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背景与目的:探讨北豆根有效成分诱导肿瘤细胞凋亡的作用及其作用机制。材料与方法:北豆根干品醇提液,经三步法将醇提液纯化,得到三种纯化提取物(Purifiedextract,PE)PE1、2、3。应用流式细胞技术分析细胞周期DNA变化,并经荧光染色和荧光显微镜观察凋亡细胞形态学变化。并用电子显微镜观察了凋亡细胞的超微结构变化。用凝胶电泳实验检测了凋亡细胞DNA变化。结果:PE2可诱导BGC823细胞凋亡,呈明显的时效和量效关系;在低浓度PE2作用下,多数细胞被阻滞在G0/G1期,高浓度PE2作用后,多数细胞被阻滞在S期。肿瘤细胞经PE2作用后发生细胞凋亡的形态学变化:体积缩小,胞质浓缩,出现核固缩、核碎裂等;在电子显微镜下可见细胞胞体缩小,核染色质高度浓缩,边集于核膜,有的呈半月形,有的出现核碎裂,偶见凋亡小体。细胞DNA经凝胶电泳后,出现典型的梯状电泳模式。PE2处理的癌细胞,bcl-2基因表达降低,而bax基因表达升高。结论:PE2诱导肿瘤细胞凋亡,通过调节bcl-2及bax基因的表达发挥作用是PE2抗肿瘤作用的机制之一。该结果可促进北豆根的二次开发和临床应用。
BACKGROUND & AIM: To investigate the effect and its mechanism of apoptosis of tumor cells induced by active ingredients of Radix et. MATERIALS AND METHODS: Three kinds of purified extracts (Purifiedextract, PE) PE1,2,3 were obtained by alcohol extraction from the dried root extract of Peony. The changes of cell cycle DNA were analyzed by flow cytometry. Morphological changes of apoptotic cells were observed by fluorescence staining and fluorescence microscope. The ultrastructural changes of apoptotic cells were observed under electron microscope. The DNA changes of apoptotic cells were detected by gel electrophoresis. Results: PE2 could induce the apoptosis of BGC823 cells, and showed obvious time-effect and dose-effect relationship. Under the action of low concentration of PE2, most cells were arrested in G0 / G1 phase. After high concentration of PE2, most cells were blocked in S period. The morphological changes of apoptosis of tumor cells after PE2: shrinking in volume, concentration of cytoplasm, nuclear pyknosis, nuclear fragmentation, etc .; under the electron microscope, cell bodies were reduced, nuclear chromatin highly condensed, Nuclear membrane, and some showed half-moon, and some nuclear fragmentation, and occasionally apoptotic bodies. After the DNA gel electrophoresis, a typical ladder electrophoresis pattern. PE2-treated cancer cells, bcl-2 gene expression decreased, while bax gene expression increased. CONCLUSION: PE2 can induce apoptosis of tumor cells and play an important role in the anti-tumor effect of PE2 by regulating the expression of bcl-2 and bax genes. This result can promote the secondary development and clinical application of Pea root.