高良姜总黄酮影响宫颈癌细胞增殖、凋亡及基因表达调控的机理研究
发布时间：2018-08-27 16:45
【摘要】：目的:研究高良姜总黄酮对人宫颈癌细胞株SiHa的增殖、形态学变化和其细胞凋亡并对相关肿瘤干细胞标记物转录表达水平的影响,探讨高良姜总黄酮的抗肿瘤作用机理,为高良姜总黄酮作为抗肿瘤药物开发提供理论和实验依据。方法:1)根据原有的工艺,采用乙醇提取,石油醚脱脂,氯仿萃取等方法得到高良姜总黄酮;2)用倒置显微镜观察不同浓度高良姜总黄酮在不同时间内作用于宫颈癌SiHa细胞后的形态学变化;3)采用四甲基偶氮唑盐(MTT)显色法,检测不同浓度高良姜总黄酮在不同时间内对宫颈癌SiHa细胞存活和生长所产生的不同影响,设抗癌药顺氯氨铂(cis-Dichlorodiamineplatinum,DDP)为阳性对照;4)通过Annexin V-FITC/PI双染色、流式细胞仪检测并分析高良姜总黄酮对SiHa细胞24h作用而引起的细胞凋亡率,设抗癌药顺氯氨铂(DDP)为阳性对照;5)Trizol一步法抽提高良姜总黄酮干预的SiHa细胞总RNA,并应用荧光定量PCR法,签定高良姜总黄酮诱导SiHa细胞的肿瘤干细胞标志物基因转录表达水平;6)统计学方法:实验数据以mean±SD表示,以SPSS17.0版专用统计分析软件对各组数据进行一般线性模型单变量方差分析。结果:1)从高良姜提取的总黄酮在0~400μg/ml浓度范围内,随着高良姜总黄酮干预SiHa细胞的剂量增加,细胞活力显著下降(12%),其IC50值为(24h的127.393μg/ml,48h的84.584μg/ml,72h为58.054μg/ml)抑制细胞活力的趋势与阳性对照基本一致,并呈浓度和时间依赖性;2)流式细胞仪检测显示,在50和100μg/ml高良姜总黄酮剂量引起SiHa细胞凋亡,其早期凋亡率升高(17.35%和22.4%),但是远远低于阳性对照组的早期凋亡率(60.4%);3)高良姜总黄酮干预宫颈癌细胞株SiHa后可使OCT4,ALDH1A1的转录表达水平有显著降低(P0.05),而对TWIST1的转录表达水平的影响均无统计学意义(P0.05)。结论:1)高良姜总黄酮能够抑制宫颈癌细胞的增殖、诱导细胞凋亡,其作用有剂量和时间依赖性;2)高良姜总黄酮干预宫颈癌细胞SiHa可能引起ALDH1A1和OCT4等肿瘤干细胞标记物的基因表达下调,是该药抗癌作用的一部分分子机制。
[Abstract]:Objective: to study the effects of total flavonoids of alpinia officinalis on the proliferation, morphological changes and apoptosis of human cervical cancer cell line SiHa, and on the transcription and expression level of related tumor stem cell markers, and to explore the mechanism of anti-tumor effect of total flavonoids of alpinia officinalis. To provide theoretical and experimental basis for the development of total flavonoids as antitumor drugs. Methods: according to the original technology, ethanol was used to extract, petroleum ether was degreased, The morphological changes of SiHa cells treated with different concentrations of total flavonoids of alpinia officinalis were observed by inverted microscope. The morphological changes of SiHa cells were observed by chloroform extraction. The method of tetramethyl azolium salt (MTT) was used. The effects of total flavonoids of alpinia officinalis at different concentrations on the survival and growth of cervical cancer SiHa cells were detected at different time. The anticancer drug cis-chloroplatin (cis-Dichlorodiamineplatinum,DDP) was used as the positive control. Annexin V-FITC/PI double staining was used to detect the effects of different concentrations of total flavonoids on the survival and growth of cervical cancer SiHa cells. Flow cytometry was used to detect and analyze the apoptosis rate induced by 24 h action of total flavonoids of alpinia officinalis on SiHa cells. The total RNA, of SiHa cells treated with total flavonoids of alpinia officinalis was extracted by one step method of Trizol, and the total RNA, of SiHa cells was extracted by fluorescence quantitative PCR method with cis-chloroplatin (DDP) as positive control. Statistical method for the expression of tumor stem cell markers gene transcription in SiHa cells induced by total flavonoids of alpinia officinarum L. was established. The experimental data were expressed as mean 卤SD. The univariate ANOVA of general linear model was carried out with the special statistical analysis software of SPSS17.0 version. Results: the total flavonoids extracted from alpinia officinalis were in the concentration range of 0 ~ 400 渭 g/ml, with the increase of the dose of total flavonoids in SiHa cells. The cell viability decreased significantly (12%), and its IC50 value (127.393 渭 g / ml / min at 24 h, 84.584 渭 g / ml / min at 48 h was 58.054 渭 g/ml) was consistent with that of the positive control, and was in a concentration and time dependent manner. Apoptosis of SiHa cells was induced by 50 渭 g/ml and 100 渭 g/ml total flavonoids. The rate of early apoptosis was increased (17.35% and 22.4%), but much lower than that of the positive control group (60.4%). The total flavonoids of alpinia officinalis treated cervical cancer cell line SiHa could significantly decrease the transcription expression of OCT4,ALDH1A1 (P0.05), while the expression of TWIST1 in the water was significantly lower than that in the control group (P < 0.05). There was no significant difference in the effect of the level (P0.05). Conclusion: 1) Total flavonoids of Ginger can inhibit the proliferation of cervical cancer cells and induce apoptosis. Its effect is dose-and time-dependent. Total flavonoids of alpinia officinalis can induce the down-regulation of gene expression of tumor stem cell markers such as ALDH1A1 and OCT4 in cervical cancer cell line SiHa, which is a part of the molecular mechanism of the anticancer effect of the drug.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R285
