發(fā)布時(shí)間：2018-11-08 10:14

【摘要】：目的研究烏苯美司對(duì)腎癌細(xì)胞株786-0和OS-RC-2體外生長(zhǎng)的影響,初步研究其對(duì)腎癌細(xì)胞作用的機(jī)制。 方法1.以腎癌細(xì)胞株786-0及OS-RC-2為研究對(duì)象研究烏苯美司對(duì)腎癌細(xì)胞體外生長(zhǎng)的影響：WST-8細(xì)胞增殖實(shí)驗(yàn)及細(xì)胞生長(zhǎng)曲線繪制檢測(cè)烏苯美司對(duì)腎癌細(xì)胞株786-0細(xì)胞及OS-RC-2細(xì)胞增殖能力的影響；細(xì)胞劃痕實(shí)驗(yàn)觀察烏苯美司對(duì)腎癌細(xì)胞株786-0細(xì)胞及OS-RC-2細(xì)胞遷移能力的影響；Transwell細(xì)胞侵襲實(shí)驗(yàn)觀察烏苯美司對(duì)腎癌細(xì)胞株786-0細(xì)胞及OS-RC-2細(xì)胞侵襲能力的影響。2.烏苯美司對(duì)腎癌細(xì)胞株786-0和OS-RC-2作用機(jī)制的初步研究：L-亮氨酸-P硝基苯胺底物法檢測(cè)烏苯美司對(duì)腎癌細(xì)胞株786-0細(xì)胞及OS-RC-2細(xì)胞氨肽酶活性的影響。Western Blot檢測(cè)烏苯美司作用于腎癌細(xì)胞株786-0細(xì)胞及OS-RC-2細(xì)胞后,兩種細(xì)胞CD13、LC-3、Caspase-3的表達(dá)水平。以不同濃度的烏苯美司、自噬誘導(dǎo)劑rapamycin、自噬抑制劑3-Methyladenine作用于腎癌細(xì)胞株786-0和OS-RC-2, LDH釋放試驗(yàn)檢測(cè)作用后細(xì)胞的死亡率。3.以腎癌患者手術(shù)取得病理組織為研究對(duì)象：Western Blot檢測(cè)氨肽酶N/CD13和LC-3在腎癌組織和癌旁正常組織的表達(dá)量；以腎癌患者及腎良性病變患者手術(shù)取得病理組織構(gòu)建腎臟病變的組織芯片,通過(guò)免疫組化染色觀察烏苯美司的作用靶點(diǎn)氨肽酶N/CD13在腎癌組織及正常組織中的表達(dá)情況。 結(jié)果WST-8細(xì)胞增殖檢測(cè)及細(xì)胞生長(zhǎng)曲線顯示經(jīng)0.lmg/ml烏苯美司作用后,腎癌細(xì)胞株786-0及OS-RC-2增殖均受到明顯抑制(P0.05),且隨著藥物濃度的增加,這種抑制作用也逐漸增強(qiáng)(P0.05)。細(xì)胞遷移實(shí)驗(yàn)結(jié)果表明0.5mg/ml烏苯美司作用12小時(shí)對(duì)腎癌細(xì)胞株786-0及OS-RC-2細(xì)胞的遷移有明顯的抑制作用(P0.05),且隨著烏苯美司藥物濃度增加,抑制作用也隨之增強(qiáng)(P0.05)。Transwell細(xì)胞侵襲實(shí)驗(yàn)結(jié)果顯示0.5mg/ml烏苯美司作用24小時(shí)能明顯抑制786-0及OS-RC-2細(xì)胞的侵襲(P0.05)。氨肽酶活性檢測(cè)試驗(yàn)證實(shí)經(jīng)0.25mg/ml烏苯美司作用24小時(shí)后,腎癌細(xì)胞株786-0及OS-RC-2細(xì)胞的氨肽酶活性都明顯降低活性(P0.05)。Western Blot結(jié)果顯示最高濃度為0.5mg/ml烏苯美司作用于腎癌細(xì)胞株786-0及OS-RC-2后,并沒(méi)有降低兩種細(xì)胞氨肽酶N/CD13蛋白的表達(dá)量(P0.05)；兩種腎癌細(xì)胞株的自噬相關(guān)蛋白LC-3B表達(dá)量明顯升高(P0.05),且這種效應(yīng)可被自噬誘導(dǎo)劑Rapamycin增強(qiáng)或被自噬抑制劑3-MA阻斷(P0.05)。對(duì)手術(shù)病理組織的Western Blot檢測(cè)結(jié)果顯示氨肽酶N/CD13和LC-3在腎癌組織及癌旁正常組織中均有表達(dá),且2種蛋白在癌旁正常組織中的表達(dá)量均明顯高于癌組織(P0.05)。腎臟組織芯片免疫組化染色顯示氨肽酶N/CD13在各種病理類(lèi)型的腎臟病變中均有表達(dá),且在不同病理類(lèi)型和分級(jí)分期中無(wú)明顯差異(P0.05)。 結(jié)論1.烏苯美司對(duì)腎癌細(xì)胞的增殖、遷移、侵襲有抑制作用。2.其機(jī)制與抑制氨肽酶N活性有關(guān)；烏苯美司作用后兩種腎癌細(xì)胞株的自噬相關(guān)蛋白LC-3B表達(dá)量都明顯升高。烏苯美司對(duì)785-0細(xì)胞系中的細(xì)胞毒性作用可被自噬誘導(dǎo)劑Rapamycin增強(qiáng)或倍自噬抑制劑3-MA阻斷(P0.05),提示其可能通過(guò)誘導(dǎo)細(xì)胞自噬發(fā)揮抗腫瘤效應(yīng)。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R737.11

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;Molecular mechanism and regulation of autophagy[J];Acta Pharmacologica Sinica;2005年12期

，

本文編號(hào)：2318166