细胞的敏感性指数（SI）。结果表明，TAT-LUC能够检测到低于10 nM的细胞外ATP，发光强度与ATP含量之间具有很强的相关性（R2 = 0.994）。在不进行细胞裂解的情况下，细胞内ATP分析的检测限为40个肿瘤细胞。此外，通过该测定成功确定了四株肿瘤细胞（Skov-3/DDP、A549/DDP、MDA-MB-231、Huh-7）的化学敏感性。TAT-LUC的细胞穿透能力使该测定不仅能够实时反映肿瘤细胞的耐药性，还能最大限度地缩短检测时间，这对个性化癌症化疗可能是一种有价值的辅助手段。
