人NRAGE基因对细胞生长与细胞迁移影响的实验研究

发布时间：2018-03-12 16:44

本文选题：MAGE-D1　切入点：NRAGE　出处：《南京师范大学》2006年硕士论文　论文类型：学位论文

【摘要】：人NRAGE基因对细胞生长与细胞迁移影响的实验研究 NRAGE(neurotrophin receptor p75-interacting MAGE homolog)，是一种神经营养蛋白受体相关的黑色素瘤抗原基因同源物，属于MAGE家族。它包含13个外显子，其中第2-12个外显子组成的开放阅读框编码出分子量约为86kDa的蛋白。一些报道显示NRAGE与细胞周期抑制及细胞凋亡相关。对于该基因在细胞内功能的研究尚处于初步阶段。我们从人胎盘cDNA文库中克隆而来的hNRAGE，与大鼠NRAGE基因同源率达89％，，它的过表达能抑制肝癌细胞的增殖。本论文利用重组腺病毒表达载体研究人NRAGE基因在体外和体内对于细胞生长及细胞迁移力的影响。结果如下：首先，用细菌内同源重组法成功构建了能在真核细胞内高效表达NRAGE基因的重组腺病毒Ad-myc-hNRAGE。并用免疫印记实验验证了该重组腺病毒的功能。其次，在体外的细胞周期检测中，利用重组腺病毒Ad-myc-hNRAGE或NRAGE真核表达质粒在HEK 293、U2 OS、B16-BL6细胞内过表达人NRAGE基因，细胞周期被阻滞在G1／G0和G2／M期，而S期细胞比率明显下降，表明细胞的生长与分裂受到明显抑制。利用C57BL／6J小鼠同源黑色素瘤细胞B16-BL6在该品系小鼠皮下能迅速成瘤的特性，我们对过表达NRAGE的B16-BL6细胞在体内的成瘤性进行检测。结果表明相对于对照腺病毒Ad-GFP感染的B16-BL6细胞，感染重组腺病毒Ad-myc-hNRAGE的B16-BL6在C57BL／6J小鼠体内成瘤速度明显下降。再次，小鼠肺高转移黑色素瘤细胞B16-BL6的体外愈伤实验及Transwell小室模拟浸润实验证实，利用重组腺病毒Ad-myc-hNRAGE在B16-BL6细胞中过表达NRAGE基因能够抑制其侵袭与迁移。尾静脉注射肺高转移黑色素瘤细胞B16-BL6至C57BL／6J小鼠体内的转移实验结果显示，重组腺病毒Ad-myc-hNRAGE感染的B16-BL6细胞在小鼠的肺和肝脏等器官形成的黑色素瘤转移瘤明显少于对照组。上述结果证明NRAGE基因的在体内外对细胞生长具有抑制作用，并且该基因的过表达能够有效抑制黑色素瘤细胞B16-BL6在体外的迁移以及在C57BL／6J
[Abstract]:Effects of human NRAGE gene on cell growth and cell migration. NRAGE(neurotrophin receptor p75-interacting MAGE homologue, a neurotrophic protein receptor-associated melanoma antigen gene homologue, belongs to the MAGE family. It contains 13 exons. The open reading frame composed of exons 2-12 encodes a protein with molecular weight of about 86kDa. Some reports have shown that NRAGE is related to cell cycle inhibition and apoptosis. The hNRAGE cloned from the human placental cDNA library has a homology of 89 with the rat NRAGE gene. Its overexpression can inhibit the proliferation of hepatoma cells. The effects of human NRAGE gene on cell growth and cell migration in vitro and in vivo were studied using recombinant adenovirus expression vector. The results are as follows:. Firstly, the recombinant adenovirus Ad-myc-hNRAGE, which can express NRAGE gene efficiently in eukaryotic cells, was successfully constructed by intra-bacterial homologous recombination, and the function of the recombinant adenovirus was verified by immunological imprinting. Secondly, in vitro cell cycle detection, the recombinant adenovirus Ad-myc-hNRAGE or NRAGE eukaryotic expression plasmid was used to overexpression the human NRAGE gene in HEK 293U2OS2OSB16-BL6 cells, and the cell cycle was blocked in the G1 / G0 and G2 / M phases, while the S phase cell ratio was significantly decreased. The results showed that the growth and division of the cells were obviously inhibited. Using C57BL / 6J murine melanoma cell line B16-BL6, it was found that B16-BL6 could rapidly develop tumor in this strain. We detected the tumorigenicity of B16-BL6 cells overexpression of NRAGE in vivo. The results showed that the tumorigenic rate of B16-BL6 infected with recombinant adenovirus Ad-myc-hNRAGE in C57BL / 6J mice was significantly lower than that of B16-BL6 cells infected with control adenovirus Ad-GFP. Thirdly, the in vitro callus test and Transwell chamber simulated infiltration test of mouse lung metastatic melanoma cell B16-BL6 were confirmed. Overexpression of NRAGE gene in B16-BL6 cells by recombinant adenovirus Ad-myc-hNRAGE could inhibit its invasion and migration. B16-BL6 cells infected with recombinant adenovirus Ad-myc-hNRAGE had fewer melanoma metastases in mice such as lung and liver. These results suggest that NRAGE gene can inhibit cell growth in vitro and in vivo, and overexpression of the gene can effectively inhibit the migration of melanoma cell B16-BL6 in vitro and C57BL / 6J.
【学位授予单位】：南京师范大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346

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