艾迪注射液对人肝癌多药耐药细胞的逆转作用研究

发布时间：2018-04-22 11:07

本文选题：艾迪注射液 + 多药耐药　；参考：《贵州医科大学》2017年硕士论文

【摘要】：目的:探讨艾迪注射液对人肝癌多药耐药细胞Bel-7402/5-FU的耐药性是否具有逆转作用及其可能的逆转机制。方法:1.采用5-氟尿嘧啶(fluorouracil,5-FU)通过浓度梯度递加法建立人肝癌耐药细胞Bel-7402/5-FU。2.待耐药细胞Bel-7402/5-FU建立成功后,将实验分为两组,即Bel-7402组和Bel-7402/5-FU组,将两组细胞种植于96孔板中,待细胞贴壁稳定后,再将每种细胞分为7个小组,每种细胞的第一组加入正常培养基,后面六组分别加入含5-FU、ADM、MMC、MTX、CTX、CDDP的培养基,每孔终体积为200ul,培养24h后,每孔加入10ul的cck-8试剂,继续培养2h后,酶标仪在450nm下读取吸光度值,使用SPSS19.0统计软件计算每种化疗药物对亲本细胞Bel-7402及耐药细胞Bel-7402/5-FU 的半数抑制浓度(half maximal inhibitory concentration,IC50),计算6种化疗药物Bel-7402细胞和Bel-7402/5-FU细胞的抑制率(IR)和Bel-7402/5-FU的耐药指数(RI)。3.通过不同浓度的艾迪注射液作用于Bel-7402/5-FU后,cck-8法检测艾迪注射液对Bel-7402/5-FU是否具有抑制作用。4.将实验分为3组,即Bel-7402组、Bel-7402/5-FU组和艾迪注射液作用过的Bel-7402/5-FU组,分别提取三组细胞中的总蛋白后,Western blot法检测三组细胞中多药耐药相干蛋白1(MRP1)、P-糖蛋白(P-gp,170kD)、程序化死亡因子5(PDCD5,15kD)蛋白的表达情况。结果:1、Bel-7402/5-FU细胞对6种化疗药物的半数抑制浓度较Bel-7402细胞明显升高,对6种化疗药物表现出不同程度的耐药性。2、艾迪注射液对Bel-7402/5-FU细胞具有抑制作用,且随着艾迪注射液浓度的增加,抑制作用越强。3、与Bel-7402细胞相比,Bel-7402/5-FU细胞株中MRP1、P-gp表达明显升高,PDCD5表达较低。使用半数抑制浓度的艾迪注射液分别作用于Bel-7402细胞及Bel-7402/5-FU细胞后,与Bel-7402/5-FU细胞相比,经过艾迪注射液作用后的Bel-7402/5-FU细胞中MRP1、P-gp表达降低,PDCD5表达增高,差异具有统计学意义(P0.05)。结论:艾迪注射液对肝癌细胞的多药耐药性具有一定的逆转作用,其机制与降低多药耐药相关蛋白1和P-糖蛋白的表达,促进凋亡相关蛋白5的表达有关。
[Abstract]:Aim: to investigate whether Aidi injection can reverse the drug resistance of multidrug resistant human hepatoma cell line Bel-7402/5-FU and its possible reversal mechanism. Method 1: 1. 5-fluorouraciline 5-FU (5-FU) was used to establish human hepatoma resistant cell line Bel-7402 / 5-FU. After the establishment of drug-resistant cell line Bel-7402/5-FU, the experiment was divided into two groups: Bel-7402 group and Bel-7402/5-FU group. The two groups of cells were implanted in 96-well plate. When the cells were stably adhered to the wall, each cell was divided into seven groups. The first group of each cell was added to the normal medium, and the latter six groups were added respectively to the medium containing 5-FUU ADMU MMCMTXTX CTX CDDP. The final volume of each pore was 200 ul.After 24 hours of culture, the cck-8 reagent of 10ul was added to each pore, and after 2 hours of culture, the absorbance value was read by the enzyme marker under 450nm. SPSS19.0 software was used to calculate the half maximal inhibitory concentration (IC50) of each chemotherapeutic drug on Bel-7402 and Bel-7402/5-FU, and to calculate the inhibitory rate of Bel-7402 and Bel-7402/5-FU cells on 6 chemotherapeutic drugs (Bel-7402 and Bel-7402/5-FU) and the resistance index of Bel-7402/5-FU (RIN. 3). The inhibitory effect of Aidi injection on Bel-7402/5-FU was detected by cck-8 method after different concentrations of Aidi injection was treated with Bel-7402/5-FU. The experiment was divided into three groups: Bel-7402 group, Bel-7402 / 5-FU group and Bel-7402/5-FU group, which were treated with Aidi injection. The total protein was extracted from the three groups of cells by Western blot method to detect the expression of multidrug resistance-associated protein (MRP1P-1), P-gpfU (170kDN) and programmed death factor 5PDCD5( 15kD) in the three groups of cells. Results the half inhibitory concentration of Bel-7402 / 5-FU cells to 6 chemotherapeutic drugs was significantly higher than that of Bel-7402 cells, and the drug resistance to 6 chemotherapeutic drugs was different. Adi injection had inhibitory effect on Bel-7402/5-FU cells, and with the increase of Aidi injection concentration, The stronger the inhibitory effect was, the lower the expression of PDCD5 was in Bel-7402 / 5-FU cell line compared with that in Bel-7402 cell line. Compared with Bel-7402/5-FU cells, the expression of MRP1P-gp in Bel-7402/5-FU cells treated with Aidi injection with 50% inhibitory concentration was significantly lower than that in Bel-7402/5-FU cells, and the difference was statistically significant (P 0.05). Conclusion: Aidi injection can reverse the multidrug resistance of hepatoma cells, and its mechanism is related to the reduction of the expression of multidrug resistance-associated protein 1 and P-glycoprotein, and the promotion of apoptosis-related protein 5 expression.
【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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