单抗偶联靶向超抗原SEA对人膀胱癌细胞株BIU-87的抑制作用及其机制

目的 研究单抗偶联靶向超抗原金黄色葡萄球菌肠毒素A(SEA)对人膀胱癌细胞株BIU-87的抑癌作用.方法 实验分对照组、SEA组及单抗靶向SEA组;以外周血单个核细胞为效应细胞,BIU-87为靶细胞,以不同效靶比接种细胞;对照组予全培养液,后2组分别予不同稀释浓度的SEA及单抗靶向SEA,以细胞计数kit-8(CCK-8)测定各组BIU-87细胞的增殖抑制率;荧光染色法观察凋亡形态学变化;流式细胞术检测凋亡率,Western印迹检测Bax、Bcl-2蛋白的表达;酶联免疫吸附试验(ELISA)法测定共培养上清白细胞介素(IL)2、肿瘤坏死因子(TNF)α、γ干扰素(IFN-γ)含量.结果 单抗靶向SEA组细胞增殖抑制率为[(31.2±2.6)%～(88.7±3.0)%],显著高于对照组[(5.8±3.0)%～(16.5±4.0)%]及SEA组[(20.7±2.1)%～(68.6±4.0)%](P＜0.05).实验组细胞均发生典型的凋亡形态学改变,其中单抗靶向SEA组凋亡率为[(14.6±0.7)%～(66.5±3.3)%],显著高于SEA组[(9.1±0.6)%～(29.9±1.3)%]及对照组[(4.6±0.7)%～(6.5±0.3)%](P＜0.05).实验组较对照组均有上调Bax/Bcl-2比值作用,其中单抗靶向SEA组为(3.29±0.34),显著高于SEA组(1.21±0.05)及对照组(0.45±0.15)(P＜0.05).单抗靶向SEA组12、24 h共培养上清中的IL-2、TNF-α、IFN-γ浓度与SEA组比较差异均无统计学意义(均P＞0.05).结论 单抗靶向SEA对膀胱肿瘤细胞显示出更强大的增殖抑制作用,这可能与其进一步上凋肿瘤细胞Bax/Bcl-2比值促进其凋亡有关.

Inhibitive effect and mechanism of monoclonal antibody targeted SEA on human bladder cancer cell line BIU-87

Objective To investigate the in vitro inhibitive effect and apoptotic mechanism of BDI-1 monoclonal antibody targeted SEA on human bladder cancer cell line BIU-87. Methods Human peripheral blood mononuclear cells (PBMC) and human bladder cancer cell line BIU-87 were used as effector and target cells respectively in three experimental groups, control group, SEA group and monoclonal antibody targeted SEA group, treated with different concentrations of SEA and targeted SEA respectively in the latter two. The inhibition rates of target cells were measured by cell counting kit-8 (CCK-8) assay. Apoptosis was determined by fluorescent Hocehst 33258 staining and flow cytometry (FCM). The expression of Bax, Bcl-2proteins and cytokine concentration of co-culture supernatants were detected by Western blot and ELISA respectively. Results Compared with those of control group (from 5.8% ± 3.0% to 16. 5% ± 4. 0% ) and SEA group ( from 20.7% ±2. 1% to 68.6% ±4.0% ), the inhibition rates in targeted SEA group increased significantly (from 3 1. 2% ± 2. 6% to 88.7% ± 3.0% ,P ＜ 0. 05 ). Characteristic changes of apoptosis were observed in both experimental treatment groups, especially in targeted SEA group. FCM also showed more prominent apoptosis induced in targeted SEA group (from 14.6% ± 0.7% to 66.5% ± 3.3% ),significantly higher than those of control group ( from 4. 6% ± 0. 7% to 6. 5% ± 0. 3% ) and SEA group (from 9. 1% ±0. 6% to 29. 9% ± 1.3% ,P ＜0.05 ). In addition, Western blot analysis indicated that the ratio of Bax/Bcl-2 significantly increased to (3. 29 ±0. 34) by targeted SEA treatment at an extremely low concentration, significantly higher than those of SEA group ( 1.21 ±0.05) and control group (0. 45 ±0. 15)( P ＜ 0. 05 ). While at the same time ELISA assay showed no significant difference of IL-2, TNF-α, IFN-γconcentrations between SEA and targeted SEA groups. Conclusion Monoclonal antibody targeted SEA significantly increases the inhibitive effect of BIU-87 cell line possibly through the up-regulation of Bax/Bcl-2 ratio to promote the cellular apoptosis.