EVn-50对人视网膜母细胞瘤细胞生长和凋亡作用的影响

发布时间：2018-06-23 08:12

本文选题：人视网膜母细胞瘤 + 黄荆子　；参考：《南华大学》2010年硕士论文

【摘要】： 目的研究黄荆子乙酸乙酯提取物(EVn-50)抑制体外培养人视网膜母细胞瘤细胞(RB细胞)生长作用以及诱导RB细胞凋亡作用,为开发和研制临床治疗人视网膜母细胞瘤候选药物提供实验和理论依据。方法体外培养RB细胞;MTT比色法测定EVn-50对RB细胞增殖活性的影响;细胞计数法检测RB细胞存活率,绘制生长曲线;软琼脂集落形成法测定EVn-50抑制体外培养RB细胞的锚定非依赖性生长作用;PI染色流式细胞术(FCM)定量分析EVn-50诱导RB细胞凋亡程度;AO/EB染色荧光显微镜观察EVn-50诱导RB细胞凋亡的形态学改变;DNA琼脂糖凝胶电泳确证EVn-50诱导RB细胞凋亡作用。结果 MTT比色法测定结果显示:EVn-50可作为黄荆子抗RB的有效提取物。EVn-50对RB细胞增殖活性抑制作用强,并呈浓度依赖性,其IC50为9.60μg/ml;细胞计数结果表明:0.3、3.0、30.0μg/ml的EVn-50均具有抑制RB细胞生长作用,呈浓度和时间依赖性;软琼脂集落形成法的结果表明:EVn-50对RB细胞锚定非依赖性生长具有明显的抑制作用,并呈浓度依赖性。PI染色流式细胞术分析的量效实验结果发现:0.3、3.0、30.0μg/ml的EVn-50作用于RB细胞48小时后的细胞凋亡率分别是(2.4±0.1)%、(17.4±0.6)%、(40.7±0.7)%;而时效实验中30.0μg/ml的EVn-50作用于RB细胞24、48、72小时后的细胞凋亡率分别是(20.8±0.4)%、(40.7±0.7)%、(63.1±0.8)%;远高于空白对照组的凋亡率(0.8±0.3)%。表明:EVn-50诱导RB细胞凋亡,并呈浓度和时间依赖性。AO/EB染色荧光显微镜观察EVn-50处理后,部分RB细胞呈现胞质和胞核呈红色,核固缩或碎裂的典型凋亡细胞形态特征。EVn-50 0.3μg/ml、3.0μg/ml、30.0μg/ml分别处理RB细胞48小时,以及EVn-50 30.0μg/ml分别作用RB细胞0、24、48和72h琼脂糖凝胶电泳出现典型“梯形”DNA条带。结论 1. EVn-50抑制体外培养人视网膜母细胞瘤细胞RB细胞生长,呈浓度和时间依赖性;EVn-50是黄荆子抗人视网膜母细胞瘤作用的有效提取物。 2. EVn-50有效诱导人视网膜母细胞瘤RB细胞凋亡,呈浓度和时间依赖性。
[Abstract]:Objective to study the inhibitory effect of ethyl acetate extract (EVn-50) on the growth and apoptosis of human retinoblastoma cells (RB cells) in vitro. To provide experimental and theoretical basis for the development and development of clinical drug candidates for the treatment of human retinoblastoma. Methods the effect of EVn-50 on the proliferation of RB cells was determined by MTT colorimetry in vitro, the survival rate of RB cells was measured by cell count method, and the growth curve was drawn. Inhibitory effect of EVn-50 on Anchorage Independent growth of RB cells in Vitro by soft Agar Colony Colony formation quantitative Analysis of apoptosis degree induced by EVn-50 by flow Cytometry (FCM) and fluorescence microscope of EVn-50 induced RB cells Morphological changes of apoptosis in RB cells induced by EVn-50 were confirmed by DNA agarose gel electrophoresis. Results the results of MTT colorimetric assay showed that WEVn-50 could inhibit the proliferation of RB cells in a concentration-dependent manner as an effective extract of Vitex chinensis L. against RB, and EVn-50 could inhibit the proliferation of RB cells in a dose-dependent manner. The IC50 of EVn-50 was 9.60 渭 g / ml, and the results of cell count showed that the EVn-50 of 30 渭 g/ml at 30 渭 g/ml could inhibit the growth of RB cells in a concentration-and time-dependent manner, and the results of soft Agar colony forming method showed that the growth of RB cells anchoring independent of EVn-50 was inhibited obviously by the method of soft Agar colony forming. The dose-effect analysis of flow cytometry in a concentration dependent manner with Pi staining showed that the apoptosis rates of RB cells treated with EVn-50 of 30.0 渭 g/ml at 30 渭 g/ml for 48 hours were (2.4 卤0.1), (17.4 卤0.6), (40.7 卤0.7), respectively, while in the aging experiment, the effects of EVn-50 of 30.0 渭 g/ml on the fine cells of RB cells after 2448 hours were (2.4 卤0.1), (17.4 卤0.6), (40.7 卤0.7), respectively. The apoptotic rate was (20.8 卤0.4), (40.7 卤0.7), (63.1 卤0.8) and much higher than that in the control group (0.8 卤0.3). The results showed that the apoptosis of RB cells was induced by w / EVn-50, and some RB cells showed red cytoplasm and nucleus after treated with EVn-50 in a concentration-and time-dependent manner. Morphological characteristics of typical apoptotic cells with nuclear pyknosis or fragmentation. EVn-50 0.3 渭 g 路ml ~ (-1) 路ml ~ (3) 渭 g / ml ~ (30) 渭 g/ml was used to treat RB cells for 48 hours, and EVn-50 ~ (30.0) 渭 g/ml was used to treat RB cells for 48 hours and 72 h after agarose gel electrophoresis, typical "trapezoid" DNA bands appeared. Conclusion 1. EVn-50 inhibits the growth of human retinoblastoma RB cells in a dose-and time-dependent manner. EVn-50 is an effective extract of Vitex chinensis against human retinoblastoma. 2. Human retinoblastoma RB cells were induced to apoptosis by EVn-50 in a concentration and time dependent manner.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R739.7

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