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目的采用反义寡核苷酸技术研究survivin与白血病细胞K562分化的关系。方法实验分为反义寡核苷酸组、无义寡核苷酸组、脂质体组、空白对照组,转染后48h观察细胞形态及超微结构的变化;转染后24、48h分别进行联苯胺染色、过氧化物酶染色及硝基四氮唑蓝(NBT)还原实验检测细胞功能;流式细胞仪检测细胞表面分化抗原CD33的表达;免疫组织化学方法检测survivin蛋白水平。结果反义寡核苷酸作用后,K562细胞出现向成熟红系及粒系分化的形态学及超微结构的改变,联苯胺染色阳性率、过氧化物酶染色阳性率、NBT还原实验阳性率均高于无义寡核苷酸组、脂质体组及空白对照组(P<0.01);细胞表面分化抗原CD33的平均荧光强度在反义寡核苷酸组降低(P<0.01);反义寡核苷酸作用24h后细胞内survivin蛋白的表达水平低于空白对照组(P<0.05),但与无义寡核苷酸组、脂质体组比较差异无统计学意义(P>0.05),而48h后survivin蛋白表达水平进一步降低,与其他各组比较差异有统计学意义(P<0.05)。结论survivin反义寡核苷酸能诱导K562细胞向成熟红系及粒系方向分化。
Objective To study the relationship between survivin and K562 differentiation in leukemia cells by using antisense oligonucleotide technique. Methods The experiment was divided into antisense oligonucleotide group, nonsense oligonucleotide group, liposome group and blank control group. The morphological and ultrastructural changes of cells were observed 48 h after transfection. At 24 h and 48 h after transfection, The cell function was detected by benzidine staining, peroxidase staining and nitroblue tetrazolium blue (NBT) reduction assay. The expression of cell surface differentiation antigen CD33 was detected by flow cytometry and the protein level of survivin was detected by immunohistochemistry. Results After antisense oligodeoxynucleotide treatment, the morphological and ultrastructural changes of erythroid and granulocytic differentiation appeared in K562 cells. The positive rate of benzidine staining, the positive rate of peroxidase staining and the positive rate of NBT reduction assay (P <0.01). The average fluorescence intensity of cell surface differentiation antigen CD33 was decreased in antisense oligonucleotide group (P <0.01), while that in non-sense oligonucleotide group, liposome group and blank control group was the highest The expression of survivin protein in the cells was lower than that in the blank control group (P <0.05) after treated with sense oligonucleotide for 24 h, but no significant difference compared with the non-sense oligonucleotide group and the liposome group (P> 0.05 ), While the expression of survivin protein decreased further after 48h, which was significantly different from other groups (P <0.05). Conclusion The survivin antisense oligonucleotide can induce K562 cells to differentiate into mature erythroid and granulocytic lines.