【摘要】：研究背景:腎細(xì)胞癌是腎實(shí)質(zhì)來(lái)源的惡性腫瘤中最常見(jiàn)的類(lèi)型,在成年人癌癥疾患中約占2%~3%。目前腎癌的治療以手術(shù)為主,其術(shù)后輔助治療尚無(wú)統(tǒng)一標(biāo)準(zhǔn)。紫杉醇(Taxol)是一種新型的抗微管劑,能夠促進(jìn)微管(MT)蛋白聚集而穩(wěn)定MT,從而抑制微管網(wǎng)絡(luò)的動(dòng)態(tài)重組,抑制腫瘤細(xì)胞增值,被用于進(jìn)展性腎癌根治性手術(shù)后的輔助化療,可使部分患者受益。研究發(fā)現(xiàn),RhoA表達(dá)在多種腫瘤中明顯增強(qiáng),對(duì)腫瘤生長(zhǎng)、增值、侵襲及轉(zhuǎn)移等發(fā)生發(fā)展的多個(gè)方面具有調(diào)節(jié)作用。但在腎癌中,活性RhoA(GTP-RhoA)與細(xì)胞骨架結(jié)構(gòu)的關(guān)系以及對(duì)細(xì)胞周期(cell cycle)的調(diào)控過(guò)程尚無(wú)相關(guān)報(bào)道。目的:探討Taxol誘導(dǎo)MT改變對(duì)cell cycle和活性RhoA表達(dá)的影響。進(jìn)一步探究MT改變,RhoA蛋白的分布及位置變化。通過(guò)破壞MT解聚,觀察活性RhoA表達(dá)和細(xì)胞周期的變化;通過(guò)抑制活性RhoA,觀察MT及cell cycle的變化。從而探究活性RhoA在Taxol誘導(dǎo)腎癌細(xì)胞骨架改變阻滯cell cycle過(guò)程中的意義。方法:以Taxol處理的腎癌細(xì)胞OS-RC-2細(xì)胞株為研究對(duì)象,用免疫熒光染色MT及RhoA蛋白,共聚焦顯微鏡觀察MT變化及RhoA蛋白分布及位置變化。用Western-blot半定量測(cè)定RhoA蛋白及GTP-RhoA蛋白表達(dá)情況,用流式法對(duì)細(xì)胞的周期改變進(jìn)行測(cè)定。實(shí)驗(yàn)所得數(shù)據(jù)以`X±SD表示,用SPSS 13.0版本分析數(shù)據(jù),多組間比較采用one-way ANOVA,組間兩兩比較采用LSD和SNK檢驗(yàn)。P0.05具有統(tǒng)計(jì)學(xué)意義結(jié)果:1.Taxol誘導(dǎo)OS-RC-2細(xì)胞周期阻滯在G2/M期,GTP-RhoA表達(dá)顯著上調(diào),且表現(xiàn)為時(shí)間正相關(guān)。2.Taxol處理誘導(dǎo)OS-RC-2細(xì)胞MT聚合,形成特異性的束狀結(jié)構(gòu),包繞在核周。3.活性RhoA伴隨著MT聚合、解聚而重新分布,但始終與MT位置保持相對(duì)一致,具有“同位效應(yīng)”。4.MT解聚劑Col破壞Taxol誘導(dǎo)的MT特異性結(jié)構(gòu),逆轉(zhuǎn)了細(xì)胞G2/M期阻滯,使活性RhoA表達(dá)下調(diào)。5.C3轉(zhuǎn)移酶抑制RhoA活性后,部分逆轉(zhuǎn)了Taxol誘導(dǎo)的細(xì)胞周期阻滯,但對(duì)聚合的MT結(jié)構(gòu)無(wú)明顯影響。結(jié)論:Taxol誘導(dǎo)MT聚合形成特異性的束狀環(huán),引起OS-RC-2細(xì)胞內(nèi)RhoA的活性增強(qiáng)。這一過(guò)程對(duì)Taxol治療腎癌中引起細(xì)胞周期阻滯,抑制腫瘤增值有重要作用。但反之活性RhoA對(duì)已聚合的MT無(wú)明顯調(diào)節(jié)作用。意義:通過(guò)研究OS-RC-2細(xì)胞內(nèi)MT、活性RhoA以及細(xì)胞周期三者間的關(guān)系,闡明了活性RhoA在MT調(diào)控腎癌細(xì)胞周期中的相關(guān)機(jī)制及重要作用。在未來(lái)臨床應(yīng)用中,Rho A對(duì)調(diào)節(jié)Taxol治療腎癌療效具有潛在價(jià)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 鹵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.
【學(xué)位授予單位】：寧波大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R737.11

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