姜黄素联合紫杉醇对人肺腺癌A549细胞株生物学效应的影响

发布时间：2018-05-05 14:21

本文选题：姜黄素 + 紫杉醇　；参考：《西南医科大学》2017年硕士论文

【摘要】：目的:通过体外研究姜黄素(curcumin)联合紫杉醇(paclitaxel,PTX)对人肺腺癌A549细胞的作用,探讨两药联合对A549细胞的生物学效应的影响,并探讨其可能的机制。方法:(1)人肺腺癌A549细胞的培养:以含10%胎牛血清的RPMI1640培养基,置于饱和湿度、CO_2浓度为5%、温度为37℃的恒温培养箱中培养。(2)分组及处理:设置对照组(不加入任何药物)、DMSO组、姜黄素组、紫杉醇组、姜黄素联合紫杉醇组,当细胞培养至对数期处理后,DMSO组给予与姜黄素组同浓度的DMSO溶液,姜黄素组给予姜黄素浓度为20μmol/L,紫杉醇组给予紫杉醇浓度为4nmol/L,联合组姜黄素浓度为20μmol/L,紫杉醇浓度为4nmol/L分别处理。(3)MTT法分别计算紫杉醇及姜黄素的IC50:取对数期A549细胞接种于96孔板,贴壁生长24小时后分别加入浓度为0.5nmol/L、1nmol/L、2nmol/L、4nmol/L、8nmol/L的紫杉醇,浓度为5umol/L、10umol/L、20umol/L、40umol/L、80umol/L的姜黄素后继续培养48小时,检测各浓度的IOD值,并计算紫杉醇及姜黄素IC50(半数抑制浓度)。(4)MTT实验:取对数期A549细胞接种于96孔板,贴壁生长24小时后按分组条件处理后继续培养48小时,检测各组IOD值,计算增殖抑制率及两药联合的Q值。(5)细胞周期及凋亡检测:将A549细胞培养至对数期,按分组条件处理后继续培养48h,采用流式细胞仪分别检测各组细胞周期分布及细胞凋亡率。(6)将A549细胞接种于6孔板,待细胞单层铺满板底时进行划痕后,按分组条件处理,继续培养48小时观察细胞迁移情况,并计算各组细胞迁移距离。(7)制好Matrigel膜后,取对数期A549细胞制成无血清细胞悬液加入Transwell小室上室,按分组条件加入对应药物处理后48小时,取出小室进行染色并计算穿膜细胞数。(8)采用酶联免疫吸附试验检测各组处理后48小时HIF-1α及VEGF表达情况。结果:(1)紫杉醇作用于A549细胞48小时的IC50为4nmol/L,姜黄素作用于A549细胞48小时的IC50为20umol/L。(2)姜黄素联合紫杉醇组对肺癌A549细胞的增殖具有明显的抑制作用,其抑制率明显高于对照组、紫杉醇组及姜黄素组;姜黄素联合紫杉醇对肺癌A549细胞的抑制作用具有协同作用,其Q值为1.072338。(3)DMSO组细胞周期分布与对照组无明显差异(P0.05),姜黄素组细胞阻滞于S期及G2/M期,紫杉醇组细胞阻滞于G2/M期,联合用药组细胞阻滞于G0/G1期及S期。(4)DMSO组凋亡率与对照组凋亡率无明显差异(P0.05),与对照组比较各实验组凋亡率均高于对照组,联合用药组凋亡率高于其他各组,差异具有统计学意义(P0.05)。(5)与对照组比较,姜黄素组、紫杉醇组及联合用药组的迁徙距离及穿膜细胞数明显小于对照组,姜黄素组及紫杉醇组的差异无统计学意义(P0.05);联合用药组的迁徙距离及穿膜细胞数明显小于姜黄素组及紫杉醇组,差异具有统计学意义(P0.05)。(6)DMSO组HIF-1α和VEGF的表达水平与对照组比较差异无统计学意义(P0.05);姜黄素组、紫杉醇组及联合用药组HIF-1α及VEGF的表达水平明显低于对照组,且各实验组间比较差异具有统计学意义(P0.05)。HIF-1α及VEGF的表达水平呈正相关(R=0.976,P0.05)。结论:(1)姜黄素联合紫杉醇能显著抑制肺癌A549细胞的增殖及侵袭转移能力,促进细胞凋亡;两药联合对肺癌A549细胞的作用具有协同效应。(2)姜黄素联合紫杉醇能显著下调HIF-1α及VEGF的表达,姜黄素联合紫杉醇对肺癌A549细胞的增殖、凋亡及侵袭转移能力的影响可能与其下调HIF-1α及VEGF的表达相关。
[Abstract]:Objective: To study the effect of curcumin (curcumin) combined with paclitaxel (PTX) on human lung adenocarcinoma A549 cells in vitro, to explore the effects of two drugs on the biological effects of A549 cells, and to explore the possible mechanisms. Methods: (1) the culture of A549 cells in human lung adenocarcinoma: RPMI1640 culture containing 10% fetal bovine serum and saturated humidity. The CO_2 concentration was 5% and the temperature was 37 C incubator. (2) group and treatment: set control group (no drugs), DMSO group, curcumin group, paclitaxel group, curcumin combined paclitaxel group. After cell culture to logarithmic treatment, group DMSO was given the same concentration of curcumin group with DMSO solution, curcumin group was given curcumin concentration. The concentration of paclitaxel in paclitaxel group was 20 mol/L, the concentration of paclitaxel was 4nmol/L, the concentration of curcumin was 20 mol/L, and the concentration of paclitaxel was 4nmol/L respectively. (3) MTT method was used to calculate the IC50: of taxol and curcumin in logarithmic phase of A549 cells inoculated to 96 hole plates, and when the adherent growth was 24 small, the concentration was 0.5nmol/L, 1nmol/L, 2nmol/L, 4nmo. L/L, 8nmol/L paclitaxel, the concentration of 5umol/L, 10umol/L, 20umol/L, 40umol/L, 80umol/L of curcumin continued to be cultured for 48 hours, detection of the IOD value of each concentration, and calculated paclitaxel and curcumin IC50 (4) MTT experiment: logarithmic A549 cells were inoculated to 96 holes, after 24 hours adherent growth and continued to be treated according to the conditions of the group. The IOD value of each group was detected for 48 hours, the proliferation inhibition rate and the combined Q value of the two drugs were calculated. (5) cell cycle and apoptosis detection: the A549 cells were cultured to the logarithmic phase, and the 