CYLD联合Livin在膀胱肿瘤化疗敏感性的研究

发布时间：2018-05-05 03:22

本文选题：膀胱癌 + 吉西他滨　；参考：《山东大学》2017年硕士论文

【摘要】：研究背景膀胱癌是西方国家和亚洲国家尿路上皮癌中最常见类型的癌症,而且膀胱癌的流行率和死亡率逐年增加。它是美国第四大常见的癌症,也是男性第九大常见的恶性肿瘤,在中国泌尿系统肿瘤的发病率中居首位。虽然随着膀胱癌早期检测手段的普及,患者5年生存率达到80%,但其5年复发率超过50%,并且会有10-20%的患者进展为浸润性膀胱癌。吉西他滨做为膀胱癌的一线化疗药物已广泛应用于临床,但是化疗耐药性的产生仍是不可避免的问题。因此,克服吉西他滨化疗的耐药性和提高化疗的有效性,寻找克服化疗耐药性的新靶点和提高药物的化疗敏感性,已经成为近期研究的热点。提高吉西他滨有效的化疗效率,并有效降低化疗的毒性,能够延长膀胱癌患者的生存时间,并改善预后,获得更好的生活质量。目的本研究首先探讨了 CYLD和Livin与膀胱癌的进展之间的关系,然后通过稳定转染CYLD和Livin,研究CYLD和Livin在吉西他滨诱导下的对肿瘤细胞毒性、自噬及凋亡中的作用,为寻求改善癌症细胞的化疗敏感性,和促进减灭癌症细胞的新的靶向分子目标,以及为当前的癌症化疗方案寻找新的科研基础。方法(1)培养膀胱癌细胞253J、T24,分别稳定转染CYLD、Livin,进行Western Blot、RT-PCR检测各细胞株中CYLD及Livin的表达水平。(2)采用CCK-8检测CYLD和Livin以及共转染对膀胱癌细胞吉西他滨化疗敏感性的影响,并计算其半数抑制浓度(50%inhibiting concentration,IC50);采用Transwell细胞侵袭及迁移实验检测CYLD和Livin抑制浸润性膀胱癌细胞株253J、T24的侵袭及迁移能力。(3)通过Western Blot方法检测联合转染后的T24及253J中NF-kB信号通路表达水平及自噬、凋亡相关蛋白的表达情况,探讨联合转染中CYLD协同Livin诱导的对肿瘤细胞毒性及凋亡的分子通路机制,并通过string-db分析测CYLD和Livin的潜在关系。(4)建立小鼠皮下肿瘤模型,采用腹腔注射的方法将吉西他滨注射到小鼠体内,并将小鼠分组:对照组(DMSO)、CYLD组、Livin组、CYLD+Livin组。观察并记录各组对膀胱癌皮下肿瘤生长状态的影响,分析CYLD和Livin对肿瘤模型增殖的抑制表现。结果(1)CCK-8细胞活性实验检测CYLD和Livin可分别提高膀胱癌细胞的化疗敏感性,而CYLD联合Livin可以大幅提高吉西他滨诱导的对肿瘤细胞毒性作用,进而促进了膀胱癌细胞对化疗药物吉西他滨的敏感性。(2)Transwell细胞实验检测CYLD和Livin可以使膀胱癌细胞株253J、T24的侵袭及迁移能力受到抑制,而CYLD联合Livin的共转染可以使膀胱癌细胞的侵袭、迁移能力受到明显抑制。(3)Western Blot检测膀胱癌细胞株253J、T24,发现膀胱癌细胞株吉西他滨化疗敏感性增强可能与抑制细胞自噬蛋白LC3、P62,促进细胞凋亡蛋白Caspase3、Smac相关,并发现CYLD可通过NF-kB信号通路调节Livin,进而提高癌细胞吉西他滨化疗敏感性。(4)体内试验结果表明联合转染组(CYLD+Livin)能明显增强吉西他滨对肿瘤的抑制作用。结论(1)过表达CYLD可以抑制膀胱癌细胞株的侵袭、迁移能力,提高其对吉西他滨的化疗敏感性。(2)敲减Livin可以抑制膀胱癌细胞株的侵袭、迁移能力,提高其对吉西他滨的化疗敏感性。(3)CYLD可通过NF-kB信号通路调节Livin(4)CYLD联合Livin可明显增加膀胱癌细胞在化疗上对吉西他滨的敏感性,这种联合治疗为改进传统的化疗方案提高新的实验基础。
[Abstract]:Background bladder cancer is the most common type of cancer in the western and Asian countries, and the prevalence and mortality of bladder cancer are increasing year by year. It is the fourth most common cancer in the United States. It is also the ninth common malignant tumor in men. It ranks first in the incidence of urological tumors in China. Although with bladder cancer, it is the most common cancer in China. The 5 year survival rate of patients with early detection is 80%, but the recurrence rate of 5 years is more than 50%, and the patients with 10-20% will develop into invasive bladder cancer. Gemcitabine as a first-line chemotherapy drug for bladder cancer is widely used in clinical, but the production of chemotherapeutic drug resistance is still an inevitable problem. Therefore, to overcome gemcitabine To improve the efficiency of chemotherapy and reduce the toxicity of chemotherapy, it can prolong the survival time of the patients with bladder cancer and improve the prognosis, and get better prognosis. The purpose of this study was to investigate the relationship between CYLD and Livin and the progression of bladder cancer. Then through stable transfection of CYLD and Livin, the effects of CYLD and Livin on cytotoxicity, autophagy and apoptosis induced by gemcitabine, in order to improve chemosensitivity of cancer cells, and promote the reduction of cancer cells, were studied. New target molecular targets and new research foundation for current cancer