Nedd化与皮肤黑素瘤发病机制的相关性研究

发布时间：2018-11-21 07:07

【摘要】：黑素瘤是皮肤科致死率最高的恶性肿瘤,细胞增殖能力强,且不易受化疗药物的干扰,易发生转移,预后不良。目前黑素瘤的发生机制仍不清楚。连接态NEDD8与多种恶性肿瘤的发生有相关性,与黑素瘤的关系尚未见研究报道。本研究旨在探讨连接态NEDD8在黑素瘤发生发展中的作用。 (1)连接态NEDD8在黑素瘤组织和细胞中的表达经western blot检测发现,与瘤旁组织和痣组织相比,连接态NEDD8在皮肤黑素瘤瘤组织中的表达明显上调。与正常人黑素细胞相比,连接态NEDD8在黑素瘤细胞株中的表达明显上调。 (2)催化连接态NEDD8形成的相关酶蛋白在黑素瘤细胞株中的表达通过real-time PCR技术检测NEDD8、APP-BP1、UBA3、UBC12以及UCH-L3在黑素瘤细胞株中的表达。结果显示与正常人黑素细胞相比,这些与连接态NEDD8的形成密切相关的蛋白在黑素瘤细胞株中的表达几乎均明显升高。 (3)shRNA-UBA3重组质粒的构建及细胞转染构建shRNA-UBA3与pRNAT-U6.2/Lenti的重组质粒载体,转染M14细胞株,单克隆筛选稳定表达shRNA-UBA3的细胞株,经免疫细胞化学法和蛋白印迹法证实转染后的干扰效果,连接态NEDD8表达下调明显。 (4)连接态NEDD8对M14细胞生物学行为的影响经细胞增殖、细胞周期及细胞侵袭实验的观察发现,干扰连接态NEDD8的形成,使M14细胞的在体外的增殖能力明显受到抑制,发生了G1期阻滞,且细胞的侵袭能力也发生了明显的下降。体内成瘤实验结果也证实,接种转染后的细胞,瘤体在裸鼠中的增长速度和程度明显受到抑制。结论 NEDD8与cullin蛋白的连接可能是黑素瘤细胞无序增殖和远处转移的原因之一。在黑素瘤细胞及组织中,连接态的NEDD8明显上调,阻断NEDD8与cullin蛋白的连接会明显降低M14细胞的增殖及侵袭能力,并且会对其细胞周期的进程产生影响。
[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.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R739.5

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