乌苯美司对肾癌细胞体外生长的影响及机制的研究
发布时间：2018-11-08 10:14
【摘要】：目的研究乌苯美司对肾癌细胞株786-0和OS-RC-2体外生长的影响,初步研究其对肾癌细胞作用的机制。 方法1.以肾癌细胞株786-0及OS-RC-2为研究对象研究乌苯美司对肾癌细胞体外生长的影响：WST-8细胞增殖实验及细胞生长曲线绘制检测乌苯美司对肾癌细胞株786-0细胞及OS-RC-2细胞增殖能力的影响；细胞划痕实验观察乌苯美司对肾癌细胞株786-0细胞及OS-RC-2细胞迁移能力的影响；Transwell细胞侵袭实验观察乌苯美司对肾癌细胞株786-0细胞及OS-RC-2细胞侵袭能力的影响。2.乌苯美司对肾癌细胞株786-0和OS-RC-2作用机制的初步研究：L-亮氨酸-P硝基苯胺底物法检测乌苯美司对肾癌细胞株786-0细胞及OS-RC-2细胞氨肽酶活性的影响。Western Blot检测乌苯美司作用于肾癌细胞株786-0细胞及OS-RC-2细胞后,两种细胞CD13、LC-3、Caspase-3的表达水平。以不同浓度的乌苯美司、自噬诱导剂rapamycin、自噬抑制剂3-Methyladenine作用于肾癌细胞株786-0和OS-RC-2, LDH释放试验检测作用后细胞的死亡率。3.以肾癌患者手术取得病理组织为研究对象：Western Blot检测氨肽酶N/CD13和LC-3在肾癌组织和癌旁正常组织的表达量；以肾癌患者及肾良性病变患者手术取得病理组织构建肾脏病变的组织芯片,通过免疫组化染色观察乌苯美司的作用靶点氨肽酶N/CD13在肾癌组织及正常组织中的表达情况。 结果WST-8细胞增殖检测及细胞生长曲线显示经0.lmg/ml乌苯美司作用后,肾癌细胞株786-0及OS-RC-2增殖均受到明显抑制(P0.05),且随着药物浓度的增加,这种抑制作用也逐渐增强(P0.05)。细胞迁移实验结果表明0.5mg/ml乌苯美司作用12小时对肾癌细胞株786-0及OS-RC-2细胞的迁移有明显的抑制作用(P0.05),且随着乌苯美司药物浓度增加,抑制作用也随之增强(P0.05)。Transwell细胞侵袭实验结果显示0.5mg/ml乌苯美司作用24小时能明显抑制786-0及OS-RC-2细胞的侵袭(P0.05)。氨肽酶活性检测试验证实经0.25mg/ml乌苯美司作用24小时后,肾癌细胞株786-0及OS-RC-2细胞的氨肽酶活性都明显降低活性(P0.05)。Western Blot结果显示最高浓度为0.5mg/ml乌苯美司作用于肾癌细胞株786-0及OS-RC-2后,并没有降低两种细胞氨肽酶N/CD13蛋白的表达量(P0.05)；两种肾癌细胞株的自噬相关蛋白LC-3B表达量明显升高(P0.05),且这种效应可被自噬诱导剂Rapamycin增强或被自噬抑制剂3-MA阻断(P0.05)。对手术病理组织的Western Blot检测结果显示氨肽酶N/CD13和LC-3在肾癌组织及癌旁正常组织中均有表达,且2种蛋白在癌旁正常组织中的表达量均明显高于癌组织(P0.05)。肾脏组织芯片免疫组化染色显示氨肽酶N/CD13在各种病理类型的肾脏病变中均有表达,且在不同病理类型和分级分期中无明显差异(P0.05)。 结论1.乌苯美司对肾癌细胞的增殖、迁移、侵袭有抑制作用。2.其机制与抑制氨肽酶N活性有关；乌苯美司作用后两种肾癌细胞株的自噬相关蛋白LC-3B表达量都明显升高。乌苯美司对785-0细胞系中的细胞毒性作用可被自噬诱导剂Rapamycin增强或倍自噬抑制剂3-MA阻断(P0.05),提示其可能通过诱导细胞自噬发挥抗肿瘤效应。
[Abstract]:Objective To study the effect of Ubenmei on the in vitro growth of renal cell carcinoma cell line 786-0 and OS-RC-2. square Method 1. The effect of Ubenmei on the in vitro growth of renal cell line 786-0 and OS-RC-2 was studied by the study of cell line 786-0 and OS-RC-2 of renal cell carcinoma. The effect of Ubenmei on the cell migration of renal cell carcinoma cell line 786-0 and OS-RC-2 cells was observed by cell scratch test, and the effect of Ubenmei on the invasion ability of cell line 786-0 cells and OS-RC-2 cells was observed by Transwell cell invasion experiment.. 2. Preliminary study on the mechanism of the action of Ubenumin on the renal cell carcinoma cell line 786-0 and OS-RC-2: L-leucine-P-nitroaniline substrate method for the detection of the activity of the Ubiome on the human renal cell line 786-0 cells and the OS-RC-2 cell aminopeptidase Effect of Western Blot on the expression of CD13, LC-3 and Caspase-3 in renal cell carcinoma cell line 786-0 and OS-RC-2 cells Level. The death rate of cells following the test of release test of renal cell carcinoma cell line 786-0 and OS-RC-2 by autophagy inhibitor, rapamycin, autophagy inhibitor 3-Methyladmine, at different concentrations.. 3. The expression of aminopeptidase N/ CD13 and LC-3 in the renal cell carcinoma and the adjacent normal tissues was detected by Western Blot. The expression of aminopeptidase N/ CD13 in the renal cell carcinoma and normal tissues was observed by immunohistochemical staining. Results The proliferation of WST-8 cells and the cell growth curve showed that the proliferation of renal cell carcinoma cell line 786-0 and OS-RC-2 was significantly inhibited after the action of 0. lmg/ ml of Ubenmei (P <0.05), and the inhibition effect was also enhanced with the increase of drug concentration (P The results of the cell migration showed that the effect of 0. 