bcl-2反义寡核苷酸联合三氧化二砷对膀胱癌细胞生长的抑制作用

The co-inhibitory effect of bcl-2 antisense oligonucleotides and As2O3 on bladder cancer cell line

目的观察bcl-2反义寡核苷酸（antisense oligonucleotides，ASODN）与三氧化二砷（As2O3）对膀胱癌细胞生长的抑制作用，比较联合或单独用药的抑制效果。方法人工合成包含bcl-2翻译起始位点的ASODN及无义核酸（NSODN），以质脂体（Lipofectamine）作为载体转染T-24膀胱癌细胞株。分为对照组（加PBS）、反义组（ASODN 1μmoL/L）、无义组（NSODN 1μmoL/L）、As2O3组（2μmoL／L）、As2O3＋反义组（联合用药组：As2O3 1μmol／L，ASODN0．5μmol／L）。采用肿瘤细胞生长曲线、四氮唑蓝（MTT）实验和集落形成实验，观察药物对肿瘤细胞生长的抑制作用，流式细胞仪检测肿瘤细胞的凋亡情况。结果从细胞生长曲线看出，与对照组相比，联合用药组肿瘤细胞数减少了62．09％，反义组减少了31．04％，As2O3组减少了39．08％，无义组减少了21．84％。MTT实验，肿瘤细胞抑制率联合用药组为（86．11±7．35）％，As2O3组为（67．21±8．99）％，反义组为（50．23±5.46）％，无义组为（8．22±0．33）％，组间比较差异有统计学意义（F=180．2，P〈0．01）。半数抑制浓度（IC50）联合用药组为（0．98±0．12）μmol／L，As2O3组为（1．53±0．32）μmol／L。反义组的为（1．96±0．22）μmol／L，无义组为（4．33±0．33）μmol／L，组间比较差异有统计学意义（F=354.0，P〈0．01）。肿瘤细胞集落生存率联合用药组为53．76％，反义组为76．32％，As2O3组为75．25％，无义组为90．33％。肿瘤细胞凋亡率联合用药组为（22．66±1．66）％，组间比较均差异有统计学意义（F=232．15，P〈0．01）。结论Bcl-2反义寡核苷酸与As2O3联合应用可明显提高对膀胱癌细胞生长的抑制作用。

Objective To investigate the co-inhibitory effect of As2O3 and antisense oligonucleotides （ASODN） on bladder cancer cell line. Methods The artificially synthesized ASODN and non-antisense oligonucleotides （NSODN） including the translation initiation site were transfected into bladder cancer cell by Lipofectamine. Then medications were given to the cell line： in control group, only PBS was added to; in ASODN group, the final concentration of ASODN was 1 μmol/L; in nonsence group, the final concentration of NSODN was 1 μmol/L; in As2O3 group, the final concentration was 2 μmol/L; in As2O3 ＋ ASODN（semi-quantity combination） group, the final concentration of As2O3 and ASODN was 1 μmol/L and 0.5 μmol/L respectively. Cell growth was observed by tumor cell growth chart, MTT assay, and eolonogenie assay. Tumor apoptosis was detected by flow cytometry. Results Compared with the control group, the cell population of combined medication decreased by 62.09% while that of ASODN, As2O3 and NSODN group decreased by 31.04%, 39.08% and 21.84%, respectively. MTT assay identified that the inhibition ratio of combined, As2O3, ASODN and NSODN group was（86.11 ± 7.35）%, （67.21 ±8.99）%, （50.23 ± 5.46）%, （8.22 ±0.33）% respectively, the difference being statistical between each group（F = 180.20,P 〈 0.01 ）. The IC50 of combined group was （0.98 ± 0.12）μmol/L, while that of As2O3, ASODN and NSODN group was （1.53 ± 0.32）μmol/L, （1.96± 0.22） μmol/L and （4.33± 0.33）μmol/L respectively. Colonogenic assay found out that the colony survival rate of the combined group, the ASODN, As2O3 and NSODN group was 53.76%, 76.32%, 75.25% and 90.33% respectively. Flow eytometry detection revealed that the apoptosis rate of combined group was （22.66 ±1.66）% ,there was a significant difference between the combined group and the two（F= 232.15,P 〈 0.01 ）. Conclusion The combination of antisense oligonucleotides and As2O3 has a stronger inhibitory effect on bladder cancer than either one used alone.