48h was continued after the group conditions, and the cell cycle distribution and the apoptosis rate were detected by flow cytometry. (6) the A549 cells were inoculated to 6 orifice plates and treated by the cells. After the single layer was covered with the bottom of the plate, the cell migration was observed for 48 hours and the migration distance was calculated for 48 hours. (7) after the preparation of the Matrigel membrane, the logarithmic A549 cells were taken into the Transwell compartment and added to the corresponding drug treatment for 48 hours after the treatment of the corresponding drug treatment. The cells were stained and the number of membrane cells was calculated. (8) the expression of HIF-1 alpha and VEGF was detected by enzyme linked immunosorbent assay for 48 hours after treatment. Results: (1) paclitaxel acted on A549 cells for 48 hours IC50 4nmol/L, and curcumin acted on the IC50 of A549 cells for 48 hours of 20umol/L. (2) combined paclitaxel group of curcumin and paclitaxel group to lung cancer A549 cells The inhibition rate of the proliferation was significantly higher than that of the control group, paclitaxel group and curcumin group. The curcumin combined paclitaxel had synergistic effect on the inhibition of lung cancer A549 cells. The Q value of the cell cycle of 1.072338. (3) DMSO group was not significantly different from that of the control group (P0.05), and the curcumin group cells were blocked at S and G2/M phase. The cells of paclitaxel group were blocked in G2/M phase, and the cells in the combination group were blocked at G0/G1 and S phase. (4) there was no significant difference between the apoptosis rate of the DMSO group and the control group (P0.05). Compared with the control group, the apoptosis rate of the experimental group was higher than that of the control group. The apoptosis rate of the combined group was higher than that of the other groups (P0.05). (5) compared with the control group, the rate of apoptosis was higher than that of the control group (P0.05). The migration distance and the number of membrane perforating cells in the curcumin group, the paclitaxel group and the combination group were significantly smaller than the control group, and the difference between the curcumin group and the paclitaxel group was not statistically significant (P0.05). The migration distance and the number of membrane cells in the combination group were significantly smaller than the curcumin group and the upura group (P0.05). (6) group DMSO HI There was no significant difference in the expression level of F-1 alpha and VEGF with the control group (P0.05); the expression level of HIF-1 alpha and VEGF in the curcumin group, the paclitaxel group and the combined drug group was significantly lower than that of the control group, and the difference between the experimental groups was statistically significant (P0.05) and the expression level of.HIF-1 A and VEGF was positively correlated (R=0.976, P0.05). (1) Curcumin combined paclitaxel can significantly inhibit the proliferation and invasion and metastasis of lung cancer A549 cells and promote cell apoptosis. Two drugs have synergistic effects on the role of A549 cells in lung cancer. (2) curcumin combined paclitaxel can significantly downregulate the expression of HIF-1 A and VEGF. The proliferation, apoptosis and invasion of curcumin combined with paclitaxel on lung cancer A549 cells The effect of metastasis may be related to its downregulation of HIF-1 and VEGF.

【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2

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