本文選題：犬乳腺腫瘤　切入點：二甲雙胍　出處：《東北農(nóng)業(yè)大學》2017年碩士論文　論文類型：學位論文

【摘要】：犬乳腺腫瘤作為危害母犬健康的常發(fā)的腫瘤性疾病之一,其受犬的年齡、品種、絕育狀態(tài)、繁殖情況以及遺傳因素等多種因素的影響。隨著犬作為伴侶動物的常態(tài)化,犬飼養(yǎng)年限越來越長,犬乳腺腫瘤的發(fā)病率在逐年升高,嚴重威脅犬的健康。犬乳腺腫瘤無論從臨床發(fā)病特點、組織病理學分型還是乳腺超聲、CT診斷上都與人類乳腺癌具有極高的相似性,可以作為人類乳腺癌研究的完美模型。因此,尋求安全而又有效的抗腫瘤藥物無論對人類還是犬都是十分必要和迫切的。二甲雙胍作為一種口服降糖藥,主要用于Ⅱ型糖尿病的治療,對大量臨床案例的統(tǒng)計分析發(fā)現(xiàn)二甲雙胍在降低糖尿病患者血糖的同時,可以顯著降低2型糖尿病患者罹患腫瘤的風險和死亡率,對腫瘤起到一定的預(yù)防和治療作用。本研究以犬轉(zhuǎn)移乳腺腫瘤細胞系CHMm和原發(fā)腫瘤細胞系CHMp為模型,進行二甲雙胍對犬乳腺腫瘤增殖抑制實驗的研究,為二甲雙胍用于犬乳腺瘤的研究和治療提供一定的實驗基礎(chǔ)和理論依據(jù),主要研究內(nèi)容及結(jié)果如下:1.通過CCK-8法和平板克隆形成實驗,檢測不同濃度二甲雙胍對犬乳腺腫瘤細胞增殖活性和克隆形成能力的影響。結(jié)果表明,二甲雙胍可顯著抑制犬乳腺腫瘤細胞CHMm和CHMp的增殖活性和克隆形成能力,并呈現(xiàn)一定的濃度和時間依賴性,根據(jù)對CHMm和CHMp細胞IC50的比較發(fā)現(xiàn):CHMm對二甲雙胍的敏感性更高。2.通過Annexin-FITC和PI染色法,檢測二甲雙胍誘導(dǎo)犬乳腺腫瘤細胞凋亡情況,并通過western blot法檢測凋亡關(guān)鍵蛋白的表達。研究證實,隨著二甲雙胍作用濃度的升高,可誘導(dǎo)犬乳腺腫瘤細胞CHMm發(fā)生細胞凋亡,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未觀察到誘導(dǎo)凋亡的發(fā)生,核心蛋白的活化片段量沒有發(fā)生顯著改變。3.通過PI染色和流式細胞儀,檢測二甲雙胍對犬乳腺腫瘤細胞周期的影響,并通過western blot法檢測周期相關(guān)蛋白的變化情況。結(jié)果發(fā)現(xiàn),二甲雙胍可將CHMm和CHMp細胞周期阻滯在G0/G1期,并能上調(diào)p21、p27和下調(diào)Cyclin D1、CDK4等G0/G1期關(guān)鍵蛋白的表達。4.通過western blot法檢測二甲雙胍對犬乳腺腫瘤細胞內(nèi)AMPK/m TOR和PI3K/Akt/m TOR信號通路上關(guān)鍵蛋白的表達,結(jié)果發(fā)現(xiàn)二甲雙胍可顯著激活CHMm細胞中的AMPK蛋白,并抑制PI3K、Akt及mTOR的表達。而在CHMp,經(jīng)二甲雙胍處理后,未觀察到AMPK的顯著性激活,但觀察到了PI3K、Akt及m TOR等相關(guān)蛋白的抑制現(xiàn)象。結(jié)論:本實驗驗證了二甲雙胍對犬乳腺腫瘤的增殖抑制作用,并且此抑制作用可能以激活A(yù)MPK和抑制PI3K/Akt/m TOR信號分子為作用靶點通過誘導(dǎo)細胞凋亡和G0/G1期細胞阻滯來實現(xiàn)。這些發(fā)現(xiàn)表明二甲雙胍有望成為犬乳腺腫瘤的潛在治療藥物,進而為人類乳腺癌的攻克提供一個完美的實驗?zāi)Ｐ秃驮鷮嵉幕A(chǔ)。
[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.
【學位授予單位】：東北農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S858.292

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