盐酸多柔比星诱导犬乳腺肿瘤CHMp、CHMm细胞凋亡

发布时间：2018-03-01 07:35

  本文关键词： 犬乳腺肿瘤细胞系 CHMp CHMm 盐酸多柔比星 细胞凋亡　出处：《华南农业大学》2016年硕士论文　论文类型：学位论文

【摘要】：犬乳腺肿瘤是仅次于皮肤肿瘤的常见肿瘤,迄今外科手术联合化学药物是最有效的治疗方法。基于兽医临床尚无理想的化疗药物、复制人医临床的化疗药物应用于治疗犬乳腺肿瘤缺乏理论及依据、研发新药时程长且成功率低等现状,本研究选用在治疗人耐药性乳腺肿瘤中取得良好效果的盐酸多柔比星进行体外抗肿瘤实验。研究该药诱导犬乳腺肿瘤细胞的凋亡机制,目的在于探求其在抗犬乳腺肿瘤的作用靶点及机理,为该药的化学疗法在应用于宠物临床提供理论依据,以解决宠物临床化疗药物匮乏的燃眉之急。选用盐酸多柔比星作为诱导剂及体外培养的犬乳腺肿瘤原发病灶及其转移灶的细胞系CHMp株和CHMm株为研究对象,利用CCK-8法分析盐酸多柔比星对两株乳腺肿瘤细胞系的抑制情况,选用不同的药物浓度诱导肿瘤细胞,进行形态学观察。同时利用AO/EB染色法考证药物对两株细胞系的促凋亡作用,并通过分光光度法检测处理后胞内凋亡相关蛋白Caspase-3、6、8、9的活性变化,Real-time PCR法检测细胞中Bcl-2 mRNA、Bax mRNA、p53 mRNA等相关凋亡因子表达量变化,Western Blotting方法检测胞浆蛋白中促凋亡蛋白Bax的表达情况,从而初步探究盐酸多柔比星诱导犬乳腺肿瘤细胞的机制。实验结果显示,不同浓度的盐酸多柔比星均能有效的诱导犬乳腺肿瘤细胞凋亡,药物浓度越高,抑制能力越强,且能显著提高CHMp胞内Caspase-3、6、8、9的表达量(P≤0.05);极显著提高CHMm胞内Caspase-3的表达量(P≤0.001),显著提高Caspase-6、8、9的表达量(P≤0.05)。与此同时,不同的药物浓度均使CHMp细胞内p53 mRNA、Bax mRNA表达量极显著提高(P≤0.001),Bcl-2 mRNA表达无明显差异;使CHMm细胞内p53 mRNA表达显著提高(P≤0.05),Bax mRNA表达量极显著提高(P≤0.001),Bcl-2 mRNA表达无明显差异或呈极显著降低(P≤0.001)。Western Blotting结果显示,药物作用诱导细胞以后,两种细胞胞内促凋亡蛋白Bax表达量提高,且表达量与药物浓度呈正相关。该实验结果表明盐酸多柔比星可通过促进细胞周期调控相关因子p53、线粒体凋亡通路相关因子Bcl-2家族成员Bax、死亡受体通路相关成员Caspase家族,激活Caspase联级反应启动细胞凋亡程序,从而诱导细胞发生凋亡。本实验初步揭示了盐酸多柔比星诱导犬乳腺肿瘤细胞凋亡的相关机制,为兽医临床应用该药物奠定科学理论基础,为筛选有效宠物临床抗肿瘤用药提供参考。
[Abstract]:The canine breast tumor is the second most common tumor after the skin tumor. So far, surgery combined with chemical drugs is the most effective treatment. The clinical chemotherapeutic drugs used in replicating human medicine for the treatment of canine breast tumors lack theory and basis, and have a long history and low success rate in the development of new drugs. In this study, doxorubicin hydrochloride, which had a good effect in the treatment of human breast cancer with drug resistance, was used to study the mechanism of apoptosis induced by doxorubicin hydrochloride. The aim of this study was to explore the target and mechanism of its antitumor effect in dogs, and to provide a theoretical basis for the application of chemotherapeutic drugs in pet clinic. In order to solve the urgent need of clinical chemotherapeutic drugs in pets, doxorubicin hydrochloride was used as inducer and CHMp and CHMm strains of canine breast tumor primary tumor and its metastatic foci were cultured in vitro. The inhibition of doxorubicin hydrochloride on two breast tumor cell lines was analyzed by CCK-8 method. The tumor cells were induced by different drug concentration and the morphological observation was carried out. Meanwhile, AO/EB staining was used to study the effect of drug on promoting apoptosis of two breast tumor cell lines. The expression of apoptosis factors such as Bcl-2 mRNA-Bax mRNA-p53 mRNA was detected by real-time PCR method. The expression of apoptosis-promoting protein Bax in cytoplasmic protein was detected by Western Blotting. The results showed that doxorubicin hydrochloride could induce the apoptosis of canine breast tumor cells effectively, and the higher the concentration of doxorubicin, the stronger the inhibition ability. In addition, the expression of Caspase-3, Caspase-3, Caspase-6, Caspase-8, Caspase-6, Caspase-6, and Caspase-6 were significantly increased in CHMp cells (P 鈮，

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