二甲双胍对犬乳腺肿瘤细胞体外增殖抑制作用的影响

发布时间：2018-03-13 19:29

本文选题：犬乳腺肿瘤　切入点：二甲双胍　出处：《东北农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：犬乳腺肿瘤作为危害母犬健康的常发的肿瘤性疾病之一,其受犬的年龄、品种、绝育状态、繁殖情况以及遗传因素等多种因素的影响。随着犬作为伴侣动物的常态化,犬饲养年限越来越长,犬乳腺肿瘤的发病率在逐年升高,严重威胁犬的健康。犬乳腺肿瘤无论从临床发病特点、组织病理学分型还是乳腺超声、CT诊断上都与人类乳腺癌具有极高的相似性,可以作为人类乳腺癌研究的完美模型。因此,寻求安全而又有效的抗肿瘤药物无论对人类还是犬都是十分必要和迫切的。二甲双胍作为一种口服降糖药,主要用于Ⅱ型糖尿病的治疗,对大量临床案例的统计分析发现二甲双胍在降低糖尿病患者血糖的同时,可以显著降低2型糖尿病患者罹患肿瘤的风险和死亡率,对肿瘤起到一定的预防和治疗作用。本研究以犬转移乳腺肿瘤细胞系CHMm和原发肿瘤细胞系CHMp为模型,进行二甲双胍对犬乳腺肿瘤增殖抑制实验的研究,为二甲双胍用于犬乳腺瘤的研究和治疗提供一定的实验基础和理论依据,主要研究内容及结果如下:1.通过CCK-8法和平板克隆形成实验,检测不同浓度二甲双胍对犬乳腺肿瘤细胞增殖活性和克隆形成能力的影响。结果表明,二甲双胍可显著抑制犬乳腺肿瘤细胞CHMm和CHMp的增殖活性和克隆形成能力,并呈现一定的浓度和时间依赖性,根据对CHMm和CHMp细胞IC50的比较发现:CHMm对二甲双胍的敏感性更高。2.通过Annexin-FITC和PI染色法,检测二甲双胍诱导犬乳腺肿瘤细胞凋亡情况,并通过western blot法检测凋亡关键蛋白的表达。研究证实,随着二甲双胍作用浓度的升高,可诱导犬乳腺肿瘤细胞CHMm发生细胞凋亡,并伴有凋亡核心蛋白caspase-3和PARP的活化切割片段的增加。在CHMp未观察到诱导凋亡的发生,核心蛋白的活化片段量没有发生显著改变。3.通过PI染色和流式细胞仪,检测二甲双胍对犬乳腺肿瘤细胞周期的影响,并通过western blot法检测周期相关蛋白的变化情况。结果发现,二甲双胍可将CHMm和CHMp细胞周期阻滞在G0/G1期,并能上调p21、p27和下调Cyclin D1、CDK4等G0/G1期关键蛋白的表达。4.通过western blot法检测二甲双胍对犬乳腺肿瘤细胞内AMPK/m TOR和PI3K/Akt/m TOR信号通路上关键蛋白的表达,结果发现二甲双胍可显著激活CHMm细胞中的AMPK蛋白,并抑制PI3K、Akt及mTOR的表达。而在CHMp,经二甲双胍处理后,未观察到AMPK的显著性激活,但观察到了PI3K、Akt及m TOR等相关蛋白的抑制现象。结论:本实验验证了二甲双胍对犬乳腺肿瘤的增殖抑制作用,并且此抑制作用可能以激活AMPK和抑制PI3K/Akt/m TOR信号分子为作用靶点通过诱导细胞凋亡和G0/G1期细胞阻滞来实现。这些发现表明二甲双胍有望成为犬乳腺肿瘤的潜在治疗药物,进而为人类乳腺癌的攻克提供一个完美的实验模型和扎实的基础。
[Abstract]:As one of the most common cancer diseases, mammary gland tumor is affected by the age, breed, sterilizing state, breeding condition and genetic factors of the dog. The years of dog breeding are getting longer and longer, and the incidence of mammary gland tumors in dogs is increasing year by year, which is a serious threat to the health of dogs. The histopathological classification and CT diagnosis of breast cancer are highly similar to those of human breast cancer, and can be used as the perfect model for the study of human breast cancer. It is necessary and urgent to seek safe and effective antineoplastic drugs for both human and canine. Metformin, as an oral hypoglycemic drug, is mainly used in the treatment of type 2 diabetes. Statistical analysis of a large number of clinical cases showed that metformin can significantly reduce the risk of cancer and mortality in patients with type 2 diabetes at the same time as reducing blood sugar in patients with diabetes. In this study, we used canine metastatic breast tumor cell line CHMm and primary tumor cell line CHMp as models to study the inhibitory effect of metformin on the proliferation of canine breast tumor. To provide some experimental and theoretical basis for the study and treatment of canine mammary neoplasms with metformin. The main contents and results are as follows: 1. By CCK-8 method and plate clone formation experiment, The effects of metformin at different concentrations on the proliferative activity and clone forming ability of canine breast tumor cells were determined. The results showed that metformin could significantly inhibit the proliferation activity and clone formation ability of canine breast tumor cells CHMm and CHMp. According to the comparison of IC50 in CHMm and CHMp cells, the sensitivity of IC50 to metformin was higher. The apoptosis of mammary gland tumor cells induced by metformin was detected by Annexin-FITC and Pi staining. The expression of apoptotic key proteins was detected by western blot assay. The results showed that with the increase of metformin concentration, apoptosis could be induced in canine breast tumor cells CHMm. The number of activated fragments of apoptotic core protein caspase-3 and PARP was not significantly changed by Pi staining and flow cytometry (FCM), but no apoptosis was observed in CHMp. The effect of metformin on the cell cycle of canine breast tumor was detected, and the changes of cycle related proteins were detected by western blot. The results showed that metformin could block the cell cycle of CHMm and CHMp at G _ 0 / G _ 1 phase. It also up-regulated the expression of p21, p27 and down-regulated G _ 0 / G _ 1 key proteins such as Cyclin D1 / CDK4. The expression of metformin on AMPK/m TOR and PI3K/Akt/m TOR signaling pathway in canine breast tumor cells was detected by western blot assay. The results showed that metformin could significantly activate AMPK protein and inhibit the expression of PI3KnAkt and mTOR in CHMm cells, but no significant activation of AMPK was observed in CHMptreated with metformin. But the inhibitory effects of PI3KPU Akt and m TOR on the proliferation of canine breast tumor were observed. Conclusion: the inhibitory effect of metformin on the proliferation of canine breast tumor was confirmed. The inhibition may be targeted at activating AMPK and inhibiting PI3K/Akt/m TOR signaling molecules by inducing apoptosis and G _ 0 / G _ 1 phase arrest. These findings suggest that metformin may be a potential therapeutic agent for canine breast tumors. And then provide a perfect experimental model and solid foundation for human breast cancer.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.292

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