本文關(guān)鍵詞：miR-185對(duì)黑色素瘤的抑制作用和輻射增敏作用研究　出處：《蘭州大學(xué)》2016年碩士論文　論文類(lèi)型：學(xué)位論文

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【摘要】：黑色素瘤是發(fā)生率、死亡率增長(zhǎng)速度最快的皮膚病癌癥,一直以來(lái)都受到人們的廣泛關(guān)注。放療作為目前臨床上最常用的抗癌常規(guī)治療手段之一,是治療晚期轉(zhuǎn)移型黑色瘤的主要方法。但是,研究發(fā)現(xiàn)黑色素瘤細(xì)胞普遍對(duì)放療具有一定程度的耐受性,這使得常規(guī)放療療效受限,而通過(guò)增大放療劑量提高療效時(shí)又會(huì)帶來(lái)較大的副作用。目前,臨床上開(kāi)發(fā)了一系列放療增敏劑用以提高放療的治療效果,主要包括化學(xué)類(lèi)、中草藥類(lèi)藥物,然而這些放療增敏劑均存在一定的弊端。因此,尋求高效、低毒的放療增敏劑對(duì)于臨床黑色素瘤的放射治療意義重大。microRNA(mi RNA)是一類(lèi)長(zhǎng)度約為22個(gè)核苷酸的小RNA分子,通過(guò)與靶基因mRNA的3'-UTR區(qū)結(jié)合而引起其降解或翻譯抑制,在轉(zhuǎn)錄后水平發(fā)揮調(diào)控功能。miRNA不僅可以調(diào)節(jié)細(xì)胞增殖、分化、周期和凋亡等細(xì)胞進(jìn)程,而且參與調(diào)控細(xì)胞的輻射應(yīng)答通路,影響細(xì)胞對(duì)射線的敏感性。多項(xiàng)研究證實(shí),miR-185通過(guò)誘導(dǎo)腫瘤細(xì)胞的周期阻滯、促進(jìn)細(xì)胞的凋亡以及改變細(xì)胞基因組表觀遺傳等方式在多種腫瘤中發(fā)揮抑瘤因子的功能。另一方面,我們的前期研究發(fā)現(xiàn)X射線輻照后的腎癌組織中mi R-185的表達(dá)水平發(fā)生顯著下調(diào),在體外和體內(nèi)水平miR-185均能顯著增強(qiáng)腎癌細(xì)胞的輻射敏感性;此外,miR-185還能降低胃癌細(xì)胞對(duì)化療藥物的耐受性。這些研究表明miR-185在調(diào)控腫瘤細(xì)胞對(duì)放療和化療的應(yīng)激響應(yīng)進(jìn)程中發(fā)揮著重要的作用,能夠影響腫瘤細(xì)胞對(duì)輻射及對(duì)化療藥物的敏感性。然而,miR-185是否在黑色素瘤中同樣能夠發(fā)揮腫瘤抑制因子的功能以及是否能夠增強(qiáng)黑色素瘤細(xì)胞的輻射敏感性仍不清楚。為了探究miR-185對(duì)黑色素瘤發(fā)生和發(fā)展的影響,我們通過(guò)轉(zhuǎn)染的方法在小鼠黑色素瘤細(xì)胞系B16中過(guò)表達(dá)miR-185,并利用細(xì)胞生長(zhǎng)曲線、劃痕及克隆形成實(shí)驗(yàn)檢測(cè)miR-185對(duì)B16細(xì)胞增殖、遷移和克隆能力的影響,結(jié)果顯示miR-185能夠顯著地抑制細(xì)胞增殖、降低克隆形成和削弱細(xì)胞遷移。此外,我們將B16細(xì)胞接種到C57小鼠皮下成瘤,構(gòu)建了同種移植瘤模型,利用PEI載體直接在瘤體內(nèi)轉(zhuǎn)染miR-185,發(fā)現(xiàn)外源性過(guò)表達(dá)miR-185能顯著抑制瘤體生長(zhǎng),而瘤體組織Ki-67及PCNA免疫組化結(jié)果也表明過(guò)表達(dá)miR-185顯著降低了瘤體內(nèi)增殖期細(xì)胞的比率。在明確了miR-185的腫瘤抑制功能后,我們繼續(xù)深入探索了miR-185對(duì)黑色素瘤細(xì)胞輻射敏感性的影響。我們發(fā)現(xiàn)4Gy的X射線能顯著降低B16細(xì)胞的增殖和遷移能力。更為重要的是與對(duì)照組相比,輻照對(duì)過(guò)表達(dá)miR-185的B16細(xì)胞具有更好的殺傷效果,過(guò)表達(dá)miR-185能顯著增強(qiáng)輻照導(dǎo)致的細(xì)胞增殖抑制和遷移抑制以及促凋亡水平。小鼠移植瘤實(shí)驗(yàn)同樣表明miR-185能增強(qiáng)X射線輻照對(duì)瘤體生長(zhǎng)的抑制效果,使得瘤細(xì)胞凋亡水平明顯增加。綜上所述,我們的研究首次證實(shí)了無(wú)論在體外還是體內(nèi)水平miR-185都能夠發(fā)揮抑制黑色素瘤生長(zhǎng)的作用,是一個(gè)強(qiáng)有力的抑癌因子;并且miR-185還能夠增強(qiáng)黑色素細(xì)胞對(duì)電離輻射的敏感性,是一個(gè)有效的放療增敏分子。我們的研究為miR-185的抑癌及輻射增敏增加了新的基礎(chǔ)數(shù)據(jù),有助于推動(dòng)miR-185的臨床應(yīng)用。
[Abstract]:Melanoma is the incidence of skin cancer mortality is the fastest growing, has been widely concerned by people. Radiotherapy as one of the most commonly used clinical anticancer conventional treatment method at present, is the main method for the treatment of advanced metastatic melanoma type. However, the study found that the tolerance of melanoma cells generally have a certain the degree of radiation, which makes the limited conventional radiotherapy, and by increasing the radiation dose to improve the curative effect and will bring more side effects. At present, the clinical development of a series of radiosensitizer to improve the therapeutic effect of radiotherapy, including chemical, herbal drugs, but these radiosensitizer there are some disadvantages. Therefore, looking for effective, low toxicity radiosensitizer for radiotherapy in the treatment of major clinical.MicroRNA melanoma (MI RNA) is a kind of about 22 nucleotides in length Small molecular RNA, through the mRNA and the 3'-UTR of target gene binding caused by the degradation or translation inhibition at the post transcriptional level play a regulatory function