[Abstract]:Objective: to study the effects of total flavonoids of alpinia officinalis on the proliferation, morphological changes and apoptosis of human cervical cancer cell line SiHa, and on the transcription and expression level of related tumor stem cell markers, and to explore the mechanism of anti-tumor effect of total flavonoids of alpinia officinalis. To provide theoretical and experimental basis for the development of total flavonoids as antitumor drugs. Methods: according to the original technology, ethanol was used to extract, petroleum ether was degreased, The morphological changes of SiHa cells treated with different concentrations of total flavonoids of alpinia officinalis were observed by inverted microscope. The morphological changes of SiHa cells were observed by chloroform extraction. The method of tetramethyl azolium salt (MTT) was used. The effects of total flavonoids of alpinia officinalis at different concentrations on the survival and growth of cervical cancer SiHa cells were detected at different time. The anticancer drug cis-chloroplatin (cis-Dichlorodiamineplatinum,DDP) was used as the positive control. Annexin V-FITC/PI double staining was used to detect the effects of different concentrations of total flavonoids on the survival and growth of cervical cancer SiHa cells. Flow cytometry was used to detect and analyze the apoptosis rate induced by 24 h action of total flavonoids of alpinia officinalis on SiHa cells. The total RNA, of SiHa cells treated with total flavonoids of alpinia officinalis was extracted by one step method of Trizol, and the total RNA, of SiHa cells was extracted by fluorescence quantitative PCR method with cis-chloroplatin (DDP) as positive control. Statistical method for the expression of tumor stem cell markers gene transcription in SiHa cells induced by total flavonoids of alpinia officinarum L. was established. The experimental data were expressed as mean 卤SD. The univariate ANOVA of general linear model was carried out with the special statistical analysis software of SPSS17.0 version. Results: the total flavonoids extracted from alpinia officinalis were in the concentration range of 0 ~ 400 渭 g/ml, with the increase of the dose of total flavonoids in SiHa cells. The cell viability decreased significantly (12%), and its IC50 value (127.393 渭 g / ml / min at 24 h, 84.584 渭 g / ml / min at 48 h was 58.054 渭 g/ml) was consistent with that of the positive control, and was in a concentration and time dependent manner. Apoptosis of SiHa cells was induced by 50 渭 g/ml and 100 渭 g/ml total flavonoids. The rate of early apoptosis was increased (17.35% and 22.4%), but much lower than that of the positive control group (60.4%). The total flavonoids of alpinia officinalis treated cervical cancer cell line SiHa could significantly decrease the transcription expression of OCT4,ALDH1A1 (P0.05), while the expression of TWIST1 in the water was significantly lower than that in the control group (P < 0.05). There was no significant difference in the effect of the level (P0.05). Conclusion: 1) Total flavonoids of Ginger can inhibit the proliferation of cervical cancer cells and induce apoptosis. Its effect is dose-and time-dependent. Total flavonoids of alpinia officinalis can induce the down-regulation of gene expression of tumor stem cell markers such as ALDH1A1 and OCT4 in cervical cancer cell line SiHa, which is a part of the molecular mechanism of the anticancer effect of the drug.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R285
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