chemotherapy scheme. Methods (1) 253J, T24, CYLD, Livin, Western Blot, RT-PCR were used to detect the expression level of CYLD and Livin in each cell line respectively. (2) CCK-8 CYLD and Livin, and co transfection of bladder cancer cells were used. The effect of chemosensitivity of Western itine, and the 50%inhibiting concentration, IC50, and Transwell cell invasion and migration test were used to detect the invasion and migration of CYLD and Livin in infiltrating bladder cancer cell line 253J, T24. (3) the Western Blot method was used to detect the T24 and 253J. The expression of autophagy and autophagy, the expression of autophagy and apoptosis related protein, and to explore the molecular pathway mechanism of CYLD synergistic Livin induced cytotoxicity and apoptosis in combined transfection, and the potential relationship between CYLD and Livin by string-db analysis. (4) to establish a mouse model of subcutaneous tumor and to injecting gemcitabine by intraperitoneal injection In mice, the mice were divided into groups: control group (DMSO), group CYLD, group Livin, group CYLD+Livin. The effects of each group on the growth of subcutaneous tumor of bladder cancer were observed and recorded, and the inhibition of CYLD and Livin on the proliferation of tumor model was analyzed. Results (1) CCK-8 cell activity testing CYLD and Livin can improve the sensitivity of bladder cancer cells to chemotherapy. CYLD combined with Livin can significantly increase the toxicity of gemcitabine on tumor cells and further promote the sensitivity of cystocarcinoma cells to the chemotherapeutic gemcitabine. (2) the detection of CYLD and Livin in Transwell cells can make the bladder cancer cell line 253J, the invasion and migration of T24, and CYLD combined with Livin. The transfection could inhibit the invasion and migration of bladder cancer cells. (3) Western Blot was used to detect the bladder cancer cell line 253J, T24. It was found that the chemosensitivity of the bladder cancer cell line gemcitabine may be associated with the inhibition of the autophagic protein LC3, P62, the promotion of apoptosis protein Caspase3, Smac, and the discovery of CYLD through NF-kB signals. Livin was used to improve the chemosensitivity of gemcitabine. (4) in vivo test results showed that the combined transfection group (CYLD+Livin) could significantly enhance the inhibitory effect of gemcitabine on tumor. Conclusion (1) overexpression of CYLD can inhibit the invasion and migration of bladder cancer cell lines and improve the chemosensitivity to gemcitabine. (2) knock down Li Vin can inhibit the invasion and migration of bladder cancer cell line and improve its chemosensitivity to gemcitabine. (3) CYLD can modulate Livin (4) CYLD combined with Livin through the NF-kB signaling pathway to significantly increase the sensitivity of bladder cancer cells to gemcitabine in chemotherapy. This combination therapy improves the traditional chemotherapy regimen to improve the new experiment Basics.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.14

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