抗PML-RAR_α反义核酸的设计、合成和对NB4细胞生长、凋亡的影响  

Design and synthesis of antiPMLRAR antisense and its effects on the growth and apoptosis of NB4 cells

作　　者：陈烨[1,2] 缪金明[1,2] 朱学宏[1,2] 邵念贤[1,2] 方智雯[1,2] 欧阳仁荣[1,2] 

机构地区：[1]上海第二医科大学附属仁济医院白血病实验室 [2]上海血液学研究所

出　　处：《中华血液学杂志》1999年第6期303-306,共4页Chinese Journal of Hematology

基　　金：国家自然科学基金

摘　　要：目的合成针对长型ＰＭＬＲＡＲα融合基因ｍＲＮＡ起始部位和融合位点的反义核酸（ＡＳ），探讨ＡＳ的稳定性、特异性及其对ＮＢ４细胞生长、凋亡的影响。方法以ＮＢ４细胞株为研究对象，采用台盼蓝拒染法进行细胞计数；聚丙烯酰胺凝胶进行寡核苷酸的电泳；流式细胞仪、ＤＮＡ电泳、细胞荧光染色进行细胞凋亡检测。结果合成的ＡＳ稳定、耐细胞内外核酸酶、无非特异性作用；起始部位ＡＳ（ＳＴＡＳ）和融合位点ＡＳ（ＦＵＡＳ）明显抑制细胞增殖，且呈剂量依赖性。浓度２０μｇ／ｍｌ时增殖抑制率０％～１１．３％；８０μｇ／ｍｌ时，抑制率５０．０％～６７．７％。ＦＵＡＳ（６０μｇ／ｍｌ）处理第７天、第９天出现明显的凋亡细胞群，处理后３天、５天、７天、９天的凋亡细胞率分别为９．３％、２４．５％、４１．０％、３４．２％。结论针对长型ＰＭＬＲＡＲα融合基因的ＡＳ设计、合成是成功的；抗ＰＭＬＲＡＲαＡＳ能抑制细胞增殖，诱导细胞凋亡。Objective To synthesize antisense oligodeoxynucleotides (AS) targeting the fusion point region (FUAS) and the start codon region (STAS) of the long type PMLRAR mRNA and investigate its stability, specificity and effects on the growth and apoptosis of NB4 cells. Methods NB4 cell apoptosis was assayed by flow cytometry, cellfluorescence and DNA gel electrophoresis. Trypan blue exclusion was used for cell counts, and polyacrylamide gel electrophoresis for oligodeoxynucleotides. Results The synthesized 18bp phosphorothioate oligodeoxynucleotides was stable, resistant to nuclease, and no unspecific effects. Both STAS and FUAS could inhibit the NB4 cell growth in a dosedependent manner. Low concentration (20g/ml) of STAS or FUAS resulted in growth inhibition of 0%11.3%, and the highest inhibition was found at concentration of 80g/ml (inhibition rate 50.0%67.7%). Cell DNA content analyzed by flow cytometry showed the typical profile of apoptotic cells at 7th day, 9th day of treatment with FUAS. Percentages of apoptotic cells in FUAStreated cells was 9.3%, 24.5%, 41.0% and 34.2% after 3,5,7 and 9 days of FUAS treatment, respectively. Conclusion STAS and FUAS successfully synthesized and both of them could inhibit the growth and induce the cell apoptosis of NB4 cells.

关 键 词：PML-RARΑ NB4细胞系 细胞 反义核酸 白血病 

分 类 号：R979.19[医药卫生—药品] R733.7[医药卫生—药学]