本文選題：腎細胞癌 + 吸煙��； 參考：《安徽醫(yī)科大學(xué)》2017年碩士論文

【摘要】：背景與研究目的腎癌是泌尿生殖系統(tǒng)常見的惡性腫瘤,發(fā)生率排名居人類惡性腫瘤第十四位。它起源于腎小管上皮細胞,以透明細胞癌最常見,占腎細胞癌的70%-80%,另外還有乳頭狀癌、嫌色細胞癌等。目前腎癌病因尚未明確,有資料顯示其發(fā)病與吸煙、解熱鎮(zhèn)痛藥、激素、病毒、咖啡鎘等有關(guān)。其中吸煙是已知最明確的危險因素,據(jù)推測20%-30%的男性和10%-20%的女性腎癌的發(fā)生是由吸煙引起。我國是世界上最大的煙草生產(chǎn)和消費國,吸煙引發(fā)的腫瘤及相關(guān)疾病,已成為國民健康的嚴重威脅。上皮間質(zhì)轉(zhuǎn)化在細胞惡轉(zhuǎn)及腫瘤轉(zhuǎn)移過程中起到至關(guān)重要的作用。本課題通過體外實驗研究吸煙暴露對人腎癌細胞上皮間質(zhì)轉(zhuǎn)化(Epithelial-mesenchymal transition,EMT)的影響,并探討ERK5信號通路在吸煙暴露誘導(dǎo)腎癌細胞的EMT中的作用。方法建立體外細胞吸煙處理模型;MTT法篩選對腎癌細胞ACHN、A498細胞活力無明顯影響的香煙懸液(cigarette smoking extract,CSE)濃度,后根據(jù)篩選結(jié)果選擇相應(yīng)濃度CSE處理腎癌細胞。(1)通過顯微鏡觀察CSE對腎癌細胞形態(tài)的影響并記錄;(2)通過transwell實驗、劃痕實驗觀察CSE能否引起腎癌細胞侵襲、轉(zhuǎn)移能力改變;(3)運用western blotting、免疫熒光檢測上皮間質(zhì)轉(zhuǎn)化相關(guān)分子標(biāo)志物的變化;q RT-PCR方法檢測CSE對腎癌細胞上皮間質(zhì)轉(zhuǎn)化相關(guān)標(biāo)志物m RNA表達水平的影響;(4)用western blotting檢測吸煙處理對腎癌細胞ERK5通路及AP-1通路活性的影響;(5)ERK5通路特異性抑制劑XMD8-92處理細胞,檢測其對吸煙誘導(dǎo)的腎癌細胞ACHN、A498上皮間質(zhì)轉(zhuǎn)化的干預(yù)效果。結(jié)果(1)通過MTT法篩選適宜濃度的CSE作為實驗濃度(0%、0.1%、0.25%、0.5%),當(dāng)CSE濃度在0.75%及以上時,細胞活力明顯受到影響,并具有統(tǒng)計學(xué)差異(P0.05);(2)CSE處理腎癌細胞6天后,鏡下觀察細胞由原來的聚集變的更為分散,形態(tài)由原來的扁橢圓形變?yōu)殚L梭形,部分細胞伸出偽足;劃痕實驗、transwell實驗表明,經(jīng)CSE處理后腎癌細胞轉(zhuǎn)移侵襲能力明顯增強。(3)western blotting、免疫熒光檢測發(fā)現(xiàn)腎癌細胞經(jīng)CSE處理后,上皮標(biāo)志物E-Cadherin、ZO-1表達水平呈上調(diào)趨勢,間質(zhì)標(biāo)志物vimentin、N-cadherin表達水平呈下調(diào)趨勢;同時,經(jīng)q RT-PCR證實,上述相關(guān)標(biāo)志物的m RNA表達水平亦表現(xiàn)相同的變化趨勢。(4)通過western blotting檢測發(fā)現(xiàn),磷酸化ERK5(phosphorylated ERK5,P-ERK5)水平呈明顯上升趨勢,而總ERK5(total ERK5,T-ERK5)呈明顯下降趨勢;AP-1通路中磷酸化c-jun(phosphorylated c-jun,p-c-jun)、磷酸化c-fos(phosphorylated c-fos,p-c-fos)表達水平呈上升趨勢�？梢�,吸煙處理可激活ERK5通路。(5)ERK5通路特異性抑制劑XMD8-92可逆轉(zhuǎn)吸煙誘導(dǎo)的腎癌細胞上皮間質(zhì)轉(zhuǎn)化過程,表現(xiàn)如下:細胞形態(tài)上,由間質(zhì)表型向上皮表型轉(zhuǎn)變,且細胞重新表現(xiàn)相互聚集狀態(tài);劃痕實驗及transwell實驗見細胞轉(zhuǎn)移侵襲能力出現(xiàn)逆轉(zhuǎn);EMT相關(guān)標(biāo)志物,上皮標(biāo)志物E-cadherin、ZO-1表達水平上升,而間質(zhì)標(biāo)志物間質(zhì)標(biāo)志物vimentin、N-cadherin表達水平下調(diào),相關(guān)標(biāo)志物m RNA表達水平也表現(xiàn)相應(yīng)的變化趨勢。結(jié)論吸煙暴露可誘導(dǎo)腎癌細胞ACHN、A498發(fā)生上皮間質(zhì)轉(zhuǎn)化,ERK5信號通路在這一變化過程中起重要的正調(diào)控作用,ERK5通路的抑制可阻止這樣變化的發(fā)生。這也進一步證實了,ERK5在吸煙引起的腎癌轉(zhuǎn)移侵襲過程中起重要作用。
[Abstract]:Background and research objective renal cancer is a common malignant tumor of the genitourinary system. The incidence of the fourteenth human malignant tumor is in the human malignant tumor. It originated from the renal tubular epithelial cells. It is the most common type of clear cell carcinoma, which accounts for 70%-80% of renal cell carcinoma. There are also papillary carcinoma and chromophobe cell carcinoma. The etiology of renal cancer is not yet clear. It is associated with smoking, antipyretic analgesics, hormones, viruses, and coffee and cadmium. Smoking is the most clear risk factor known. It is presumed that the incidence of kidney cancer in 20%-30% men and 10%-20% is caused by smoking. China is the largest tobacco producing and consumer country in the world, smoking caused cancer and related diseases, and has become the national health. The transformation of epithelial mesenchymal transition plays a vital role in the process of malignant transformation of cells and tumor metastasis. In this study, the effects of smoking exposure on Epithelial-mesenchymal transition (EMT) in human renal cell carcinoma cells were studied in vitro, and the EMT of ERK5 signaling pathway in the induced EMT of renal cancer cells in smoking exposure was explored. Methods to establish an in vitro cell smoking treatment model, the MTT method was used to screen the concentration of cigarette smoking extract (CSE), which had no significant influence on the activity of ACHN and A498 cells in renal cell carcinoma cells, and then selected the corresponding concentration CSE to treat the renal cell carcinoma cells according to the screening results. (1) the effects of CSE on the morphology of renal cell carcinoma cells were observed and recorded. (2) through the Transwell experiment, the scratch test was used to observe whether CSE could cause the invasion of renal cell carcinoma cells and change the metastasis ability; (3) the changes of epithelial mesenchymal transition markers were detected by Western blotting and immunofluorescence; and the Q RT-PCR method was used to detect the effect of CSE on the RNA expression level of the epithelial mesenchymal transition related markers of renal cell carcinoma cells; (4) weste. RN blotting was used to detect the effects of smoking treatment on ERK5 pathway and AP-1 pathway activity in renal cell carcinoma cells; (5) ERK5 pathway specific inhibitor XMD8-92 cells were used to detect the interference effect on ACHN and A498 epithelial mesenchymal transition in renal cancer cells induced by smoking. Results (1) the concentration of CSE was screened by MTT method as the experimental concentration (0%, 0.1%, 0.25%, 0.5%). When the concentration of CSE was 0.75% or more, the cell vitality was significantly affected and had statistical difference (P0.05). (2) after 6 days of CSE treatment of renal cell carcinoma cells, the cells were observed to be more dispersed from the original aggregation, the shape changed from the original oblate ellipsoid to the long spindle shape, the part of the cells extended to the pseudo foot; the scratch test, the Transwell experiment showed that CSE was found. The metastasis and invasion ability of the renal cell carcinoma cells was obviously enhanced. (3) Western blotting, the immunofluorescence detection showed that the epithelial markers E-Cadherin, the expression level of ZO-1 were up trend, the interstitial marker vimentin, the N-cadherin expression level downregulated, and the m RNA table of the above related markers was confirmed by Q RT-PCR. (4) through Western blotting detection, the level of phosphorylated ERK5 (phosphorylated ERK5, P-ERK5) showed an obvious upward trend, while the total ERK5 (total ERK5, T-ERK5) showed a significant downward trend, and the AP-1 pathway was phosphorylated. The expression level showed an upward trend. Smoking treatment can activate the ERK5 pathway. (5) the ERK5 pathway specific inhibitor XMD8-92 can reverse the process of epithelial mesenchymal transition in renal carcinoma cells induced by smoking, as follows: cell morphology changes from interstitial phenotype to epitheliality, and cell reacts with mutual aggregation; scratch test and Transwell It was found that the invasion ability of cell metastasis was reversed, EMT related markers, epithelial markers E-cadherin, ZO-1 expression level increased, the interstitial marker vimentin, N-cadherin expression level decreased, and the related markers m RNA expression level also showed the corresponding change trend. Conclusion smoking exposure can induce renal cancer cell ACHN, A498 hair. The ERK5 signaling pathway plays an important positive role in the transformation of epithelial mesenchymal transition, and the inhibition of ERK5 pathway can prevent such changes. This further confirms that ERK5 plays an important role in the process of metastasis and invasion of kidney cancer.

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.11

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