本文選題：5-FU + 犬乳腺腫瘤　； 參考：《中國農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：研究目的:乳腺腫瘤是母犬易發(fā)的腫瘤之一,且多數(shù)腫瘤是惡性的,僅通過外科手術(shù)切除的方法不能完全治愈此類疾病。為了提高患病動物的生存時間和生活質(zhì)量,需要在手術(shù)切除后進行輔助化療。不幸的是,隨著化療進程的深入,腫瘤細(xì)胞會對化療藥物產(chǎn)生耐藥性。為了研究犬乳腺腫瘤化療抵抗的機制,我們利用體外培養(yǎng)的方法獲得耐5-FU犬乳腺腫瘤細(xì)胞系,并通過研究該細(xì)胞系的生長特性,耐藥性與腫瘤干細(xì)胞之間的聯(lián)系,Notch1通路在腫瘤耐藥性中發(fā)揮的作用等對耐藥細(xì)胞進行研究。研究方法:1、利用濃度梯度法建立體外耐5-FU犬乳腺腫瘤細(xì)胞系。從細(xì)胞形態(tài)、交叉耐藥性、生長特性、耐藥蛋白表達(dá)、體內(nèi)試驗等多方面分析耐藥腫瘤細(xì)胞的特性。2、利用原代培養(yǎng)的方法,從實驗動物體獲得化療后的原代乳腺腫瘤細(xì)胞。利用差速貼壁法和有限稀釋克隆法對分離的原代腫瘤細(xì)胞進行分離鑒定,并對純化后的細(xì)胞進行耐藥性評估。3、通過對耐藥犬乳腺腫瘤細(xì)胞干細(xì)胞特性和轉(zhuǎn)移特性的分析,探究耐藥性與腫瘤干細(xì)胞性之間的聯(lián)系。4、分析Notch1通路在耐藥細(xì)胞中的激活狀況,及特異性抑制劑能否反轉(zhuǎn)細(xì)胞耐藥性及其反轉(zhuǎn)機制。研究結(jié)果:1、通過8個月的體外培養(yǎng),我們成功獲得了耐5-FU犬乳腺腫瘤細(xì)胞系,并將其命名為CMT7364/5-FU。與親代敏感細(xì)胞相比,耐藥細(xì)胞更傾向于成簇生長,并出現(xiàn)多核現(xiàn)象;細(xì)胞的群體倍增時間更長;細(xì)胞對除5-FU外的四種化療藥物產(chǎn)生交叉耐藥性;ABCB1和ABCG2蛋白表達(dá)上調(diào);荷瘤小鼠同樣對5-FU產(chǎn)生抗性。2、原代細(xì)胞經(jīng)純化后,利用染色體核型分析,確認(rèn)純化的細(xì)胞來源于犬。該細(xì)胞群體倍增時間類似于耐藥細(xì)胞,且對5-FU的耐藥性比CMT7364/5-FU更強。3、通過流式細(xì)胞分析,耐藥細(xì)胞中CD24/CD44+細(xì)胞亞群比例大于親代敏感細(xì)胞;耐藥細(xì)胞通過懸浮培養(yǎng)形成的微球體數(shù)量也多于親代敏感細(xì)胞;耐藥細(xì)胞貼壁培養(yǎng)克隆形成能力也更強;干細(xì)胞通路相關(guān)蛋白均出現(xiàn)表達(dá)上調(diào);104個耐藥細(xì)胞即可在小鼠皮膚成瘤。通過劃痕修復(fù)試驗及transwell小室分析,耐藥細(xì)胞的遷移和侵襲能力更強;同時與轉(zhuǎn)移相關(guān)的蛋白MMP-2及vimentin均出現(xiàn)過表達(dá)現(xiàn)象;動物實驗表明耐藥細(xì)胞具有更強的向遠(yuǎn)端肺臟轉(zhuǎn)移的能力。4、Notch1通路在耐藥細(xì)胞出現(xiàn)上調(diào);特異性抑制劑DAPT可以一定程度干擾細(xì)胞的自我更新和轉(zhuǎn)移,從而反轉(zhuǎn)耐藥性。結(jié)論:濃度梯度遞增法可以成功用于體外建立耐藥犬乳腺腫瘤細(xì)胞系,與親代細(xì)胞相比其生物學(xué)特性差異明顯;具備更強的干細(xì)胞特性;出現(xiàn)Notch1通路的過度激活。靶向抑制Notch1通路可以一定程度逆轉(zhuǎn)細(xì)胞的耐藥性,從而為臨床治療提供新的依據(jù)。
[Abstract]:Objective: breast tumor is one of the most susceptible tumors in female dogs, and most of the tumors are malignant, which can not be completely cured by surgical resection. In order to improve the survival time and quality of life of infected animals, adjuvant chemotherapy should be performed after surgical resection. Unfortunately, as chemotherapy progresses, cancer cells become resistant to chemotherapy drugs. In order to study the mechanism of chemotherapeutic resistance of canine breast tumor, we obtained 5-FU resistant canine breast tumor cell line in vitro, and studied the growth characteristics of the cell line. Relationship between drug resistance and tumor stem cells the role of Notch1 pathway in tumor resistance was studied. Methods: the 5-Fu canine mammary tumor cell line was established by concentration gradient method. The characteristics of drug-resistant tumor cells were analyzed from the aspects of cell morphology, cross-resistance, growth characteristics, expression of drug-resistant proteins, and in vivo test. The primary breast tumor cells after chemotherapy were obtained from experimental animals by primary culture. The primary tumor cells were isolated and identified by differential adherent method and limited dilution clone method, and the purified cells were evaluated for drug resistance. 