骨肉瘤MG-63细胞中Survivin的表达及其反义寡核苷酸对细胞增殖与凋亡的作用

The expression of survivin in osteosarcoma MG-63 cells and the effect of survivin antisense oligonucleotide on its proliferation and apoptosis

目的探讨Survivin在骨肉瘤细胞中的表达及Survivin反义寡核苷酸(ASODN)对骨肉瘤MG-63细胞增殖和凋亡的影响。方法设计针对Survivin的ASODN和正义核苷酸(SODN)序列,制备脂质体-寡核苷酸复合物并转染MG-63细胞,分为空脂质体(Lip)组、脂质体介导的SODN(Lip-SODN)组和脂质体介导的ASODN(Lip-ASODN)组。采用免疫细胞染色法检测MG-63细胞中Survivin的表达情况,Western blotting检测各组转染48h后的Survivin蛋白水平,MTT法检测转染24、48和72h后MG-63细胞的增殖情况,流式细胞仪检测转染48h后MG-63细胞的凋亡情况。结果 Survivin主要表达于MG-63细胞的胞核。Lip-ASODN组的Survivin蛋白水平均低于Lip-SODN组和Lip组。不同浓度Survivin ASODN对MG-63细胞的增殖抑制程度不同,Lip-ASODN组的细胞增殖抑制率高于Lip组,且呈浓度依赖性;而Lip-SODN组的增殖抑制率与Lip组的差异无统计学意义(P>0.05)。Lip-ASODN组的凋亡率为(88.47±0.79)%,高于Lip组的(10.01±0.90)%和Lip-SODN组的(4.12±0.25)%,差异有统计学意义(P<0.05)。结论靶向Survivin的ASODN可以显著抑制骨肉瘤MG-63细胞的增殖能力,并促进其凋亡。

Objective To investigate the expression of survivin in osteosarcoma MG-63 cells and the effect of survivin anti- sense oligonucleotide（ASODN） on its proliferation and apoptosis. Methods The ASODN and sense oligonucleotide（SODN） targeting survivin were designed and constructed. The liposome/oligonucleotide complexes were prepared by adding oligonucleotide and then transferred into MG-63 cells. According to the protocol, the MG-63 cells were assigned into Lip group （empty liposome） , Lip-SODN group （liposome-mediated SODN） and Lip-ASODN group （liposome-mediated ASODN）. The immunohistochemieal staining was used to detect the expression of survivin in MG-63 cells. Western blotting assay was employed to measure the protein levels of survivin at 48th hour after transfection of each group. The proliferation at 24th, 48th and 72nd hour and apoptosis at 48th hour after transfection were measured by MTT and flow cytometry, respectively. Results The survivin was mainly expressed in the nucleus of MG-63 cells. The protein level of Lip-ASODN group was lower than that of Lip group and Lip-SODN group. The Lip-ASODN group＇s proliferation inhibi- tion rate was superior to Lip group in a dose-dependent manner, while the difference between Lip-SODN group and Lip group was not significant（P〉0. 05）. The apoptotie rate of Lip-ASODN group was（ 88.47±0. 79） %, higher than（ 10.01±0. 90） % of Lip group and （4. 12±0. 25） % of Lip-SODN group with significant difference（P〈0. 05）. Conclusion The ASODN targeting survivin can inhibit the proliferative capacity and promote the apoptosis of osteosarcoma MG-63 cells.