5 mg/ ml of UBENZUS on the migration of cell line 786-0 and OS-RC-2 in renal cell carcinoma was significantly inhibited (P0.05). The results of the invasion and experiment of Transwell cells showed that the effects of 0. 5 mg/ ml of Ubenzol on the cells of 786-0 and OS-RC-2 were significantly inhibited (P <0.05). The results showed that the activity of aminopeptidase of the cell line 786-0 and OS-RC-2 in the renal cell carcinoma cell line 786-0 and the OS-RC-2 cells decreased significantly after 24 hours after the action of 0. 25mg/ ml of the UBENZUME (P0.05). Western Blot showed that the highest concentration was 0.5mg/ ml. After RC-2, the expression of the two-cell aminopeptidase N/ CD13 protein was not reduced (P0.05); the expression of the autophagy-related protein LC-3B in both renal cell lines was significantly higher (P0.05), and the effect could be enhanced by the autophagy-inducing agent Rapamycin or blocked by the autophagy inhibitor 3-MA (P The results showed that the expression of aminopeptidase N/ CD13 and LC-3 in the normal tissues of the renal cell carcinoma and the adjacent normal tissues of the carcinoma was significantly higher than that of the cancer tissues (P The expression of aminopeptidase N/ CD13 in the renal lesions of various pathological types was shown by the immunohistochemical staining of the renal tissue chip, and there was no significant difference in the different pathological types and stage stages (P 0. 05 Conclusion 1. The proliferation, migration and invasion of renal cancer cells in 1. 2. The mechanism is related to the inhibition of the activity of aminopeptidase N; the autophagy-related protein LC-3B of the two renal cell lines after the action of the Ubenzo-Mast The amount of expression was significantly increased. The cytotoxic effect of the Ubiome on the 785-0 cell line could be blocked by the autophagy-inducing agent Rapamycin (P. 05), suggesting that it could be induced by the induction of the cell.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.11
本文编号：2318166
[Abstract]:Objective To study the effect of Ubenmei on the in vitro growth of renal cell carcinoma cell line 786-0 and OS-RC-2. square Method 1. The effect of Ubenmei on the in vitro growth of renal cell line 786-0 and OS-RC-2 was studied by the study of cell line 786-0 and OS-RC-2 of renal cell carcinoma. The effect of Ubenmei on the cell migration of renal cell carcinoma cell line 786-0 and OS-RC-2 cells was observed by cell scratch test, and the effect of Ubenmei on the invasion ability of cell line 786-0 cells and OS-RC-2 cells was observed by Transwell cell invasion experiment.. 2. Preliminary study on the mechanism of the action of Ubenumin on the renal cell carcinoma cell line 786-0 and OS-RC-2: L-leucine-P-nitroaniline substrate method for the detection of the activity of the Ubiome on the human renal cell line 786-0 cells and the OS-RC-2 cell aminopeptidase Effect of Western Blot on the expression of CD13, LC-3 and Caspase-3 in renal cell carcinoma cell line 786-0 and OS-RC-2 cells Level. The death rate of cells following the test of release test of renal cell carcinoma cell line 786-0 and OS-RC-2 by autophagy inhibitor, rapamycin, autophagy inhibitor 3-Methyladmine, at different concentrations.. 