3. The characteristics of breast cancer stem cells and metastasis characteristics of drug-resistant canine mammary cancer cells were analyzed. To explore the relationship between drug resistance and tumor stem cell sex, to analyze the activation of Notch1 pathway in drug-resistant cells, and whether the specific inhibitor can reverse the drug resistance and its reversal mechanism. After 8 months of culture, we successfully obtained 5-FU resistant canine mammary tumor cell line and named it CMT7364 / 5-FU. Compared with the parental sensitive cells, the drug-resistant cells were more prone to cluster growth and appeared multicore phenomenon, the population doubling time of the cells was longer, and the expression of ABCB1 and ABCG2 proteins was up-regulated by the cross-resistance of the cells to the four chemotherapeutic drugs except 5-FU, and the expression of ABCB1 and ABCG2 protein was up-regulated in the four chemotherapeutic drugs except 5-FU. The tumor-bearing mice were also resistant to 5-FU. After purification, the purified cells were confirmed to be derived from dogs by chromosome karyotype analysis. The cell population doubling time was similar to that of drug-resistant cells, and its resistance to 5-FU was stronger than that of CMT7364 / 5-FU. By flow cytometry analysis, the percentage of CD24% CD44 cells in resistant cells was higher than that of parent sensitive cells. The number of microspheres formed by suspension culture of drug-resistant cells was more than that of parental sensitive cells, and the clone forming ability of drug-resistant cells in adherent culture was also stronger. The expression of stem cell pathway related proteins was up-regulated, and 104 drug resistant cells were able to develop tumor in mouse skin. By scratch repair test and transwell chamber analysis, the ability of migration and invasion of drug-resistant cells was stronger, and the expression of MMP-2 and vimentin associated with metastasis was also observed. Animal experiments showed that drug-resistant cells had a stronger ability to transfer to the distal lung. 4) Notch1 pathway was up-regulated in drug-resistant cells, and DAPT, a specific inhibitor, could interfere with the self-renewal and metastasis of the cells to some extent, thus reversing drug resistance. Conclusion: the method of increasing concentration gradient can be successfully used in the establishment of drug-resistant canine breast tumor cell lines in vitro. Compared with the parental cells, the biological characteristics of the cells are significantly different, the stem cell characteristics are stronger, and the Notch1 pathway is over-activated. Targeting inhibition of Notch1 pathway can reverse cell drug resistance to a certain extent and provide a new basis for clinical treatment.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S858.292

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相關(guān)期刊論文 前2條

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