本文選題：MAGE-D1　切入點(diǎn)：NRAGE　出處：《南京師范大學(xué)》2006年碩士論文　論文類型：學(xué)位論文

【摘要】：人NRAGE基因?qū)?xì)胞生長(zhǎng)與細(xì)胞遷移影響的實(shí)驗(yàn)研究 NRAGE(neurotrophin receptor p75-interacting MAGE homolog)，是一種神經(jīng)營(yíng)養(yǎng)蛋白受體相關(guān)的黑色素瘤抗原基因同源物，屬于MAGE家族。它包含13個(gè)外顯子，其中第2-12個(gè)外顯子組成的開(kāi)放閱讀框編碼出分子量約為86kDa的蛋白。一些報(bào)道顯示NRAGE與細(xì)胞周期抑制及細(xì)胞凋亡相關(guān)。對(duì)于該基因在細(xì)胞內(nèi)功能的研究尚處于初步階段。我們從人胎盤(pán)cDNA文庫(kù)中克隆而來(lái)的hNRAGE，與大鼠NRAGE基因同源率達(dá)89％，，它的過(guò)表達(dá)能抑制肝癌細(xì)胞的增殖。 本論文利用重組腺病毒表達(dá)載體研究人NRAGE基因在體外和體內(nèi)對(duì)于細(xì)胞生長(zhǎng)及細(xì)胞遷移力的影響。結(jié)果如下： 首先，用細(xì)菌內(nèi)同源重組法成功構(gòu)建了能在真核細(xì)胞內(nèi)高效表達(dá)NRAGE基因的重組腺病毒Ad-myc-hNRAGE。并用免疫印記實(shí)驗(yàn)驗(yàn)證了該重組腺病毒的功能。 其次，在體外的細(xì)胞周期檢測(cè)中，利用重組腺病毒Ad-myc-hNRAGE或NRAGE真核表達(dá)質(zhì)粒在HEK 293、U2 OS、B16-BL6細(xì)胞內(nèi)過(guò)表達(dá)人NRAGE基因，細(xì)胞周期被阻滯在G1／G0和G2／M期，而S期細(xì)胞比率明顯下降，表明細(xì)胞的生長(zhǎng)與分裂受到明顯抑制。利用C57BL／6J小鼠同源黑色素瘤細(xì)胞B16-BL6在該品系小鼠皮下能迅速成瘤的特性，我們對(duì)過(guò)表達(dá)NRAGE的B16-BL6細(xì)胞在體內(nèi)的成瘤性進(jìn)行檢測(cè)。結(jié)果表明相對(duì)于對(duì)照腺病毒Ad-GFP感染的B16-BL6細(xì)胞，感染重組腺病毒Ad-myc-hNRAGE的B16-BL6在C57BL／6J小鼠體內(nèi)成瘤速度明顯下降。 再次，小鼠肺高轉(zhuǎn)移黑色素瘤細(xì)胞B16-BL6的體外愈傷實(shí)驗(yàn)及Transwell小室模擬浸潤(rùn)實(shí)驗(yàn)證實(shí)，利用重組腺病毒Ad-myc-hNRAGE在B16-BL6細(xì)胞中過(guò)表達(dá)NRAGE基因能夠抑制其侵襲與遷移。尾靜脈注射肺高轉(zhuǎn)移黑色素瘤細(xì)胞B16-BL6至C57BL／6J小鼠體內(nèi)的轉(zhuǎn)移實(shí)驗(yàn)結(jié)果顯示，重組腺病毒Ad-myc-hNRAGE感染的B16-BL6細(xì)胞在小鼠的肺和肝臟等器官形成的黑色素瘤轉(zhuǎn)移瘤明顯少于對(duì)照組。 上述結(jié)果證明NRAGE基因的在體內(nèi)外對(duì)細(xì)胞生長(zhǎng)具有抑制作用，并且該基因的過(guò)表達(dá)能夠有效抑制黑色素瘤細(xì)胞B16-BL6在體外的遷移以及在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.
【學(xué)位授予單位】：南京師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R346

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 張燕,劉夢(mèng)蕾,沈茜;可調(diào)控性鼠PLP-Ig嵌合體重組腺病毒載體的構(gòu)建和表達(dá)[J];細(xì)胞與分子免疫學(xué)雜志;2004年06期

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