活性RhoA在紫杉醇诱导肾癌周期阻滞中的作用及机制研究

发布时间：2018-08-26 11:52

【摘要】：研究背景:肾细胞癌是肾实质来源的恶性肿瘤中最常见的类型,在成年人癌症疾患中约占2%~3%。目前肾癌的治疗以手术为主,其术后辅助治疗尚无统一标准。紫杉醇(Taxol)是一种新型的抗微管剂,能够促进微管(MT)蛋白聚集而稳定MT,从而抑制微管网络的动态重组,抑制肿瘤细胞增值,被用于进展性肾癌根治性手术后的辅助化疗,可使部分患者受益。研究发现,RhoA表达在多种肿瘤中明显增强,对肿瘤生长、增值、侵袭及转移等发生发展的多个方面具有调节作用。但在肾癌中,活性RhoA(GTP-RhoA)与细胞骨架结构的关系以及对细胞周期(cell cycle)的调控过程尚无相关报道。目的:探讨Taxol诱导MT改变对cell cycle和活性RhoA表达的影响。进一步探究MT改变,RhoA蛋白的分布及位置变化。通过破坏MT解聚,观察活性RhoA表达和细胞周期的变化;通过抑制活性RhoA,观察MT及cell cycle的变化。从而探究活性RhoA在Taxol诱导肾癌细胞骨架改变阻滞cell cycle过程中的意义。方法:以Taxol处理的肾癌细胞OS-RC-2细胞株为研究对象,用免疫荧光染色MT及RhoA蛋白,共聚焦显微镜观察MT变化及RhoA蛋白分布及位置变化。用Western-blot半定量测定RhoA蛋白及GTP-RhoA蛋白表达情况,用流式法对细胞的周期改变进行测定。实验所得数据以`X±SD表示,用SPSS 13.0版本分析数据,多组间比较采用one-way ANOVA,组间两两比较采用LSD和SNK检验。P0.05具有统计学意义结果:1.Taxol诱导OS-RC-2细胞周期阻滞在G2/M期,GTP-RhoA表达显著上调,且表现为时间正相关。2.Taxol处理诱导OS-RC-2细胞MT聚合,形成特异性的束状结构,包绕在核周。3.活性RhoA伴随着MT聚合、解聚而重新分布,但始终与MT位置保持相对一致,具有“同位效应”。4.MT解聚剂Col破坏Taxol诱导的MT特异性结构,逆转了细胞G2/M期阻滞,使活性RhoA表达下调。5.C3转移酶抑制RhoA活性后,部分逆转了Taxol诱导的细胞周期阻滞,但对聚合的MT结构无明显影响。结论:Taxol诱导MT聚合形成特异性的束状环,引起OS-RC-2细胞内RhoA的活性增强。这一过程对Taxol治疗肾癌中引起细胞周期阻滞,抑制肿瘤增值有重要作用。但反之活性RhoA对已聚合的MT无明显调节作用。意义:通过研究OS-RC-2细胞内MT、活性RhoA以及细胞周期三者间的关系,阐明了活性RhoA在MT调控肾癌细胞周期中的相关机制及重要作用。在未来临床应用中,Rho A对调节Taxol治疗肾癌疗效具有潜在价值。
[Abstract]:Background: renal cell carcinoma (RCC) is the most common type of malignant tumor of renal parenchyma, accounting for about 2% of adult cancer diseases. At present, surgery is the main treatment of renal cell carcinoma, and there is no uniform standard for postoperative adjuvant treatment. Paclitaxel (Taxol) is a new antitubule agent, which can promote the aggregation of microtubule (MT) protein and stabilize MT, thus inhibit the dynamic recombination of microtubule network, inhibit the proliferation of tumor cells, and be used in adjuvant chemotherapy after radical surgery for progressive renal cell carcinoma. It can benefit some patients. It was found that RhoA expression was significantly increased in many kinds of tumors, which could regulate tumor growth, proliferation, invasion and metastasis. However, the relationship between active RhoA (GTP-RhoA) and cytoskeleton structure and the regulation of cell cycle (cell cycle) have not been reported. Objective: to investigate the effect of MT changes induced by Taxol on the expression of cell cycle and active RhoA. To further explore the MT changes in the distribution and location of RhoA protein. The expression of active RhoA and the changes of cell cycle were observed by destroying the depolymerization of MT, and the changes of MT and cell cycle were observed by inhibiting the activity of RhoA,. To explore the significance of active RhoA in Taxol induced renal cancer cell cytoskeleton block cell cycle process. Methods: Taxol treated renal cancer cell line OS-RC-2 was used as the study object, MT and RhoA protein were stained with immunofluorescence, and the changes of MT and RhoA protein distribution and location were observed by confocal microscope. The expression of RhoA protein and GTP-RhoA protein were measured by Western-blot, and the cell cycle was determined by flow cytometry. The experimental data were expressed as'X 卤SD 'and analyzed with SPSS version 13.0. The results showed that the expression of GTP-RhoA in OS-RC-2 cell cycle arrest induced by taxol in G 2 / M phase was significantly up-regulated by LSD and SNK test (P 0.05). The results showed that the MT polymerization of OS-RC-2 cells induced by Taxol was positively time-dependent and formed a specific bunchy structure, which was wrapped around the nucleus. 3. The active RhoA was redistributed with the polymerization of MT and depolymerization, but remained relatively consistent with the position of MT. 4. The depolymerization agent Col of MT destroyed the specific structure of MT induced by Taxol and reversed the G 2 / M phase arrest of cells. After down-regulation of RhoA activity by down-regulation of active RhoA expression, the cell cycle arrest induced by Taxol was partially reversed, but there was no significant effect on the structure of polymerized MT. Conclusion MT polymerization induced by 1: Taxol could induce the formation of specific fascicular rings and increase the activity of RhoA in OS-RC-2 cells. This process plays an important role in cell cycle arrest and inhibition of tumor proliferation in the treatment of renal cell carcinoma with Taxol. On the contrary, the active RhoA had no obvious effect on the MT that had been polymerized. Significance: by studying the relationship between MT, activity RhoA and cell cycle in OS-RC-2 cells, the mechanism and important role of active RhoA in the regulation of RCC cell cycle by MT were elucidated. Rho A has potential value in regulating the efficacy of Taxol in the treatment of renal cell carcinoma.
【学位授予单位】：宁波大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.11

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