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目的:研究腺病毒携带CD20启动子调控的TRAIL基因特异性杀伤B-NHL细胞的作用。方法:人工合成含有CD20启动子片段-分泌信号-异亮氨酸拉链-s TRAIL(aa114~aa281)-真核poly(A)序列的融合基因,装入腺病毒载体Ad Easy系统,包装出携带P20-st TRAIL序列的腺病毒感染细胞,采用荧光素酶报告基因法研究CD20启动子在CD20+细胞系中的特异性转录,Western blotting验证TRAIL蛋白在细胞内的特异性表达,体外MTT法检测TRAIL特异性抑制CD20+细胞生长的作用。结果:成功构建携带P20-st TRAIL序列的腺病毒载体并分别感染CD20阳性和阴性淋巴瘤细胞系后发现,CD20启动子仅在CD20+的BJAB淋巴瘤细胞中具有转录活性、启动s TRAIL表达并在体外形成具有生物学功能的同源三聚体结构;在细胞中和培养上清中均检测到st TRAIL在mRNA和蛋白水平的表达,但在CD20-细胞系中则无明显表达。体外功能实验显示,BJAB细胞在被Ad P20-st TRAIL感染后自身生长受到明显抑制,而Ad P20-st TRAIL感染的CD20阴性细胞以及空白载体感染的细胞未见生长抑制现象。结论:CD20启动子可以特异性调控腺病毒携带的治疗基因st TRAIL的表达,实现TRAIL对CD20+B-NHL细胞的靶向杀伤。
Objective: To study the effect of adenovirus carrying CDAIL promoter on TRAIL gene-specific killing of B-NHL cells. Methods: The fusion gene containing CD20 promoter fragment, secreted signal-isoleucine zipper-s TRAIL (aa114 ~ aa281) - eukaryotic poly (A) sequence was synthesized and packaged into the adenovirus vector Ad Easy system P20-st TRAIL sequence was used to infect cells. The transcription of CD20 promoter in CD20 + cell line was detected by luciferase reporter assay. The specific expression of TRAIL protein in cells was detected by Western blotting. The expression of TRAIL Specific inhibition of CD20 + cell growth. RESULTS: The successful construction of the adenovirus vector carrying the P20-st TRAIL sequence and the subsequent infection of CD20-positive and -negative lymphoma cell lines revealed that the CD20 promoter was transcriptionally active only in CD20 + BJAB lymphoma cells, initiating the expression of s TRAIL and in The biotin-like structure was found in vitro. The expression of st TRAIL mRNA and protein was detected both in the cell and in the culture supernatant, but not in the CD20-cell line. In vitro functional experiments showed that the growth of BJAB cells was significantly inhibited after Ad P20-st TRAIL infection, but no growth inhibition was observed in cells infected with Ad P20-st TRAIL infected CD20-negative cells and blank vector. Conclusion: CD20 promoter can specifically regulate the expression of st TRAIL, a therapeutic gene carried by adenovirus, and achieve targeted killing of TRAIL on CD20 + B-NHL cells.