【摘要】：黑素瘤是皮膚科致死率最高的惡性腫瘤,細(xì)胞增殖能力強(qiáng),且不易受化療藥物的干擾,易發(fā)生轉(zhuǎn)移,預(yù)后不良。目前黑素瘤的發(fā)生機(jī)制仍不清楚。連接態(tài)NEDD8與多種惡性腫瘤的發(fā)生有相關(guān)性,與黑素瘤的關(guān)系尚未見研究報道。本研究旨在探討連接態(tài)NEDD8在黑素瘤發(fā)生發(fā)展中的作用。 (1)連接態(tài)NEDD8在黑素瘤組織和細(xì)胞中的表達(dá) 經(jīng)western blot檢測發(fā)現(xiàn),與瘤旁組織和痣組織相比,連接態(tài)NEDD8在皮膚黑素瘤瘤組織中的表達(dá)明顯上調(diào)。與正常人黑素細(xì)胞相比,連接態(tài)NEDD8在黑素瘤細(xì)胞株中的表達(dá)明顯上調(diào)。 (2)催化連接態(tài)NEDD8形成的相關(guān)酶蛋白在黑素瘤細(xì)胞株中的表達(dá) 通過real-time PCR技術(shù)檢測NEDD8、APP-BP1、UBA3、UBC12以及UCH-L3在黑素瘤細(xì)胞株中的表達(dá)。結(jié)果顯示與正常人黑素細(xì)胞相比,這些與連接態(tài)NEDD8的形成密切相關(guān)的蛋白在黑素瘤細(xì)胞株中的表達(dá)幾乎均明顯升高。 (3)shRNA-UBA3重組質(zhì)粒的構(gòu)建及細(xì)胞轉(zhuǎn)染 構(gòu)建shRNA-UBA3與pRNAT-U6.2/Lenti的重組質(zhì)粒載體,轉(zhuǎn)染M14細(xì)胞株,單克隆篩選穩(wěn)定表達(dá)shRNA-UBA3的細(xì)胞株,經(jīng)免疫細(xì)胞化學(xué)法和蛋白印跡法證實轉(zhuǎn)染后的干擾效果,連接態(tài)NEDD8表達(dá)下調(diào)明顯。 (4)連接態(tài)NEDD8對M14細(xì)胞生物學(xué)行為的影響 經(jīng)細(xì)胞增殖、細(xì)胞周期及細(xì)胞侵襲實驗的觀察發(fā)現(xiàn),干擾連接態(tài)NEDD8的形成,使M14細(xì)胞的在體外的增殖能力明顯受到抑制,發(fā)生了G1期阻滯,且細(xì)胞的侵襲能力也發(fā)生了明顯的下降。體內(nèi)成瘤實驗結(jié)果也證實,接種轉(zhuǎn)染后的細(xì)胞,瘤體在裸鼠中的增長速度和程度明顯受到抑制。 結(jié)論 NEDD8與cullin蛋白的連接可能是黑素瘤細(xì)胞無序增殖和遠(yuǎn)處轉(zhuǎn)移的原因之一。在黑素瘤細(xì)胞及組織中,連接態(tài)的NEDD8明顯上調(diào),阻斷NEDD8與cullin蛋白的連接會明顯降低M14細(xì)胞的增殖及侵襲能力,并且會對其細(xì)胞周期的進(jìn)程產(chǎn)生影響。
[Abstract]:Melanoma is a malignant tumor with the highest fatality rate in dermatology. It has strong cell proliferation ability and is not easily interfered by chemotherapeutic drugs. It is easy to metastasize and has poor prognosis. At present, the mechanism of melanoma is still unclear. Connective NEDD8 is associated with the occurrence of many kinds of malignant tumors, and the relationship with melanoma has not been reported. The purpose of this study was to investigate the role of connective NEDD8 in the development of melanoma. (1) the expression of connective NEDD8 in melanoma tissues and cells was detected by western blot. Compared with adjacent tissues and nevus tissues, the expression of connective NEDD8 in skin melanoma tissues was significantly up-regulated. Compared with normal human melanocytes, the expression of connective NEDD8 in melanoma cells was significantly up-regulated. (2) the expression of NEDD8,APP-BP1,UBA3,UBC12 and UCH-L3 in melanoma cell line was detected by real-time PCR technique. The results showed that the expression of these proteins closely related to the formation of connective NEDD8 in melanoma cell lines was significantly higher than that in normal human melanocytes. (3) Construction of shRNA-UBA3 recombinant plasmid and cell transfection construction of shRNA-UBA3 and pRNAT-U6.2/Lenti recombinant plasmid vector, transfection of M14 cell line, monoclonal screening of stable expression of shRNA-UBA3 cell line; The interference effect of transfection was confirmed by immunocytochemistry and Western blotting, and the expression of connective NEDD8 was down-regulated. (4) the effect of connective NEDD8 on the biological behavior of M14 cells was observed by cell proliferation, cell cycle and cell invasion experiments. It was found that the ability of M14 cells to proliferate in vitro was significantly inhibited by interfering with the formation of connected NEDD8. G 1 arrest occurred and the invasiveness of the cells decreased significantly. The results of in vivo tumorigenesis also confirmed that the growth rate and degree of tumor in nude mice were significantly inhibited by inoculation of transfected cells. Conclusion the connection between NEDD8 and cullin protein may be one of the reasons for the disorder proliferation and distant metastasis of melanoma cells. In melanoma cells and tissues, connective NEDD8 was up-regulated. Blocking the connection between NEDD8 and cullin protein significantly reduced the proliferation and invasion ability of M14 cells, and affected the cell cycle progression of M14 cells.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R739.5

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 彭再梅;王惠芳;山長婷;;肺部良惡性病變組織中泛素和cullin-1表達(dá)及臨床病理意義(英文)[J];中南大學(xué)學(xué)報(醫(yī)學(xué)版);2009年03期

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本文編號：2346265