小豆蔻明联合顺铂抑制卵巢癌SKOV3细胞增殖及其作用机制研究

发布时间：2018-05-07 13:28

  本文选题：顺铂 + 小豆蔻明　； 参考：《福建医科大学》2014年硕士论文

【摘要】：目的 铂类药物化疗为卵巢癌治疗的重要方法，，但化疗过程中肿瘤细胞耐药性的产生严重影响药物的疗效。目前，采用其它抗肿瘤药物与铂类联用而达到治疗效果是临床常用的治疗方案。因此，寻找能提高铂类治疗卵巢癌疗效的药物临床意义重大。小豆蔻明（Cardamonin, CAR）为姜科植物草豆蔻中提取的黄酮类单体。国内外的研究表明，CAR具有抗肿瘤、抗炎等多种药理活性，能增强TRAIL的抗肿瘤作用，其作用机制与增强死亡受体以及凋亡蛋白的表达有关。然而，CAR能否增强顺铂（cisplatin, Cis）的肿瘤抑制作用尚不明确。本课题旨在考察CAR联合Cis对卵巢癌SKOV3细胞增殖的作用，并探明其作用机制，为联合铂类抗肿瘤化疗药物的研发提供新方向，为CAR进一步开发奠定理论基础及提供实验依据。 方法 1分组与给药 SKOV3分为正常对照组（control），溶剂对照组（0.1%DMSO），Cis组（2μg/mL）、CAR组（20μM）和Cis+CAR组（2μg/mL+20μM）。 2测定指标及方法 2.1MTT法和克隆形成实验测定细胞增殖； 2.2PI染色法检测细胞周期； 2.3DNA碎片法检测细胞凋亡； 2.4MDC染色法检测细胞自噬； 2.5RT-PCR法检测谷胱甘肽-S-转移酶-π （glutathione S-transferase-π, GST-π)、多药耐药蛋白（multidrug resistance protein, MRP）的mRNA表达； 2.6Western Blot法检测Bcl-2、XIAP、survivin和LC3的蛋白表达。 结果 1CAR联合Cis用药能够显著性抑制SKOV3细胞增殖，其抑制率明显高于Cis单独用药（P 0.01），20μM CAR与2μg/mL Cis具有协同作用； 2CAR联合Cis对SKOV3细胞克隆形成的抑制显著强于单独用药（P 0.01）； 3与单独用药相比，CAR联合Cis对DNA碎片的生成无显著性差异； 4Cis能够诱导GST-π mRNA表达的增加（P 0.01），而对MRP的表达无明显影响；CAR能够降低MRP mRNA的表达（P 0.01），而对GST-π的表达无明显影响；两药联合时，CAR能够降低Cis诱导的GST-π高表达，同时降低MRP的表达（P 0.01）； 5Cis和CAR单独处理后，XIAP与survivin的表达降低（P 0.01）；而联合给药组Bcl-2、XIAP、survivin的表达显著性降低，其抑制作用明显高于单独给药组（P 0.01）。 6Cis和CAR处理过的细胞内MDC染色颗粒数增加；而Cis和CAR联用后，细胞内MDC染色颗粒数目显著性增加，二者具有协同诱导自噬的作用。 结论 1CAR与Cis具有协同抑制卵巢癌SKOV3细胞增殖的作用； 2CAR联合Cis具有增强卵巢癌SKOV3细胞自噬，抑制细胞周期的作用； 3CAR增强Cis抗肿瘤的作用与抗凋亡蛋白和耐药基因表达的降低密切相关。
[Abstract]:Purpose Chemotherapy with platinum drugs is an important method in the treatment of ovarian cancer, but the drug resistance of tumor cells in the course of chemotherapy seriously affects the efficacy of the drugs. At present, the use of other antitumor drugs combined with platinum to achieve therapeutic effect is commonly used in clinical treatment. Therefore, it is of great clinical significance to find drugs that can improve the efficacy of platinum in the treatment of ovarian cancer. Cardamonin (Carr) is a flavonoid monomer extracted from Curcumaceae. Studies at home and abroad have shown that car has many pharmacological activities, such as anti-tumor, anti-inflammatory and so on, which can enhance the anti-tumor effect of TRAIL, and its mechanism is related to the enhancement of the expression of death receptor and apoptotic protein. However, whether car can enhance the tumor inhibitory effect of cisplatin (Cis) is not clear. The purpose of this study was to investigate the effect of CAR combined with Cis on the proliferation of ovarian cancer SKOV3 cells, and to explore its mechanism, to provide a new direction for the research and development of combined platinum antitumor chemotherapeutic agents, and to lay a theoretical foundation and experimental basis for the further development of CAR. Method 1 grouping and administration SKOV3 was divided into normal control group (n = 20), solvent control group (n = 0.1) and Cis CAR group (n = 2 渭 g / mL) and Cis CAR group (n = 2 渭 g/mL = 20 渭 M). 2 determination index and method Cell proliferation was determined by 2.1MTT assay and clone formation assay. Cell cycle was detected by 2.2PI staining. Apoptosis was detected by 2.3DNA fragment assay. Autophagy was detected by 2.4MDC staining. The mRNA expression of glutathione S-transferase- 蟺, glutathione S-transferase- 蟺 and multidrug resistance protein, MRP) was detected by 2.5RT-PCR. The expression of survivin and LC3 were detected by 2.6Western Blot. Result 1CAR combined with Cis could significantly inhibit the proliferation of SKOV3 cells, and the inhibitory rate was significantly higher than that of 20 渭 M CAR and 2 渭 g/mL Cis of Cis alone. The inhibitory effect of 2CAR combined with Cis on SKOV3 cell clone formation was significantly stronger than that of single drug (P 0.01). (3) there was no significant difference in the formation of DNA fragments between car and Cis alone. 4Cis could induce the increase of mRNA expression of GST- 蟺, but had no effect on the expression of MRP. Car could decrease the expression of GST- 蟺, but not the expression of GST- 蟺, and the combination of two drugs could decrease the overexpression of GST- 蟺 induced by Cis. At the same time, the expression of MRP was decreased (P 0.01). The expression of survivin and XIAP decreased significantly in 5Cis and CAR alone, but the expression of Bcl-2TXIAPP in combination group was significantly lower than that in control group (P 0.01). The number of MDC staining granules treated with 6Cis and CAR increased, while that of Cis and CAR increased significantly, both of them could induce autophagy synergistically. Conclusion 1CAR and Cis could inhibit the proliferation of ovarian cancer SKOV3 cells. 2CAR combined with Cis can enhance autophagy of SKOV3 cells and inhibit cell cycle. The role of 3CAR in enhancing the anti-tumor effect of Cis is closely related to the decrease of anti-apoptotic protein and drug resistance gene expression.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31


本文编号：1857104