新型金雀异黄素衍生物5-羟基-4’-硝基-7-丙酰氧基异黄酮对人宫颈癌HeLa细胞增殖和凋亡的影响

发布时间：2018-03-12 14:19

本文选题：宫颈肿瘤　切入点：-羟基-’-硝基--丙酰氧基异黄酮　出处：《肿瘤》2017年09期 　论文类型：期刊论文

【摘要】：目的:研究新型金雀异黄素衍生物5-羟基-4’-硝基-7-丙酰氧基异黄酮(5-hydroxy-4’-nitro-7-propionyloxy-isoflavone,HNPI)体外对人宫颈癌HeLa细胞增殖抑制及诱导凋亡的作用,并探讨可能的分子生物学机制。方法:采用不同浓度的HNPI(5、10、20、40、60和80μmol/L)处理HeLa细胞48 h,MTT检测HeLa细胞的增殖抑制率。原子力显微镜下观测HeLa细胞表面形貌、细胞高度、细胞宽度、细胞均方根粗糙度(rootmean-squared roughness,Rq)及细胞平均粗糙度(average roughness,Ra)的变化。采用DAPI荧光染色法在激光扫描共聚焦显微镜下观察HeLa细胞核形态的改变。FCM检测HeLa细胞凋亡率、细胞周期、活性氧的生成及线粒体膜电位的变化。结果 :MTT法检测结果显示,当HNPI浓度10μmol/L后,细胞的增殖抑制率呈浓度依赖性增高,差异均有统计学意义(P值均0.05);HNPI对HeLa细胞的半数抑制浓度为46.83μmol/L。浓度为20和60μmol/L的HNPI处理HeLa细胞48 h后,原子力显微镜纳米级超高分辨率成像结果显示,细胞皱缩变圆,细胞质发生浓缩,细胞核隆起,细胞膜表面的粗糙度降低,变得平整光滑,丝状伪足减少、稀疏、变短、萎缩,甚至完全破坏;膜超微结构成像结果显示,HeLa细胞膜表面超微结构突起稀疏,分布平整单一,汇合成较大的突起和隆起;对单细胞的定量分析结果显示,细胞的高度呈浓度依赖性增加,而细胞的宽度、Rq和Ra均呈浓度依赖性降低,与0.9%氯化钠溶液对照组比较,差异均有统计学意义(P值均0.05)。DAPI法检测结果显示,细胞核发生固缩、染色体聚集、凋亡小体形成。FCM法检测结果显示,HNPI可呈依赖性诱导HeLa细胞发生凋亡并阻滞HeLa细胞于G_0/G_1期,与0.9%氯化钠溶液对照组比较,差异均有统计学意义(P值均0.05);HeLa细胞内活性氧水平呈浓度依赖性升高,而线粒体膜电位呈浓度依赖性降低,与对照组比较,差异均有统计学意义(P值均0.05)。结论 :HNPI在体外可显著抑制HeLa细胞增殖并诱导凋亡,其机制可能与HNPI改变细胞超微结构,引起细胞损伤,使细胞内活性氧蓄积和线粒体膜电位降低,使细胞周期阻滞于G_0/G_1期进而发生凋亡相关。
[Abstract]:Aim: to study the inhibitory effect of a novel genistein derivative 5-hydroxy-4- nitro-7-propionyloxy-isoflavone HNPI on the proliferation and apoptosis of human cervical cancer HeLa cells in vitro, and to investigate the effects of 5-hydroxy-4-nitro-7-propionyloxy-isoflavone HNPI on the proliferation and apoptosis of human cervical cancer HeLa cells in vitro. Methods: HeLa cells were treated with different concentrations of HNPI51010C20C4060 and 80 渭 mol / L for 48 h to determine the inhibitory rate of HeLa proliferation. The surface morphology, cell height and cell width of HeLa cells were observed under atomic force microscope. The changes of root-squared roughnessen (RQ) and cell average average roughnessRa(). DAPI fluorescence staining was used to observe the morphologic changes of HeLa nuclei under laser scanning confocal microscope. FCM was used to detect the apoptosis rate and cell cycle of HeLa cells. Results the cell proliferation inhibition rate was increased in a dose-dependent manner when the concentration of HNPI was 10 渭 mol/L. The difference was statistically significant (P < 0.05). The median inhibitory concentration of HNPI on HeLa cells was 46.83 渭 mol / L. After 48 h treatment of HeLa cells with 20 and 60 渭 mol/L HNPI, the results of ultrahigh resolution atomic force microscopy (AFM) showed that the cells shrank and became round. The cytoplasm is concentrated, the nucleus bulges, the surface roughness of cell membrane is reduced, the surface of cell membrane becomes smooth and smooth, the filamentous pseudopodia is reduced, sparse, shorter, atrophic, even completely destroyed. The ultrastructural imaging results showed that the ultrastructural processes on the surface of HeLa cell membrane were sparse, distributed flat and single, and synthesized large protrusions and bulges, and the quantitative analysis of single cells showed that the cell height was increased in a concentration dependent manner. The width of RQ and Ra decreased in a concentration-dependent manner. Compared with the control group of 0.9% sodium chloride solution, the difference was statistically significant (P < 0.05). The results of DAPI method showed that the nuclei were pyknosis and chromosome aggregation. The results of apoptotic corpuscle formation. FCM assay showed that HNPI could induce apoptosis of HeLa cells in a dependent manner and block HeLa cells in the G _ 0 / G _ 1 phase, compared with the control group of 0.9% sodium chloride solution. The difference was statistically significant (P < 0.05). The level of reactive oxygen species in HeLa cells was increased in a concentration-dependent manner, while the mitochondrial membrane potential was decreased in a concentration-dependent manner, compared with the control group. Conclusion the proliferation and apoptosis of HeLa cells can be significantly inhibited and apoptosis induced by HNPI in vitro, and the mechanism may be similar to that of HNPI, which may change the ultrastructure of the cells and cause cell damage. The accumulation of reactive oxygen species in cells and mitochondrial membrane potential were decreased, and the cell cycle was blocked in the first phase of G _ (0) / G _ (1), which was related to apoptosis.
【作者单位】：湖南省妇幼保健院妇一科;
【分类号】：R737.33

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