耐5-FU犬乳腺肿瘤细胞系的建立及其生物学特性的研究

发布时间：2018-06-26 14:11

本文选题：5-FU + 犬乳腺肿瘤　；参考：《中国农业大学》2017年博士论文

【摘要】：研究目的:乳腺肿瘤是母犬易发的肿瘤之一,且多数肿瘤是恶性的,仅通过外科手术切除的方法不能完全治愈此类疾病。为了提高患病动物的生存时间和生活质量,需要在手术切除后进行辅助化疗。不幸的是,随着化疗进程的深入,肿瘤细胞会对化疗药物产生耐药性。为了研究犬乳腺肿瘤化疗抵抗的机制,我们利用体外培养的方法获得耐5-FU犬乳腺肿瘤细胞系,并通过研究该细胞系的生长特性,耐药性与肿瘤干细胞之间的联系,Notch1通路在肿瘤耐药性中发挥的作用等对耐药细胞进行研究。研究方法:1、利用浓度梯度法建立体外耐5-FU犬乳腺肿瘤细胞系。从细胞形态、交叉耐药性、生长特性、耐药蛋白表达、体内试验等多方面分析耐药肿瘤细胞的特性。2、利用原代培养的方法,从实验动物体获得化疗后的原代乳腺肿瘤细胞。利用差速贴壁法和有限稀释克隆法对分离的原代肿瘤细胞进行分离鉴定,并对纯化后的细胞进行耐药性评估。3、通过对耐药犬乳腺肿瘤细胞干细胞特性和转移特性的分析,探究耐药性与肿瘤干细胞性之间的联系。4、分析Notch1通路在耐药细胞中的激活状况,及特异性抑制剂能否反转细胞耐药性及其反转机制。研究结果:1、通过8个月的体外培养,我们成功获得了耐5-FU犬乳腺肿瘤细胞系,并将其命名为CMT7364/5-FU。与亲代敏感细胞相比,耐药细胞更倾向于成簇生长,并出现多核现象;细胞的群体倍增时间更长;细胞对除5-FU外的四种化疗药物产生交叉耐药性;ABCB1和ABCG2蛋白表达上调;荷瘤小鼠同样对5-FU产生抗性。2、原代细胞经纯化后,利用染色体核型分析,确认纯化的细胞来源于犬。该细胞群体倍增时间类似于耐药细胞,且对5-FU的耐药性比CMT7364/5-FU更强。3、通过流式细胞分析,耐药细胞中CD24/CD44+细胞亚群比例大于亲代敏感细胞;耐药细胞通过悬浮培养形成的微球体数量也多于亲代敏感细胞;耐药细胞贴壁培养克隆形成能力也更强;干细胞通路相关蛋白均出现表达上调;104个耐药细胞即可在小鼠皮肤成瘤。通过划痕修复试验及transwell小室分析,耐药细胞的迁移和侵袭能力更强;同时与转移相关的蛋白MMP-2及vimentin均出现过表达现象;动物实验表明耐药细胞具有更强的向远端肺脏转移的能力。4、Notch1通路在耐药细胞出现上调;特异性抑制剂DAPT可以一定程度干扰细胞的自我更新和转移,从而反转耐药性。结论:浓度梯度递增法可以成功用于体外建立耐药犬乳腺肿瘤细胞系,与亲代细胞相比其生物学特性差异明显;具备更强的干细胞特性;出现Notch1通路的过度激活。靶向抑制Notch1通路可以一定程度逆转细胞的耐药性,从而为临床治疗提供新的依据。
[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.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S858.292

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