3. The expression of aminopeptidase N/ CD13 and LC-3 in the renal cell carcinoma and the adjacent normal tissues was detected by Western Blot. The expression of aminopeptidase N/ CD13 in the renal cell carcinoma and normal tissues was observed by immunohistochemical staining. Results The proliferation of WST-8 cells and the cell growth curve showed that the proliferation of renal cell carcinoma cell line 786-0 and OS-RC-2 was significantly inhibited after the action of 0. lmg/ ml of Ubenmei (P <0.05), and the inhibition effect was also enhanced with the increase of drug concentration (P The results of the cell migration showed that the effect of 0. 5 mg/ ml of UBENZUS on the migration of cell line 786-0 and OS-RC-2 in renal cell carcinoma was significantly inhibited (P0.05). The results of the invasion and experiment of Transwell cells showed that the effects of 0. 5 mg/ ml of Ubenzol on the cells of 786-0 and OS-RC-2 were significantly inhibited (P <0.05). The results showed that the activity of aminopeptidase of the cell line 786-0 and OS-RC-2 in the renal cell carcinoma cell line 786-0 and the OS-RC-2 cells decreased significantly after 24 hours after the action of 0. 25mg/ ml of the UBENZUME (P0.05). Western Blot showed that the highest concentration was 0.5mg/ ml. After RC-2, the expression of the two-cell aminopeptidase N/ CD13 protein was not reduced (P0.05); the expression of the autophagy-related protein LC-3B in both renal cell lines was significantly higher (P0.05), and the effect could be enhanced by the autophagy-inducing agent Rapamycin or blocked by the autophagy inhibitor 3-MA (P The results showed that the expression of aminopeptidase N/ CD13 and LC-3 in the normal tissues of the renal cell carcinoma and the adjacent normal tissues of the carcinoma was significantly higher than that of the cancer tissues (P The expression of aminopeptidase N/ CD13 in the renal lesions of various pathological types was shown by the immunohistochemical staining of the renal tissue chip, and there was no significant difference in the different pathological types and stage stages (P 0. 05 Conclusion 1. The proliferation, migration and invasion of renal cancer cells in 1. 2. The mechanism is related to the inhibition of the activity of aminopeptidase N; the autophagy-related protein LC-3B of the two renal cell lines after the action of the Ubenzo-Mast The amount of expression was significantly increased. The cytotoxic effect of the Ubiome on the 785-0 cell line could be blocked by the autophagy-inducing agent Rapamycin (P. 05), suggesting that it could be induced by the induction of the cell.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.11
【参考文献】
相关期刊论文 前1条
1 ;Molecular mechanism and regulation of autophagy[J];Acta Pharmacologica Sinica;2005年12期
，本文编号：2318166
本文链接：https://www.wllwen.com/yixuelunwen/mjlw/2318166.html
最近更新
教材专著
