华蟾素在骨肉瘤细胞中的抗肿瘤作用及其机制实验研究

发布时间：2018-05-20 04:50

本文选题：华蟾素 + U2OS细胞　；参考：《华中科技大学》2015年博士论文

【摘要】：第一部分华蟾素诱导骨肉瘤细胞凋亡及其可能机制实验研究 [目的]：探讨华蟾素诱导骨肉瘤U20S细胞凋亡的效果及其可能机制。 [方法]：将不同浓度华蟾素作用于骨肉瘤U20S细胞,MTT法被用来检测每组骨肉瘤细胞的生长抑制率,TUNEL染色法被用来观察细胞凋亡程度并计算其凋亡率,免疫荧光染色、RT-PCR、以及Western Blot被用来检测凋亡相关以及Wnt/β-catenin通路相关蛋白。 [结果]：严格按照上述方法处理骨肉瘤U20S细胞后,MTT结果显示,随着华蟾素浓度和作用于细胞时间的增加,细胞生长抑制率明显增加,并呈时间-浓度依赖性关系。当华蟾素作用于骨肉瘤细胞不同时间后,细胞凋亡以及凋亡率随着作用时间的延长,其变化程度增加也更加明显。与作用于细胞24、48h相比,华蟾素处理细胞72h后细胞中凋亡相关蛋白bax的表达逐渐增加,而bcl-2逐渐减少,并且bax/bcl-2比值也增加明显(P0.05)。Wnt/β-catenin通路相关蛋白β-catenin的表达量与华蟾素作用时间也呈正相关。 [结论]：华蟾素能诱导骨肉瘤细胞凋亡,其效果呈浓度和时间依赖性,而其机制可能与激活Wnt/β-catenin通路有关。第二部分华蟾素联合化疗药物在骨肉瘤细胞中抗肿瘤作用的实验研究 [目的]：探讨华蟾素联合化疗药物在骨肉瘤细胞中的抗肿瘤作用。 [方法]：将实验分为四组,即对照组、华蟾素组、紫杉醇组、华蟾素+紫杉醇(联合组)。MTT法用来衡量各组骨肉瘤细胞的生长抑制情况,TUNEL染色用来观察细胞凋亡程度并计算其凋亡率,免疫荧光染色、RT-PCR、以及Western Blot用来检测凋亡相关以及’Wnt/β-catenin通路相关蛋白。 [结果]：MTT试验结果显示,当不同组别药物作用于骨肉瘤U20S细胞后,华蟾素组细胞生长抑制率低于紫杉醇组,但联合组细胞生长抑制率明显增加。当华蟾素联合紫杉醇作用于骨肉瘤U20S细胞48h后,其凋亡率增加的程度较单独用药更加明显(P0.05)。免疫荧光染色显示：在华蟾素、紫杉醇、以及二者联合用药分别作用于骨肉瘤细胞后,被测蛋白bax、β-catenin的荧光强度较对照组均有所增强,而bcl-2荧光强度有所减弱,但联合组上述蛋白荧光强度变化更明显。免疫荧光、RT-PCR、以及Western Blot结果显示：华蟾素、紫杉醇、及二者联合用药均能使bax、β-catenin表达增加,而使bcl-2表达减少,但联合用药对上调bax和β-catenin表达及下调bcl-2表达能力均强于单独用药(P0.05)。 [结论]：华蟾素联合紫杉醇对骨肉瘤具有明显的抗肿瘤效果,能促进bax和抑制bcl-2的表达,其机制可能与激活Wnt/β-catenin通路有关。
[Abstract]:Part I Experimental study on apoptosis of osteosarcoma cells induced by cinobufacin and its possible mechanism Objective: to investigate the effect of cinobufacin on apoptosis of osteosarcoma U20S cells and its possible mechanism. [methods] different concentrations of cinobufacin were applied to U20S cells of osteosarcoma. MTT method was used to detect the growth inhibition rate of each group of osteosarcoma cells. Tunel staining was used to observe the degree of apoptosis of osteosarcoma cells and calculate the apoptosis rate of U20S cells. Immunofluorescence staining was used to detect apoptosis-related and Wnt/ 尾 -catenin pathway related proteins, as well as Western Blot. [results]: after U20S cells were treated strictly according to the above methods, MTT results showed that with the increase of the concentration of cinobufacin and the time of action on the cells, the growth inhibition rate of U20S cells increased significantly in a time-concentration-dependent manner. When cinobufacin was exposed to osteosarcoma cells for different time, apoptosis and apoptosis rate increased significantly with the prolongation of time. The expression of apoptosis-related protein (bax) was gradually increased after 72 h treatment with cinobufacin, but the expression of bcl-2 decreased gradually, compared with that of 24 cells treated with cinobufacin for 48 h. The expression of 尾 -catenin, a protein associated with the P0.05N / 尾 -catenin pathway, was also positively correlated with the time of action of cinobufacin. [conclusion]: cinobufacin can induce apoptosis of osteosarcoma cells in a concentration and time dependent manner, and its mechanism may be related to the activation of Wnt/ 尾 -catenin pathway. The second part of the experimental study on the anti-tumor effect of cinobufacin combined with chemotherapeutic drugs in osteosarcoma cells Objective: to investigate the anti-tumor effect of cinobufacin combined with chemotherapeutic drugs in osteosarcoma cells. [methods] the experiment was divided into four groups: control group, cinobufacin group and paclitaxel group. MTT assay was used to measure the growth inhibition of osteosarcoma cells in each group. Tunel staining was used to observe the degree of apoptosis and calculate the apoptosis rate of osteosarcoma cells. Immunofluorescence staining was used to detect apoptosis-related and 尾 -catenin pathway related proteins by RT-PCR and Western Blot. [results] the cell growth inhibition rate of cinobufacin group was lower than that of paclitaxel group, but the cell growth inhibition rate of combination group was significantly higher than that of paclitaxel group. When cinobufacin combined with paclitaxel was treated with osteosarcoma U20S cells for 48 h, the apoptotic rate of U20S cells increased more significantly than that of P0.05 alone. Immunofluorescence staining showed that the fluorescence intensity of Baxand 尾 -catenin in osteosarcoma cells treated with cinobufacin, paclitaxel and taxol were increased, but the fluorescence intensity of bcl-2 was decreased. However, the fluorescence intensity of the above mentioned proteins in the combined group was more obvious. The results of immunofluorescence RT-PCRand Western Blot showed that cinobufacin, paclitaxel and their combination could increase the expression of baxand 尾 -catenin, but decrease the expression of bcl-2, but the ability of combination therapy to up-regulate the expression of bax and 尾 -catenin and down-regulate the expression of bcl-2 was stronger than that of P0.05. [conclusion]: cinobufacin combined with paclitaxel has obvious antitumor effect on osteosarcoma, can promote bax and inhibit the expression of bcl-2, and its mechanism may be related to the activation of Wnt/ 尾 -catenin pathway.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R738.1

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