姜黄素对阻断NF-κB信号通路抑制人卵巢癌细胞株3AO增殖
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摘要
研究目的
1.姜黄素作用卵巢癌细胞株3AO后生长情况
2.初步研究姜黄素作用卵巢癌细胞株3AO后NF-κB家族蛋白表达的变化。
研究方法
1.体外培养卵巢癌细胞株3AO。
2.采用MTT方法检测对照组、不同浓度(20、40、80μg/L)Cur干预组(干预24、48小时)吸光值,计算细胞抑制率值。
3.应用流式细胞术检测不同浓度(20、40、80μg/L)Cur干预24h后细胞周期的改变。
4.应用光学显微镜观察不同浓度(20、40、80μg/L)Cur干预细胞24h后卵巢癌细胞形态改变。
5.利用Western blot方法检测Cur(20、40、80μg/L)干预24小时后NF-κB、p-NF-κB在转录及翻译水平的表达以及其抑制物IκB、p-IκB在翻译水平的表达,与对照组进行比较。
6.免疫荧光化学方法检测Cur(20、40、80μg/L)干预3AO细胞24小时后NF-κB表达。
7.RT-PCR技术检测Cur(20、40、80μg/L)干预3AO细胞24小时后NF-κBmRNA的表达。
研究结果
1.姜黄素对人卵巢癌细胞株3AO生长的抑制作用,呈剂量时间依赖性。
2.流式细胞仪分析证实姜黄素能使3AO细胞周期阻滞于G0/G1期。
3.光学显微镜观察显示:随着姜黄素浓度的增高,卵巢癌细胞株形态逐渐发生变化,透光性及折光性降低,细胞皱缩,细胞数量减少,凋亡细胞增多。
4.Western blot结果显示随姜黄素作用浓度的增加,NF-κB、p-NF-κB蛋白表达水平明显下降,IκB、p-IκB蛋白表达有增加。
5.免疫荧光结果显示随着姜黄素浓度的增高,细胞内NF-κB表达逐渐减少。
6.RT-PCR结果显示不同浓度姜黄素培养的卵巢癌细胞,随着药物浓度增高,NF-κB mRNA表达逐渐减弱,差异有统计学意义。
研究结论
姜黄素能对3AO细胞有明显的抑制增殖作用,阻止细胞进入有丝分裂的S期,此作用可以通过下调NF-κB蛋白表达及磷酸化实现,这是姜黄素抑制卵巢癌细胞生长分子机制之一。
Objective:
1.To investigate the effects of curcumin on proliferation of human ovarian cancercell line 3AO in vitro.
2.Preliminary study NF-κB family protein expression changes in 3AO ovariancancer cell after applying curcumin.
Methods:
1.The human ovarian cancer cell line 3AO was cultured in vitro and treated withcurcumin of different concentration(20μg/L、40μg/L、80μg/L);
2.The inhibition rates of cells were detected through MTT assay;
3.the cell cycle distribution were detected by flow cytometry(FCM);
4.Morphological changes of different concentrations (20,40,80μg/L) Curintervention in ovarian cancer cells after 24 hours;
5.the expression of NF-κB、p-NF-κB、IκB、p-IκB in 3AO cell was observedby western bolt;
6.The expression of NF-κB in 3AO cell was observed byimmunofluorescence technique.
7.The expression of NF-κB mRNA in 3AO cell was observed byreal-time polymerase chain reaction .
Results:
1.Curcumin inhibited the proliferation of 3AO cells in a dose-and time-dependentmanner.
2.The cell growth was arrested at G0/G1 stage.
3.With the curcumin concentration increased gradually occurred in form ofovarian cancer cell line , light transmittance and refractive index decreased, cellshrinkage , cell number reduce ,the apoptotic cells increased.
4.The expression of NF-κB、p-NF-κB was increased in a dose dependentmanner, the expression of IκB、p-IκB is increased.
5.Immunofluorescence results showed that with increased concentrations ofcurcumin, cells gradually reduced expression of NF-κB.
6.Real-time polymerase chain reaction results showed that with increasedconcentrations of curcumin, cells gradually reduced expression of NF-κBmRNA.
Conclusion:The curcumin can inhibits the proliferation of 3AO,prevent the cellsentering the mitotic S-phase, it is related to the down-regulation of NF-κB andp-NF-κB.
