本文選題：姜黃素 + 紫杉醇�。� 參考：《西南醫(yī)科大學》2017年碩士論文

【摘要】：目的:通過體外研究姜黃素(curcumin)聯(lián)合紫杉醇(paclitaxel,PTX)對人肺腺癌A549細胞的作用,探討兩藥聯(lián)合對A549細胞的生物學效應的影響,并探討其可能的機制。方法:(1)人肺腺癌A549細胞的培養(yǎng):以含10%胎牛血清的RPMI1640培養(yǎng)基,置于飽和濕度、CO_2濃度為5%、溫度為37℃的恒溫培養(yǎng)箱中培養(yǎng)。(2)分組及處理:設置對照組(不加入任何藥物)、DMSO組、姜黃素組、紫杉醇組、姜黃素聯(lián)合紫杉醇組,當細胞培養(yǎng)至對數(shù)期處理后,DMSO組給予與姜黃素組同濃度的DMSO溶液,姜黃素組給予姜黃素濃度為20μmol/L,紫杉醇組給予紫杉醇濃度為4nmol/L,聯(lián)合組姜黃素濃度為20μmol/L,紫杉醇濃度為4nmol/L分別處理。(3)MTT法分別計算紫杉醇及姜黃素的IC50:取對數(shù)期A549細胞接種于96孔板,貼壁生長24小時后分別加入濃度為0.5nmol/L、1nmol/L、2nmol/L、4nmol/L、8nmol/L的紫杉醇,濃度為5umol/L、10umol/L、20umol/L、40umol/L、80umol/L的姜黃素后繼續(xù)培養(yǎng)48小時,檢測各濃度的IOD值,并計算紫杉醇及姜黃素IC50(半數(shù)抑制濃度)。(4)MTT實驗:取對數(shù)期A549細胞接種于96孔板,貼壁生長24小時后按分組條件處理后繼續(xù)培養(yǎng)48小時,檢測各組IOD值,計算增殖抑制率及兩藥聯(lián)合的Q值。(5)細胞周期及凋亡檢測:將A549細胞培養(yǎng)至對數(shù)期,按分組條件處理后繼續(xù)培養(yǎng)48h,采用流式細胞儀分別檢測各組細胞周期分布及細胞凋亡率。(6)將A549細胞接種于6孔板,待細胞單層鋪滿板底時進行劃痕后,按分組條件處理,繼續(xù)培養(yǎng)48小時觀察細胞遷移情況,并計算各組細胞遷移距離。(7)制好Matrigel膜后,取對數(shù)期A549細胞制成無血清細胞懸液加入Transwell小室上室,按分組條件加入對應藥物處理后48小時,取出小室進行染色并計算穿膜細胞數(shù)。(8)采用酶聯(lián)免疫吸附試驗檢測各組處理后48小時HIF-1α及VEGF表達情況。結(jié)果:(1)紫杉醇作用于A549細胞48小時的IC50為4nmol/L,姜黃素作用于A549細胞48小時的IC50為20umol/L。(2)姜黃素聯(lián)合紫杉醇組對肺癌A549細胞的增殖具有明顯的抑制作用,其抑制率明顯高于對照組、紫杉醇組及姜黃素組;姜黃素聯(lián)合紫杉醇對肺癌A549細胞的抑制作用具有協(xié)同作用,其Q值為1.072338。(3)DMSO組細胞周期分布與對照組無明顯差異(P0.05),姜黃素組細胞阻滯于S期及G2/M期,紫杉醇組細胞阻滯于G2/M期,聯(lián)合用藥組細胞阻滯于G0/G1期及S期。(4)DMSO組凋亡率與對照組凋亡率無明顯差異(P0.05),與對照組比較各實驗組凋亡率均高于對照組,聯(lián)合用藥組凋亡率高于其他各組,差異具有統(tǒng)計學意義(P0.05)。(5)與對照組比較,姜黃素組、紫杉醇組及聯(lián)合用藥組的遷徙距離及穿膜細胞數(shù)明顯小于對照組,姜黃素組及紫杉醇組的差異無統(tǒng)計學意義(P0.05);聯(lián)合用藥組的遷徙距離及穿膜細胞數(shù)明顯小于姜黃素組及紫杉醇組,差異具有統(tǒng)計學意義(P0.05)。(6)DMSO組HIF-1α和VEGF的表達水平與對照組比較差異無統(tǒng)計學意義(P0.05);姜黃素組、紫杉醇組及聯(lián)合用藥組HIF-1α及VEGF的表達水平明顯低于對照組,且各實驗組間比較差異具有統(tǒng)計學意義(P0.05)。HIF-1α及VEGF的表達水平呈正相關(R=0.976,P0.05)。結(jié)論:(1)姜黃素聯(lián)合紫杉醇能顯著抑制肺癌A549細胞的增殖及侵襲轉(zhuǎn)移能力,促進細胞凋亡;兩藥聯(lián)合對肺癌A549細胞的作用具有協(xié)同效應。(2)姜黃素聯(lián)合紫杉醇能顯著下調(diào)HIF-1α及VEGF的表達,姜黃素聯(lián)合紫杉醇對肺癌A549細胞的增殖、凋亡及侵襲轉(zhuǎn)移能力的影響可能與其下調(diào)HIF-1α及VEGF的表達相關。
[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.

【學位授予單位】：西南醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R734.2

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