重组神经生长因子受体基因在受体缺乏的神经母细胞瘤细胞系中的表达

神经母细胞瘤是儿童期的常见恶性肿瘤之一。临床上,半数以上神经母细胞瘤有明显的N-myc癌基因扩增。神经生长因子(NGF)可使某些神经母细胞瘤细胞分化成神经元样的细胞。但对有N-myc扩增的神经母细胞瘤则可能因NGF受体缺乏而无明显促分化反应。本研究运用重组DNA技术将人NGF受体基因重组到有N-myc扩增,且NGF受体表达很低的神经母细胞瘤系(IMR-32)中,经克隆化培养、筛选,建立了稳定的细胞系—IMR-32/NGFR和对照细胞系IMR-32/NEO。经用抗NGFR的单克隆抗体检测用Flow cytometry技术证实IMR-32/NGFR系中有明显的NGF受体表达,而其母系IMR-32和空病毒对照系IMR-32/NEO则无表达迹象,说明NGF受体表达在IMR-32/NGFR系中是特异的,并能同抗NGFR单克隆抗体特异结合。用Northern B10ting技术亦测得IMR-32/NGFR中NGFR的mRN A明显表达。而其母系IMR-32和对照系IMR-32/NEO则无明显表达。这说明IMR-32/NGFR系中NGFR在mRNA水平上亦是特异的。N-myc和K-ras癌基因在IMR-32、IMR-32/NEO、IMR-32/NGFR三系中无明显变化。在NGF处理后,形态上三系均无明显的分化迹象。但IMR-32/NGFR在神经原纤维轻链的表达上轻度增高。c-fos癌基因的表达见于所有三个细胞系,IMR-32/NGFR略强些。这些说明,在恢复了NGFR基因和表达之后,对N-myc和K-ra

Expression of Recorobinant NGF Receptor in Human Neuroblastoma Cell Line without NGF Receptor

Neuroblastoma is one of the most common pediatric solid tumors. In approximately 50% of clinically advanced cases N-myc oncogene is amplified. Nerve gro-wth factor (NGF)is capable to induce differentiation of some neuroblastoma cell lines. But the cerl lines with amplified N-myc usually failed to respond to NGF. It may be due to the shortage of NGF receptors in these cell lines. The amplification of N-myc oncogene may also be responsible. We introduced the recombinant NGF receptor gene into the human neuroblastoma cell line (IMR-32),which was lack of NGF receptor and had N-myc amplification. The trans-formant line (IMR-32/NGFR) expressed NGF receptor both in mRNA and receptor protein levels. The parent line (IMR-32) and the control transformant line (IMR-32/NEO) , obtained by infection with empty viral vector were all negative for NGFR expression in mRNA and receptor protein levels as shown by Northern Blotting and Flow Cytometry using fluorescent labeled monoclonal antibodies against NGFR. After NGF treatment, transformant cell line (IMR-32/NGFR) did not show significant change in morphology. The expression of neurofilament light chain mRNA only increased slightly. No significant alteration was found in the expression of "N-myc and K-ras oncogenes among IMR-32, IMR-32/NEO and IMR-32/NGFR before and after NGF treatment. These results indicate the amplification of N-myc oncogene may still limit NGF responce in this cell line even after the restoration of NGF receptor.