本文關鍵詞：羽扇豆醇誘導microRNAs調(diào)控HeLa細胞耐藥響應通路分析　出處：《哈爾濱工業(yè)大學》2015年碩士論文　論文類型：學位論文

  更多相關文章： HeLa細胞 羽扇豆醇 microRNAs 抗藥性 信號通路

【摘要】：化療是目前癌癥治療的主要手段,順鉑是癌癥化療中最常使用的一種鉑類化合藥物。但是,腫瘤細胞耐藥性的形成卻成為長期治療惡性腫瘤的障礙。近年來,越來越多的證據(jù)表明micro RNAs與腫瘤形成過程中的多種生物學活動有關,同時,其在腫瘤細胞耐藥性獲得過程中也起著重要作用。研究顯示,通過膳食控制能預防超過三分之二的腫瘤發(fā)生,蔬菜、水果、膳食纖維和某些微營養(yǎng)成分均具有一定的防癌作用,果蔬中含有一種三萜類生活性物質(zhì)——羽扇豆醇,其抗氧化、誘導凋亡、抗惡性腫瘤增殖、抗基因突變以及抗炎癥和抑制腫瘤細胞生長等多種功效使得其在腫瘤的預防方面引起廣泛關注。目前,部分micro RNAs對腫瘤耐藥性的作用已逐漸被揭示,與此同時,羽扇豆醇對腫瘤細胞的抑制機制也需要不斷完善,因此,研究耐藥細胞中羽扇豆醇對micro RNAs的表達調(diào)控作用對腫瘤的治療具有一定的指導意義,同時也能進一步完善羽扇豆醇的抗腫瘤機制。本研究通過濃度梯度法誘導出四種不同程度的順鉑抗性的He La細胞(He La/CR1、He La/CR2、He La/CR3和He La/CR4),檢測其耐藥倍數(shù),對He La細胞耐藥性活動過程進行模擬。根據(jù)前期研究和已有理論知識,挑選了五種候選耐藥性相關micro RNAs(mi R-183、mi R-182、mi R-30a、mi R-15b和mi R-16)以及其潛在靶標作為研究對象。采用RT-q PCR對micro RNAs和相應的m RNAs表達量進行檢測。隨后用高劑量的羽扇豆醇處理挑選出耐藥性較好的He La/CR4,并設置四個不同的時間節(jié)點(6h、12h、18h以及24h),并對藥物處理細胞中的候選micro RNAs及其靶標進行熒光定量PCR,對其表達變化進行檢測,進而對羽扇豆醇誘導耐藥細胞中micro RNAs相關的抑癌通路進行分析。結(jié)果表明,在四種耐藥細胞中,mi R-182和mi R-15b表達上調(diào);但是mi R-183和mi R-30a表達卻出現(xiàn)顯著下調(diào)。與此同時,根據(jù)靶標RT-PCR結(jié)果可以得出KIAA1199是mi R-183的主要靶標,PDCD4為mi R-182的靶標,但是mi R-30a的調(diào)節(jié)靶標還需要進一步驗證。與非耐藥細胞相比,耐藥細胞中micro RNAs表達量的顯著變化,表明He La細胞耐藥性的發(fā)展與一部分micro RNAs相關。羽扇豆醇處理耐藥細胞后的結(jié)果表明,相關的micro RNAs及其靶標表達再次出現(xiàn)顯著變化,而且變化趨勢耐藥性發(fā)展過程中的變化趨勢相反,這表明羽扇豆醇可能通過調(diào)節(jié)相關micro RNAs的表達而達到抑制腫瘤的目的,同時,micro RNAs可能通過調(diào)節(jié)細胞對化療的敏感性而成為腫瘤治療的研究方向。結(jié)合實驗結(jié)果和已有理論知識得出,在耐藥He La細胞中,羽扇豆醇調(diào)節(jié)micro RNAs參與的相關抑癌信號通路及下游的信號分子有Wnt/β-catenin、PI3K/PTEN/AKT、BAX、YB-1、NF-κB、Caspase家族、PGRMC1、ATG以及TGF-TGF-β等,從分析可以看出,羽扇豆醇能通過多靶標,多信號通路來抑制腫瘤細胞遷移和細胞增殖、促進細胞凋亡以及增強藥物敏感性等。
[Abstract]:Chemotherapy is the main means of cancer treatment at present, and cisplatin is one of the most commonly used platinum - like drugs in cancer chemotherapy. However, the formation of drug resistance in tumor cells is an obstacle to the long-term treatment of malignant tumors. In recent years, more and more evidences show that micro RNAs is related to many biological activities in tumor formation, and it plays an important role in the process of drug resistance acquisition. Research shows that, through dietary control could prevent more than 2/3 of the tumor, vegetables, fruits, dietary fiber and some micro nutrients have anti-cancer effects, fruits and vegetables contain a three terpenoid material life -- lupeol, its antioxidant, inducing apoptosis, anti proliferation, anti malignant tumor and gene mutation the anti-inflammatory and inhibiting tumor cell growth and other effects which caused widespread concern in the prevention of tumor. At present, part of micro RNAs on tumor drug resistance has been gradually revealed. At the same time, the inhibition mechanism of lupeol on tumor cells also need to constantly improve, therefore, has a certain guiding significance for research on expression regulation effect of lupeol in resistant cells on micro RNAs of tumor, but also to further improve the mechanism of tumor anti lupeol. In this study, four different levels of cisplatin resistant He La cells (He La/CR1, He La/CR2, He La/CR3 and He La/CR4) were induced by concentration gradient method. The drug resistance times were detected, and the process of drug resistance activity of He cells was simulated. According to previous studies and existing theoretical knowledge, five candidate resistance related micro RNAs (MI R-183, MI R-182, MI R-30a, MI R-15b and MI R-16) and their potential targets were selected as research objects. RT-q PCR was used to detect the expression of micro RNAs and corresponding M RNAs. Followed by high doses of lupeol treatment selected better He resistance La/CR4, and set the four different time points (6h, 12h, 18h and 24h), and fluorescence quantitative PCR on drug candidate micro in RNAs cells and on the expression of target, change detection, analysis of tumor suppressor pathway and then the lupeol induced micro resistant cells related to RNAs. The results showed that the expression of MI R-182 and MI R-15b was up-regulated in four kinds of drug-resistant cells, but the expression of MI R-183 and MI R-30a decreased significantly. Meanwhile, according to the result of target RT-PCR, it can be concluded that KIAA1199 is the main target of MI R-183, PDCD4 is the target of MI R-182, but the target of MI R-30a needs further verification. Compared with non drug resistant cells, the significant changes in the expression of micro RNAs in drug-resistant cells showed that the development of He La cell resistance was associated with a part of micro RNAs. The results of lupeol treatment resistant cells after the indicated that the expression of micro RNAs and its related target again significant changes, and the changes of development trend in the trend of drug resistance instead, suggesting that lupeol may lead to inhibition of tumor by regulating expression of micro, RNAs and micro RNAs may become a research direction treatment of cancer by regulating cell sensitivity to chemotherapy. According to the experimental results and theoretical knowledge obtained in He resistant La cells, lupeol regulation related inhibitory pathway and the downstream signal molecule micro RNAs cancer in Wnt/, PI3K/PTEN/AKT, BAX, -catenin beta YB-1, NF-, Caspase, PGRMC1 kappa B family, ATG and TGF-TGF- beta, as can be seen from the analysis lupeol, through multi targets, multi signal pathway to inhibit tumor cell migration and cell proliferation, promote cell apoptosis and enhance the drug sensitivity.
【學位授予單位】：哈爾濱工業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R73-36

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