發(fā)布時(shí)間：2018-08-27 16:45

【摘要】：目的:研究高良姜總黃酮對(duì)人宮頸癌細(xì)胞株SiHa的增殖、形態(tài)學(xué)變化和其細(xì)胞凋亡并對(duì)相關(guān)腫瘤干細(xì)胞標(biāo)記物轉(zhuǎn)錄表達(dá)水平的影響,探討高良姜總黃酮的抗腫瘤作用機(jī)理,為高良姜總黃酮作為抗腫瘤藥物開發(fā)提供理論和實(shí)驗(yàn)依據(jù)。方法:1)根據(jù)原有的工藝,采用乙醇提取,石油醚脫脂,氯仿萃取等方法得到高良姜總黃酮;2)用倒置顯微鏡觀察不同濃度高良姜總黃酮在不同時(shí)間內(nèi)作用于宮頸癌SiHa細(xì)胞后的形態(tài)學(xué)變化;3)采用四甲基偶氮唑鹽(MTT)顯色法,檢測不同濃度高良姜總黃酮在不同時(shí)間內(nèi)對(duì)宮頸癌SiHa細(xì)胞存活和生長所產(chǎn)生的不同影響,設(shè)抗癌藥順氯氨鉑(cis-Dichlorodiamineplatinum,DDP)為陽性對(duì)照;4)通過Annexin V-FITC/PI雙染色、流式細(xì)胞儀檢測并分析高良姜總黃酮對(duì)SiHa細(xì)胞24h作用而引起的細(xì)胞凋亡率,設(shè)抗癌藥順氯氨鉑(DDP)為陽性對(duì)照;5)Trizol一步法抽提高良姜總黃酮干預(yù)的SiHa細(xì)胞總RNA,并應(yīng)用熒光定量PCR法,簽定高良姜總黃酮誘導(dǎo)SiHa細(xì)胞的腫瘤干細(xì)胞標(biāo)志物基因轉(zhuǎn)錄表達(dá)水平;6)統(tǒng)計(jì)學(xué)方法:實(shí)驗(yàn)數(shù)據(jù)以mean±SD表示,以SPSS17.0版專用統(tǒng)計(jì)分析軟件對(duì)各組數(shù)據(jù)進(jìn)行一般線性模型單變量方差分析。結(jié)果:1)從高良姜提取的總黃酮在0~400μg/ml濃度范圍內(nèi),隨著高良姜總黃酮干預(yù)SiHa細(xì)胞的劑量增加,細(xì)胞活力顯著下降(12%),其IC50值為(24h的127.393μg/ml,48h的84.584μg/ml,72h為58.054μg/ml)抑制細(xì)胞活力的趨勢與陽性對(duì)照基本一致,并呈濃度和時(shí)間依賴性;2)流式細(xì)胞儀檢測顯示,在50和100μg/ml高良姜總黃酮?jiǎng)┝恳餝iHa細(xì)胞凋亡,其早期凋亡率升高(17.35%和22.4%),但是遠(yuǎn)遠(yuǎn)低于陽性對(duì)照組的早期凋亡率(60.4%);3)高良姜總黃酮干預(yù)宮頸癌細(xì)胞株SiHa后可使OCT4,ALDH1A1的轉(zhuǎn)錄表達(dá)水平有顯著降低(P0.05),而對(duì)TWIST1的轉(zhuǎn)錄表達(dá)水平的影響均無統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:1)高良姜總黃酮能夠抑制宮頸癌細(xì)胞的增殖、誘導(dǎo)細(xì)胞凋亡,其作用有劑量和時(shí)間依賴性;2)高良姜總黃酮干預(yù)宮頸癌細(xì)胞SiHa可能引起ALDH1A1和OCT4等腫瘤干細(xì)胞標(biāo)記物的基因表達(dá)下調(diào),是該藥抗癌作用的一部分分子機(jī)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 鹵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.
【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R285

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本文編號(hào)：2207851