表达反义N-myc逆转录病毒载体对神经生长因子诱导神经母细胞瘤细胞分化的影响

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。

Effects of Antisense N myc Gene on Nerve Growth Factor induced Differentiation of Neuroblastoma Cell Line

Objective The purpose of this study is to investigate the effects of antisense N myc gene transfection on nerve growth factor induced differentiation of neuroblastoma cell line. Methods Recombinant retroviral vector expressing antisense N myc gene was constructed. Using the Transfectam Reagent, the recombinant vector was transduced into the human neuroblastoma cell line, IMR 32/NGFR, which was previously transfected by nerve growth factor receptor gene. The transformant cell line expressing antisense N myc was established and studied by single chain RNA probe hybridization, immunocytochemistry, and nerve growth factor treatment to see whether nerve growth factor can induce differentiation in this transformant cell line and inhibition of N myc expression. The TUNEL technique and electromicroscopy were used to detect apoptosis of the tumor cells. Results The cell line transduced by antisense N myc expressed much less amount of N myc both on mRNA and protein levels. After nerve growth factor treatment, remarkable morphological differentiation appeared in these cells. Apoptosis was also enhanced in these cells. Conclusions Antisense N myc transfection could specifically inhibit the expression of N myc and promoted the nerve growth factor induced differentiation of neuroblastoma cell line. Transfection of antisense N myc could also enhance the process of apoptosis of the tumor cells.