摘要
目的研究钾通道阻断剂四氨基吡啶(4-amino-pynidine,4-AP)对宫颈癌HeLa细胞中子照射的影响。方法宫颈癌HeLa细胞分别设不同浓度(0、1、5、10、20mmol/L)4-AP组,0.67Gy252Cf中子照射后48h,用MTT法和流式细胞仪检测各组细胞活性、凋亡率。0、20mmol/L4-AP组自照射后24、48、72h分别用RT-PCR法、Western blot法检测HIF-1αmRNA及蛋白表达情况。结果在中子照射48h后,4-AP浓度为0、1、5、10、20mmol/L时,对HeLa细胞增殖的抑制率分别为0、(38.81±3.45)%、(46.63±3.88)%、(63.58±6.19)%、(77.51±8.87)%,凋亡率(%)分别为(3.15±0.85)%、(8.01±1.19)%、(16.00±1.58)%、(47.20±3.18)%、(62.56±4.27)%,差异有统计学意义(P<0.05)。说明4-AP能抑制HeLa细胞的增殖,诱导凋亡,并呈现明显的剂量依赖性。当4-AP浓度大于5mmol/L时,其作用明显(P<0.01)。0.67Gy252Cf中子照射0、24、48、72h后,4-AP浓度为0mmol/L组,HIF-1αmRNA/β-actin值分别为(0.423±0.116)、(0.464±0.111)、(0.487±0.121)、(0.415±0.099),Western blot灰度扫描结果HIF-1α/β-actin分别为(0.723±0.116)、(0.664±0.111)、(0.617±0.121)、(0.515±0.099),HIF-1αmRNA及蛋白表达均无明显降低,差异均无统计学意义(P>0.05)。4-AP浓度为20mmol/L组,HIF-1αmRNA/β-actin值分别为(0.401±0.121)、(0.364±0.142)、(0.257±0.137)、(0.165±0.099),Western blot灰度扫描结果HIF-1αmRNA/β-actin分别为(0.879±0.117)、(0.645±0.115)、(0.312±0.114)、(0.130±0.118),HIF-1αmRNA及蛋白表达均明显降低,差异有统计学意义(P<0.01)。结论钾通道阻断剂4-AP能降低中子放射治疗后缺氧诱导因子(HIF-1α)表达,降低HeLa细胞增殖,促进凋亡,提高HeLa细胞中子射线的敏感性。
Objective To investigate the potential effect of 4-amino-pynidine (4-AP) on human cervical carcinoma cell line HeLa to ^252 Cf neutron ray. Methods HeLa cells were treated with 4-AP(0, 1,5, 10, 20 mmol/L, respectively) and then exposed to 0.67 Gy ^252 Cf neutron ray. The effect of 4-AP on proliferation of HeLa ceils was assessed by MTT assay. Cell apoptosis were detected by flow cytometry. Reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were used to investigate the levels of HIF-1 a mRNA and protein of HeLa cells treated with 4-AP (0, 20 mmol/L) and 0.67 Gy ^252 Cf neutron ray. Results After HeLa cells treated with 0, 1, 5, 10, 20 mmol/L 4-AP were exposed to 0.67 Gy ^252 Cf neutron ray for 48 h, their inhibitory rate was 0, (38.81±3.45)%, (46.63±3.88)%, (63.58±6.19)%, (77.51±8.87)%, and their apoptotic rate was (3.15 ±0.85)%, (8.01±l. 19)%, (16.00 ±1.58)%, (47.20 ±3.18)%, (62. 56 ± 4.27 ) %, respectively, suggesting 4-AP may inhibit the proliferation of HeLa cells and induce their apoptosis in a dose-dependent manner ( P 〈 0. 05 ). Of HeLa cells treated with 0 mmol/L 4-AP, the optical density ratio of HIF-la mRNA/β-actin was (0.423 ±0. 116), (0.464 ±0. 111), (0.487±0. 121), (0.415 ±0.099) after 24-, 48- and 72-hour exposure to 0.67 Gy ^252 Cf neutron ray, while the optical density ratio of HIF-la protein/β-actin was (0. 723 ±0. 116), (0. 664 ±0. 111 ), (0. 617±0. 121 ), (0. 515 ±0. 099) ( both P 〉 0. 05 ). However, after treated with 20 mmol/L 4-AP and exposed to 0.67 Gy ^252 Cf neutron ray for 24, 48 and 72 h, the optical density ratio of HIF-la mRNA/β-actin of HeLa cells was (0. 401 ± 0. 121 ), (0. 364 ±0. 142), (0. 257 ±0. 137), (0. 165±0. 099), while the optical density ratio of HIF-lct protain/[5- actin were (0.879 ±-0. 117), (0.645 ±0. 115), (0. 312 ±0. 114), (0. 130 ±0. 118), showing that the lev- els of HIF-1 a mRNA and protein both declined (P 〈 0. 01 ). Gonelusion To HeLa cells exposed to ^252 Cf neutron ray, potassium channel blocker (4-AP) pretreatment inhibits their HIF-la expression and their prolifera- tion and promote their apoptosis. The potassium channel blocker (4-AP) significantly enhances the sensitivity of HeLa cells to neutron radiotherapy.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2008年第10期953-956,共4页
Journal of Third Military Medical University