莪术油对阿霉素耐药的人甲状腺未分化癌细胞株HTh74Rdox的作用研究

发布时间：2018-09-17 12:31

【摘要】：目的:探讨莪术油对阿霉素诱导耐药的人甲状腺未分化癌细胞株HTh74Rdox增殖和耐药性的影响及机制。方法:用不同浓度莪术油(0、60、90、120mg/L)处理HTh74Rdox24小时后,于倒置显微镜下观察细胞形态;四甲基偶氮唑蓝(MTT)法检测莪术油对HTh74Rdox细胞增殖的抑制作用;细胞流式术(Flow cytomety,FCM)检测HTh74Rdox细胞周期变化和凋亡;蛋白质印迹法(Western Blot,WB)检测抑凋亡蛋白(Bcl-2)和促凋亡蛋白(Bax)的表达;荧光定量(Quantitative polymerase chain reaction,qPCR)检测多药耐药性基因 1(Multi-drug resistance gene1,MDR1)和三磷酸腺苷结合盒转运蛋白 G2(ATP-binding cassette superfamily G member 2,ABCG2)信使核糖核酸(Messenger RNA,mRNA)的变化。结果:用不同浓度莪术油(0、60、90、120mg/L)处理HTh74Rdox24小时后,倒置显微镜下观察细胞形态可见,从莪术油浓度为60mg/L起有部分细胞脱离培养皿;随着莪术油处理浓度的增加,细胞密度系数降低,贴壁细胞数量减少,细胞形状由梭形变为碎片、颗粒,待莪术油浓度增加至120mg/L,细胞几乎全部脱离培养皿,细胞形状完全变为不规则形状,死亡的细胞凝结成团,细胞液可见浑浊。MTT结果显示:随着莪术油刺激浓度的升高,莪术油抑制HTh74Rdox细胞增殖的作用越来越强,具有时间依赖性(P0.05)。流式结果显示:莪术油可诱导细胞凋亡,细胞表现为早期凋亡(P0.01);Western Blot结果显示:莪术油可以增加Bax蛋白表达,可以抑制Bcl-2蛋白表达,使Bax/Bcl-2比值增加(P0.05);qPCR:莪术油可使MDR1和ABCG2的mRNA表达减少。用单药莪术油、单药阿霉素或联合用药刺激HTh74Rdox细胞24小时后,MTT法分析示,2.0mg/L阿霉素+90ml/L莪术油组与2.0mg/L阿霉素+0ml/L莪术油组比较,均具有统计学差异(P0.05),提示阿霉素与莪术油联用后,可增强阿霉素的细胞毒作用;当阿霉素用药浓度减少至1.0mg/L的时候,加入低浓度(45ml/L)莪术油后,与1.5mg/L阿霉素+45mg/L莪术油组比较,其细胞毒作用没有统计学意义(P0.05),提示加入低浓度莪术油可减少阿霉素的用量,由此可见,莪术油对阿霉素具有明显的联合协同作用。结论:莪术油可以抑制HTh74Rdox细胞的生长、诱导其凋亡并改善其耐药作用,其机制可能分别与提高Bax/Bcl-2比值和降低MDR1、ABCG2 mRNA表达有关。
[Abstract]:Aim: to investigate the effect and mechanism of zedoary turmeric oil on the proliferation and drug resistance of adriamycin-resistant human thyroid undifferentiated carcinoma cell line HTh74Rdox. Methods: HTh74Rdox24 cells were treated with different concentrations of Zedoary turmeric oil (060,90120mg / L) for hours, and the cell morphology was observed under inverted microscope, and the inhibitory effect of Zedoary turmeric oil on proliferation of HTh74Rdox cells was detected by (MTT) method. Cell cycle changes and apoptosis of HTh74Rdox cells were detected by flow cytometry (Flow cytomety,FCM), and the expression of Bcl-2 and (Bax) were detected by Western blot (Western Blot,WB). The changes of multidrug resistance gene 1 (Multi-drug resistance gene1,MDR1) and adenosine triphosphate binding cassette transporter G2 (ATP-binding cassette superfamily G member 2 ABCG2) messenger ribonucleic acid (Messenger RNA,mRNA) were detected by fluorescence quantitative (Quantitative polymerase chain reaction,qPCR. Results: after treated with different concentrations of Zedoary turmeric oil (060 ~ 90120mg / L) for HTh74Rdox24 hours, the morphology of cells was observed under inverted microscope. Some cells were separated from petri dish from the concentration of 60mg/L, and the cell density coefficient decreased with the increase of the concentration of zedoary turmeric oil. The number of adherent cells is reduced, the shape of cells is changed from fusiform to fragments, particles, when the concentration of zedoary turmeric oil increases to 120 mg / L, the cells are almost completely detached from the petri dish, the shape of the cells becomes completely irregular, and the dead cells coagulate into clusters. The results showed that with the increase of the concentration of zedoary turmeric oil the inhibitory effect of zedoary turbid oil on the proliferation of HTh74Rdox cells was stronger and stronger in a time dependent manner (P0.05). The results of flow cytometry showed that Zedoary turmeric oil could induce cell apoptosis. The results of early apoptosis (P0.01) and Western Blot showed that curcuma oil could increase the expression of Bax protein and inhibit the expression of Bcl-2 protein. The ratio of Bax/Bcl-2 was increased (P0.05) QPCR: zedoary turmeric oil decreased the mRNA expression of MDR1 and ABCG2. Compared with 2.0mg/L adriamycin 0ml/L turmeric oil group and 2.0mg/L adriamycin 0ml/L turmeric oil group, HTh74Rdox cells were stimulated with single drug, adriamycin or adriamycin for 24 hours. The results showed that doxorubicin and zedoary oil combined with doxorubicin were significantly different (P0.05). When the concentration of doxorubicin was reduced to 1.0mg/L, the low concentration (45ml/L) of zedoary turmeric oil was added, which was compared with 1.5mg/L adriamycin 45mg/L zedoary oil group. Its cytotoxic effect was not statistically significant (P0.05), suggesting that adding low concentration of zedoary oil can reduce the amount of Adriamycin, it can be seen that zedoary oil has a significant synergistic effect on doxorubicin. Conclusion: Zedoary turmeric oil can inhibit the growth of HTh74Rdox cells, induce its apoptosis and improve its drug resistance. The mechanism may be related to the increase of Bax/Bcl-2 ratio and the decrease of MDR1,ABCG2 mRNA expression.
【学位授予单位】：南京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285

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