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目的研究反义Ｎ－ｍｙｃ基因转染后人神经母细胞瘤细胞在神经生长因子处理后的诱导分化情况，探讨Ｎ－ｍｙｃ在神经生长因子诱导分化中的意义。方法用基因重组技术构建表达反义Ｎ－ｍｙｃ的逆转录病毒载体。用ＴｒａｎｓｆｅｃｔａｍＲｅａｇｅｎｔ等多种基因转染方法，转染已经基因转染恢复了神经生长因子受体表达的人神经母细胞瘤系，建成表达反义Ｎ－ｍｙｃ的转化细胞系。用神经生长因子处理转化细胞系，观察ＮＧＦ是否能诱导分化。同时用ＴＵＮＥＬ原位凋亡检测技术及超微结构技术研究表达反义Ｎ－ｍｙｃ的转化细胞系凋亡的情况。结果表达反义Ｎ－ｍｙｃ的转化细胞系经单链ＲＮＡ探针杂交，证实其Ｎ－ｍｙｃ的表达明显降低。用神经生长因子处理这些转化细胞系，细胞出现明显的形态学分化。ＴＵＮＥＬ技术及超微结构研究表明，经反义Ｎ－ｍｙｃ转染的细胞系细胞凋亡增多。结论反义Ｎ－ｍｙｃ基因转染能有效抑制Ｎ－ｍｙｃ的表达，进而促进神经生长因子诱导人神经母细胞瘤细胞的分化。反义Ｎ－ｍｙｃ基因转染能使神经母细胞瘤细胞凋亡增多。
Objective To investigate the differentiation of human neuroblastoma cells treated with antisense N-myc gene after nerve growth factor treatment and to explore the significance of N-myc in the differentiation of nerve growth factor. Methods The antisense N-myc retrovirus vector was constructed by gene recombination technology. Using TransfectamReagent and other gene transfection methods, transfected human neuroblastoma lines that have recovered the expression of the nerve growth factor receptor have been transfected and a transformed cell line expressing antisense N-myc was constructed. Transformed cell lines were treated with nerve growth factor to see if NGF could induce differentiation. At the same time, apoptosis of the transformed cell line expressing antisense N-myc was studied by TUNEL in situ apoptosis assay and ultrastructure technique. Results The transformed cell line expressing antisense N-myc was hybridized with a single-stranded RNA probe and confirmed that the expression of N-myc was significantly reduced. Treatment of these transformed cell lines with nerve growth factor resulted in distinct morphological differentiation of the cells. The TUNEL technique and ultrastructural studies have shown that apoptosis of cell lines transfected with antisense N-myc increases. Conclusion The transfection of antisense N-myc gene can effectively inhibit the expression of N-myc and further promote the differentiation of human neuroblastoma cells induced by NGF. Antisense N-myc gene transfection can increase the apoptosis of neuroblastoma cells.