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期 刊 ：中国中药杂志 2008年 33卷 16期 页码：2040.

关键词：鸡血藤；A549；细胞周期；非凋亡性程序化细胞死亡；

Keywords:Spatholobus suberctus, A549, cell cycle, programmed cell death,

摘 要 ：目的：探讨鸡血藤抗肿瘤有效部位(SSCE)对人非小细胞肺癌A549细胞的杀伤特点和作用机制。方法：采用四氮唑溴盐(MTT)法、绘制生长曲线观察药物对A549细胞增殖的影响。采用流式细胞术分析药物对肿瘤细胞周期的影响。通过HE染色和激光共聚焦扫描荧光显微镜技术、AnnexinV-PI双标法，Caspase-3活性检测以及DNA琼脂糖凝胶电泳，观察药物对细胞凋亡的诱导作用。结果：SSCE对A549细胞有明显杀伤作用，24 h药效最强，IC₅₀为25.54 mg·L^-1。细胞周期分析显示，药物作用12 h，S期细胞增多，G₀-G₁和G₂-M期细胞减少，细胞周期变化在24 h后明显减弱，48 h恢复正常。形态学观察可见，药物作用短时间内(8～12 h)，细胞核发生皱缩扭曲，胞浆内出现较多空泡及颗粒状物，胞膜完整；随后细胞核固缩。细胞膜磷脂酰丝氨酸(PS)外翻在用药后12 h最显著，具有时间-剂量依赖关系。Caspase-3活性无明显升高，且与剂量成反比。DNA琼脂糖凝胶电泳未见片断化梯形条带。结论：SSCE对A549细胞具有直接杀伤作用，药效迅速，细胞周期受到干扰(S delay)，主要表现为非凋亡性程序化细胞死亡。

Abstract:Objective: The goal of study is to explore the cytotoxic activity and its underlying mechanisms of the extract of Spatholobus suberctus in human lung cancer A549 cells. Method: The inhibitory effects of the extract on proliferation of human lung cancer cell line A549 was measured by MTT cell viability assay and growth curve. Cell cycle was analyzed using flow cytometry. Apoptotic morphological changes was observed by HE staining technique and AO/PI double-staining confocal laser scanning microscope (CLSM). Employing agarose gel electrophoresis and Annexin V-PI assay, we examed the presence of cytoplasmic histone-associated DNA fragments, and membrane phosphatidylserine (PS) externalization as well as caspase-3 activation. Result: The extract of S. suberctus shows strong cytotoxic power on A549 cells during 24 hours and IC₅₀ is 25.54 mg·L^-1. The cells in S-phase increase while the cells in G₀-G₁ and G₂-M decrease. These changes recovered after 48 hours. The nucleus became pyknosis between 8 to 12 hours and many vacuoles and granules in cytoplasm can be seen. Membrane phosphatidylserine externalization occurs in a dose-dependent and time-dependent manner afer 12 hours. Caspase-3 activity has no more changes in a converse dose-dependent manner. No cytoplasmic histone-associated DNA fragments was detected by agarose gel electrophoresis. Conclusion: The extract of S. suberctus shows a direct anti-tumor activity. The drug acts quickly and causes S delay in one cell cycle. The main cell death feature appears to be non-apoptotic programmed cell death.

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