survivin反义寡核苷酸对K562细胞诱导分化的研究

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。

Differentiation of K562 cells induced by antisense oligonucleotide targeting survivin

OBJECTIVE: Gene therapy of leukemia is a new and effective method. It is known to all that the pathogenesis and development of leukemia are related to a variety of genes. Survivin is a member of inhibitors of apoptotic proteins (IAP). Its cDNA was cloned from target cell protease receptor-1 (EPR-1). It is expressed in common tumors, but there is no expression in normal and mature tissues. High expression of survivin was detected in leukemic cells. The present study was conducted to examine the role of survivin in the differentiation of leukemic cells by using antisense-oligonucleotides. METHODS: Human leukemic cell K562 was used as the model for the study. K562 cells were divided into 4 groups randomly: antisense oligonucleuotide (ASON) group, nonsense oligonucleotide (NSON) group, lipofectin group and control group. There were 5 samples in each group, and the experiment was repeated for three times. ASON was designed with the reference to targeting survivin mRNA. K562 cells were cultured in RPMI1640 contained fetal cattle serum at a concentration of about 10 percent. Cell transfection was induced by lipofectin. Forty-eight hours after thansfection, the morphology and ultrastructure were observed. Twenty-four hours and 48 hours after thansfection, the function of K562 cells was detected by benzidine staining, POX staining and NBT staining, respectively. The mean fluorescence intensity of CD33 was determined by flow cytometry. The method of immunohistochemistry was used to examine the protein level of survivin. RESULTS: After thansfection with ASON, the size of K562 cells was reduced, but the cytoplasm was increased. The metarubricyte, segment granulocyte, apoptotic cells could be found. Morphologically and ultrastructurally, erythroid and myelocytic differentiation was observed. The positive level of benzidine staining in ASON group (11.90 +/- 2.30 at 24 h and 18.20 +/- 2.93 at 48 h) was higher than that of NSON group, lipofectin group and control group, respectively. The positive level of POX staining in ASON group (17.40 +/- 3.54 at 24 h and 29.40 +/- 3.70 at 48 h) was also higher than that of any other groups. The positive level of NBT staining in ASON group (7.50 +/- 2.26 at 24 h and 12.10 +/- 2.63 at 48 h) was significantly higher than that of NSON group, lipofectin group and control group, respectively (P < 0.01). In ASON group, the mean fluorescence intensity of CD33 (21.43 +/- 1.61 at 24 h and 14.86 +/- 1.20 at 48 h) was significantly lower than that of any other groups (P < 0.01). After thansfection for 24 h, the protein level of survivin in ASON group was decreased significantly compared to that of control group. There was no difference in survivin protein level amongst ASON group, NSON group and lipofectin group at 24 h (P > 0.05). Forty-eight hours after thansfection, the protein level of survivin was decreased significantly. CONCLUSIONS: ASON targeting survivin can induce K562 to erythroid and myelocytic differentiation. Survivin is related to differentiation of K562 cells, and it can be a target of gene therapy for leukemia.