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运用拉曼光谱法对两面针活性成分诱导的肝癌细胞凋亡进行分析,肝癌细胞7404分别经10 mg·L~(-1)氯化两面针碱及3 g·L~(-1)两面针提取液处理后,收集经药液处理12,24,36,48 h的各组细胞的拉曼光谱后,通过Hochest33342/PI荧光染色法鉴定细胞并保留荧光染色阳性(发生凋亡活细胞)的光谱,并在Origin Pro8.0系统中比较各组平均光谱的差异。收集肝癌细胞的拉曼光谱依次进行背景扣除、平滑、归一化等方法处理。Hochest荧光染色后空白组细胞核染色均匀,而药物处理48 h后,核碎裂,拉曼光谱结果显示两面针提取液处理肝癌细胞12,24,36,48 h后,与核酸及蛋白质相关的峰均有降低,其中785,1 002,1 175,1 660 cm~(-1)峰强度随两面针药物作用时间的延长而降低,表明两面针活性成分能够诱导肝癌细胞凋亡,凋亡的肝癌细胞中核酸和蛋白质的含量均低于活细胞。两面针活性成分作用时间与药效呈一定的相关性。拉曼光谱能够反映中药两面针活性成分作用后的肝癌细胞内物质变化的信息,对实时监测细胞凋亡过程及药物的临床应用具有重要意义。
Raman spectroscopy was used to analyze the apoptosis of hepatocarcinoma cells induced by the active ingredients of two-needle acupuncture. Liver cancer cell 7404 was treated with 10 mg · L -1 nitidine chloride and 3 g · L -1 double needle After the extracts were processed, the Raman spectra of each group of cells treated with the drug solution for 12, 24, 36, and 48 h were collected, and the cells were identified by Hochest 33342 / PI fluorescence staining and the positive staining of apoptotic cells Spectra were compared with the average spectral differences of each group in the Origin Pro 8.0 system. Raman spectra of hepatocellular carcinoma cells were collected, followed by background subtraction, smoothing, normalization and other methods. After Hochest staining, the nuclei of blank control group were stained evenly. After 48 h of treatment, the nuclear fragmentation and Raman spectra showed that the peaks associated with nucleic acid and protein were detected after 12, 24, 36 and 48 h treatment The peak intensities at 785, 1002, 175, 660 cm -1 decreased with the prolongation of the action time of the two-way needle, which indicated that the active ingredients of Aconitum acutifolia could induce the apoptosis of hepatocellular carcinoma and the apoptosis of hepatocellular carcinoma The content of nucleic acid and protein in the cell is lower than the living cell. The action time of the two-needle acupuncture points and the pharmacodynamics showed a certain correlation. Raman spectroscopy can reflect the changes of substance in hepatocellular carcinoma cells after the active ingredients of traditional Chinese medicine needle acupoints are active, which is of great significance for real-time monitoring of apoptosis process and clinical application of drugs.