本文關(guān)鍵詞：羽扇豆醇誘導(dǎo)microRNAs調(diào)控HeLa細(xì)胞耐藥響應(yīng)通路分析　出處：《哈爾濱工業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： HeLa細(xì)胞 羽扇豆醇 microRNAs 抗藥性 信號(hào)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(xì)胞耐藥性的形成卻成為長(zhǎng)期治療惡性腫瘤的障礙。近年來(lái),越來(lái)越多的證據(jù)表明micro RNAs與腫瘤形成過(guò)程中的多種生物學(xué)活動(dòng)有關(guān),同時(shí),其在腫瘤細(xì)胞耐藥性獲得過(guò)程中也起著重要作用。研究顯示,通過(guò)膳食控制能預(yù)防超過(guò)三分之二的腫瘤發(fā)生,蔬菜、水果、膳食纖維和某些微營(yíng)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(dǎo)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(xì)胞生長(zhǎng)等多種功效使得其在腫瘤的預(yù)防方面引起廣泛關(guān)注。目前,部分micro RNAs對(duì)腫瘤耐藥性的作用已逐漸被揭示,與此同時(shí),羽扇豆醇對(duì)腫瘤細(xì)胞的抑制機(jī)制也需要不斷完善,因此,研究耐藥細(xì)胞中羽扇豆醇對(duì)micro RNAs的表達(dá)調(diào)控作用對(duì)腫瘤的治療具有一定的指導(dǎo)意義,同時(shí)也能進(jìn)一步完善羽扇豆醇的抗腫瘤機(jī)制。本研究通過(guò)濃度梯度法誘導(dǎo)出四種不同程度的順鉑抗性的He La細(xì)胞(He La/CR1、He La/CR2、He La/CR3和He La/CR4),檢測(cè)其耐藥倍數(shù),對(duì)He La細(xì)胞耐藥性活動(dòng)過(guò)程進(jìn)行模擬。根據(jù)前期研究和已有理論知識(shí),挑選了五種候選耐藥性相關(guān)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(biāo)作為研究對(duì)象。采用RT-q PCR對(duì)micro RNAs和相應(yīng)的m RNAs表達(dá)量進(jìn)行檢測(cè)。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(shè)置四個(gè)不同的時(shí)間節(jié)點(diǎn)(6h、12h、18h以及24h),并對(duì)藥物處理細(xì)胞中的候選micro RNAs及其靶標(biāo)進(jìn)行熒光定量PCR,對(duì)其表達(dá)變化進(jìn)行檢測(cè),進(jìn)而對(duì)羽扇豆醇誘導(dǎo)耐藥細(xì)胞中micro RNAs相關(guān)的抑癌通路進(jìn)行分析。結(jié)果表明,在四種耐藥細(xì)胞中,mi R-182和mi R-15b表達(dá)上調(diào);但是mi R-183和mi R-30a表達(dá)卻出現(xiàn)顯著下調(diào)。與此同時(shí),根據(jù)靶標(biāo)RT-PCR結(jié)果可以得出KIAA1199是mi R-183的主要靶標(biāo),PDCD4為mi R-182的靶標(biāo),但是mi R-30a的調(diào)節(jié)靶標(biāo)還需要進(jìn)一步驗(yàn)證。與非耐藥細(xì)胞相比,耐藥細(xì)胞中micro RNAs表達(dá)量的顯著變化,表明He La細(xì)胞耐藥性的發(fā)展與一部分micro RNAs相關(guān)。羽扇豆醇處理耐藥細(xì)胞后的結(jié)果表明,相關(guān)的micro RNAs及其靶標(biāo)表達(dá)再次出現(xiàn)顯著變化,而且變化趨勢(shì)耐藥性發(fā)展過(guò)程中的變化趨勢(shì)相反,這表明羽扇豆醇可能通過(guò)調(diào)節(jié)相關(guān)micro RNAs的表達(dá)而達(dá)到抑制腫瘤的目的,同時(shí),micro RNAs可能通過(guò)調(diào)節(jié)細(xì)胞對(duì)化療的敏感性而成為腫瘤治療的研究方向。結(jié)合實(shí)驗(yàn)結(jié)果和已有理論知識(shí)得出,在耐藥He La細(xì)胞中,羽扇豆醇調(diào)節(jié)micro RNAs參與的相關(guān)抑癌信號(hào)通路及下游的信號(hào)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(guò)多靶標(biāo),多信號(hào)通路來(lái)抑制腫瘤細(xì)胞遷移和細(xì)胞增殖、促進(jìn)細(xì)胞凋亡以及增強(qiáng)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(xué)位授予單位】：哈爾濱工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R73-36

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