of.MiRNA can not only regulate cell proliferation, differentiation, cell cycle and apoptosis process, and the radiation response pathway involved in the regulation of cell sensitivity to radiation, affecting cell. A number of studies have confirmed that miR-185 through the induction of tumor cell cycle arrest, cell apoptosis and changes of cell genome epigenetic play a tumor suppressor function in a variety of tumors. On the other hand, our previous study found that the expression level of X ray irradiation in renal cell carcinoma mi R-185 was significantly down regulated, can significantly enhance the radiation the sensitivity of renal cell carcinoma cells in vitro and in vivo levels of miR-185; moreover, miR-185 can reduce the resistance of gastric cancer cells to chemotherapeutic agents. These studies show that miR-185 in the Stress regulation of tumor cells to radiotherapy and chemotherapy response plays an important role in the process, can affect the sensitivity of tumor cells to radiation and chemotherapeutic drugs. However, whether miR-185 in melanoma can also play a tumor suppressor function and whether it can enhance the radiation sensitivity of melanoma cells to remain unclear. To explore the influence of miR-185 on the occurrence and development of melanoma, we transfected in mouse melanoma cell line B16 overexpression of miR-185, and the cell growth curve, colony formation assay and scratch and miR-185 on B16 cell proliferation, migration and effect of cloning ability, the result shows that miR-185 can significantly inhibit cell proliferation reduce, clone formation and weaken cell migration. In addition, we B16 cells were inoculated subcutaneously into C57 mice tumor, construct transplantation tumor model by P EI vector transfection in vivo directly in the miR-185, found that overexpression of miR-185 can inhibit the growth of tumor, and the tumor tissue Ki-67 and PCNA immunohistochemistry results also showed that overexpression of miR-185 significantly reduced the rate of tumor cell proliferation period. The tumor suppressor function of miR-185, we continue to explore the effect of miR-185 on radiation sensitivity of melanoma cells. We found that X - 4Gy can significantly reduce the proliferation and migration of B16 cells. The more important is compared with the control group, irradiation on the expression of miR-185 B16 cells has a good killing effect, overexpression of miR-185 can significantly enhance the irradiation induced cell inhibition of proliferation and migration inhibition and apoptosis level. Mouse xenograft experiments also showed that miR-185 can enhance X radiation inhibition effect on tumor growth, the tumor cell apoptosis level was significantly increased Plus. In summary, our study confirms for the first time to play a role in inhibiting the growth of melanoma are either in vitro or in vivo levels of miR-185, is a powerful anti cancer factor; and miR-185 can also increase the sensitivity to ionizing radiation of melanoma cells, is an effective radiosensitizer molecule. Our study the miR-185 tumor suppressor and radiation sensitivity increases the basic data, contribute to the clinical application to promote the miR-185.

【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R739.5

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