抗hnRNP B1单克隆抗体-顺铂交联物对人肺腺癌细胞A549的体外靶向生长抑制作用

The Proliferation Inhibitory Effects of Cisplatin Cross-Linked Anti-hnRNP B1 Monoclonal Antibody on A549 Lung Cancer Cells

目的证实hnRNP B1单克隆抗体与顺铂(DDP)的交联物可与肺癌细胞发生特异性结合,并研究该交联物在体外的靶向抗癌作用。方法采用人肺腺癌细胞株A549常规体外培养传代。细胞分为DDP组、hnRNP B1单抗组、hnRNP B1单抗-DDP交联物组,分别加入DDP 3、5及7μg/mL,hnRNP B1单抗2.5、5及10μg/mL,交联物1.5、3及6μg/mL,培养2 h后,用培养基洗涤(仅对单抗组和交联物组进行洗涤),再分别作用12、24及48 h。以DDP组为阳性对照,同时设空白对照。每组设3个复孔。应用3H胸腺嘧啶(3H-TdR)掺入法在液闪仪上测定每分钟细胞计数(CPM),了解不同干预以及不同剂量、作用时间交联物对细胞增殖的影响;采用TUNEL技术观察细胞凋亡情况;应用流式细胞计数测定交联物对细胞周期分布的影响。结果DDP(7μg/mL)、hnRNP B1单抗(5μg/mL)、交联物(3μg/mL)作用48 h对A549细胞均有生长抑制作用,生长抑制率分别为49.7%、53.3%、59.2%。3μg/mL交联物组(30.4%、46.2%及59.2%)和6μg/mL交联物组(41.3%、52.5%及66.8%)作用12、24及48 h后的生长抑制率均强于同时间点7μg/mL DDP组(25.0%、41.3%及49.7%)。DDP(7μg/mL)、hnRNP B1单抗(5μg/mL)、交联物(3μg/mL)作用48 h后均可诱导A549肺癌细胞凋亡。A549细胞经交联物作用后,G0/G1期细胞增多(占53.9%),S期细胞减少(占43.2%),与DDP组(46.8%和47.1%)和单抗组(46.4%和46.3%)比较均有显著差异(P均<0.01)。结论hnRNP B1单克隆抗体与DDP的交联物可与肺癌细胞发生特异性结合,具有靶向抗癌作用。

Objective To observe the cell proliferation inhibitory effects of cisplatin （DDP） cross-linked anti-hnRNP B1 monoclonal antibody on human lung adenocarcinoma A549 cell lines. Methods The A549 cells was cultured in vitro and divided into four groups,ie, blank group, DDP group, anti-hnRNP BI antibody group and hnRNP BI antibody-DDP conjugate group. They were co-cuhured with DDP （ 3,5, 7 μg/mL） ,antibody（2. 5,5,10 μg/mL） and conjugate（ 1.5,3,6 μg/mL） respectively. The antibody group and the conjugate group were washed with culture medium after cultured for 2 hours, then cultured for another 12,24 and 48 hours. The use of ^3H thymidine （^3H-TdR） incorporation measured in liquid scintillation counter on the cell counts per minute （CPM） was to evaluate the inhibitory effect on cell proliferation with different interventions and different dose and time. The apoptosis was observed by TUNEL. And the cell cycle was determined by flow cytometry. Results The growth inhibition rates of the DDP （7 μg/mL）, antibody （5μg/mL） and conjugate（3 μg/mL） groups were 49. 7% ,53.3% and 59. 2% in 48 hours respectively. The growth inhibition rates in 12,24, 48 hours were 30. 4%, 46. 2%, 59. 2% in conjugate group at 3μg/mL,and 41.3% ,52. 5% ,66. 8% in conjugate group at 6 μg/mL, those were all significantly higher than those in DDP group at 7 μg/mL （25.0% ,41.3% ,49.7%）. All of DDP（7μg/mL）, antibody （5μg/mL） and conjugate（ 3μg/mL） could induce cell apoptosis in A549 cells. After conjugate treatment, the cells were blocked in G0/Gl phase which accounts for 53.9%, higher than DDP group （46. 8% ） and antibody group （46.4%） （P 〈 0. 01 ） , and decreased in S phase which accounts for 43.2% , lower than DDP group（47.1% ） and antibody group（46. 3% ） （ P 〈 0.01 ）. Conclusion DDP cross-linked anti-hnRNP B1 monoclonal antibody can conjugate to A549 ceils, and has target anticancer effects in vitro.