毛晓丽+覃禹+陈相宜+邝晓聪+黄庶识+刘华钢

[摘要]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OriginPro8.0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。

[关键词]拉曼光谱； 肝癌细胞； 两面针

[Abstract]The apoptosis of mono-hepatocellular induced by the active ingredients of the Zanthoxyli Radix was investigated using laser Raman spectroscopy. Hepatoma cells （BEL-7404） were treated with 10 mg·L^-1 nitidine chloride and 3 g·L^-1 the extracts of Zanthoxyli Radix， respectively， then were divided into two parts， one for fluorescence staining， the other for determination of Raman spectroscopy. The acquired spectra were then processed by background elimination， smoothing， and normalization. Fluorescence staining results showed that the nucleuses from untreated group were uniformly stained， while those from the group treated for 48 hours were densely stained and broken. The spectra results revealed that the intensity of peaks associated with nucleic acid and protein decreased after the cells were incubated with the extracts of Zanthoxyli Radix for 12， 24， 36 and 48 hours. The intensity of peaks at 785，1 002，1 175，1 660 cm^-1 was decreased with the time of the cells were incubated by the extracts of Zanthoxyli Radix. The results indicated that the extracts of Zanthoxyli Radix could induce the apoptosis of hepatoma cells and reduce the amount of nucleic acid and protein in the cells. There is a certain relevance between the drug treatment time and the efficacy. The above results suggest that Raman spectra can provide abundant information about the changes in biological macromolecules within the cells after incubated by the extracts of Zanthoxyli Radix and serve as an effective method for the real time measurement of apoptosis.

[Key words]Raman spectroscopy； hepatoma cell； Zanthoxyli Radix

doi：10.4268/cjcmm20162119

兩面针系芸香科花椒属藤本植物两面针Zanthoxylum nitidum（Roxb.）DC.的干燥根，又名入地金牛、蔓椒、双面针、双背针等。两面针是广西道地药材，富含生物碱、木质素、香豆素和挥发油等，具有活血化瘀，行气止痛，祛风通络，解毒消肿等功效，用于跌扑损伤，胃痛，牙痛，风湿麻痹，毒蛇咬伤等^[1]。现代研究发现氯化两面针碱为主的生物碱活性成分发挥抗肿瘤作用，其主要作用机制是抑制拓扑异构酶活性、阻滞细胞周期、逆转肿瘤细胞多药耐药、诱导肿瘤细胞凋亡等^[2]。……