1.姜黄素作用卵巢癌细胞株3AO后生长情况
2.初步研究姜黄素作用卵巢癌细胞株3AO后NF-κB家族蛋白表达的变化。
研究方法
1.体外培养卵巢癌细胞株3AO。
2.采用MTT方法检测对照组、不同浓度(20、40、80μg/L)Cur干预组(干预24、48小时)吸光值,计算细胞抑制率值。
3.应用流式细胞术检测不同浓度(20、40、80μg/L)Cur干预24h后细胞周期的改变。
4.应用光学显微镜观察不同浓度(20、40、80μg/L)Cur干预细胞24h后卵巢癌细胞形态改变。
5.利用Western blot方法检测Cur(20、40、80μg/L)干预24小时后NF-κB、p-NF-κB在转录及翻译水平的表达以及其抑制物IκB、p-IκB在翻译水平的表达,与对照组进行比较。
6.免疫荧光化学方法检测Cur(20、40、80μg/L)干预3AO细胞24小时后NF-κB表达。
7.RT-PCR技术检测Cur(20、40、80μg/L)干预3AO细胞24小时后NF-κBmRNA的表达。
研究结果
1.姜黄素对人卵巢癌细胞株3AO生长的抑制作用,呈剂量时间依赖性。
2.流式细胞仪分析证实姜黄素能使3AO细胞周期阻滞于G0/G1期。
3.光学显微镜观察显示:随着姜黄素浓度的增高,卵巢癌细胞株形态逐渐发生变化,透光性及折光性降低,细胞皱缩,细胞数量减少,凋亡细胞增多。
4.Western blot结果显示随姜黄素作用浓度的增加,NF-κB、p-NF-κB蛋白表达水平明显下降,IκB、p-IκB蛋白表达有增加。
5.免疫荧光结果显示随着姜黄素浓度的增高,细胞内NF-κB表达逐渐减少。
6.RT-PCR结果显示不同浓度姜黄素培养的卵巢癌细胞,随着药物浓度增高,NF-κB mRNA表达逐渐减弱,差异有统计学意义。
研究结论
姜黄素能对3AO细胞有明显的抑制增殖作用,阻止细胞进入有丝分裂的S期,此作用可以通过下调NF-κB蛋白表达及磷酸化实现,这是姜黄素抑制卵巢癌细胞生长分子机制之一。
Objective:
1.To investigate the effects of curcumin on proliferation of human ovarian cancercell line 3AO in vitro.
2.Preliminary study NF-κB family protein expression changes in 3AO ovariancancer cell after applying curcumin.
Methods:
1.The human ovarian cancer cell line 3AO was cultured in vitro and treated withcurcumin of different concentration(20μg/L、40μg/L、80μg/L);
2.The inhibition rates of cells were detected through MTT assay;
3.the cell cycle distribution were detected by flow cytometry(FCM);
4.Morphological changes of different concentrations (20,40,80μg/L) Curintervention in ovarian cancer cells after 24 hours;
5.the expression of NF-κB、p-NF-κB、IκB、p-IκB in 3AO cell was observedby western bolt;
6.The expression of NF-κB in 3AO cell was observed byimmunofluorescence technique.
7.The expression of NF-κB mRNA in 3AO cell was observed byreal-time polymerase chain reaction .
Results:
1.Curcumin inhibited the proliferation of 3AO cells in a dose-and time-dependentmanner.
2.The cell growth was arrested at G0/G1 stage.
3.With the curcumin concentration increased gradually occurred in form ofovarian cancer cell line , light transmittance and refractive index decreased, cellshrinkage , cell number reduce ,the apoptotic cells increased.
4.The expression of NF-κB、p-NF-κB was increased in a dose dependentmanner, the expression of IκB、p-IκB is increased.
5.Immunofluorescence results showed that with increased concentrations ofcurcumin, cells gradually reduced expression of NF-κB.
6.Real-time polymerase chain reaction results showed that with increasedconcentrations of curcumin, cells gradually reduced expression of NF-κBmRNA.
Conclusion:The curcumin can inhibits the proliferation of 3AO,prevent the cellsentering the mitotic S-phase, it is related to the down-regulation of NF-κB andp-NF-κB.
引文
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