ASTL抗体中和ASTL对人宫颈癌ME-180细胞放射敏感性的影响

目的 探讨龙虾肽酶样金属内肽酶（ASTL）抗体中和ASTL对人宫颈癌ME-180细胞放射敏感性的影响。 方法 培养人宫颈癌ME-180、HeLa细胞，根据细胞处理方法的不同，按以下方式进行分组。（1）将ME-180细胞分为4组:对照组、照射组（4 Gy）、ASTL抗体组、ASTL抗体+照射组（4 Gy），采用5-乙炔基-2′脱氧尿嘧啶核苷（EdU）染色、3-(4，5-二甲基噻唑-2)-2，5-二苯基四氮唑溴盐（MTT）实验检测每组细胞的增殖能力与存活情况，并于照射后0、24、48、72 h采用免疫荧光、Western blot实验检测ASTL在细胞中的定位情况。（2）将ME-180细胞分为2组:照射组、ASTL抗体+照射组，分别进行0、2、4、8 Gy照射，并采用克隆形成实验检测每组细胞的增殖能力。（3）将HeLa细胞分为2组:照射组、ASTL抗体+照射组，分别进行0、2、4、8 Gy照射，采用MTT实验检测细胞的存活情况。（4）将ME-180细胞分为2组:短发夹RNA对照组（sh-对照组）、短发夹RNA ASTL转染组（sh-ASTL组），采用实时荧光定量聚合酶链反应（qRT-PCR）、Western blot实验检测蛋白激酶B（AKT）等靶基因的表达情况；同时，分别用0、5、10 nmoL/L的抗体处理ME-180细胞，采用Western blot实验检测抗体中和对靶基因表达情况的影响。（5）将ME-180细胞分为2组:对照组、照射组（4 Gy），采用Western blot实验检测ASTL、AKT等靶基因的表达情况。以上照射均使用137Cs γ射线照射源，剂量率为1 Gy/min。组间比较采用独立样本 t检验。 结果 （1）EdU染色实验结果显示，与照射组相比，ASTL抗体+照射组（4 Gy）抑制了ME-180细胞的增殖，差异有统计学意义（t=9.25，P<0.05）；MTT实验结果显示，与照射组相比，ASTL抗体+照射组（4 Gy）在照射后24、48、72 h时，ME-180细胞生长速度明显降低，且差异均有统计学意义（t=5.17、10.32、14.27，均P<0.05)。免疫荧光、Western blot实验结果显示，4 Gy照射后24 h时，ME-180细胞中ASTL在细胞质的定位显著增加，照射后48~72 h，ASTL重新定位于细胞膜上。（2）克隆形成实验结果显示，与照射组相比，ASTL抗体+照射组能够抑制8 Gy照射后ME-180细胞的增殖，且差异有统计学意义（t=7.63，P<0.05）。（3）HeLa细胞MTT实验结果显示，与照射组相比，ASTL抗体+照射组能够降低2、4、8 Gy照射后HeLa细胞的存活率，且差异均有统计学意义（t=4.27、9.66、15.71，均P<0.05）。（4）qRT-PCR实验结果显示，ASTL干扰片段转入后，AKT的表达水平下调（t=13.94，P<0.05），同时，Western blot实验结果表明，ASTL干扰或加入ASTL抗体能够降低AKT等靶基因的表达水平。（5）Western blot实验结果显示，与对照组相比，照射组（4 Gy）ASTL、AKT等靶基因的表达水平显著升高。 结论 人宫颈癌ME-180细胞的放射敏感性与ASTL的水平相关，ASTL抗体或sh-ASTL能够增强照射后ME-180细胞的放射敏感性。

Effect of neutralizing ASTL with ASTL antibody on radiosensitivity of human cervical carcinoma ME-180 cells

Objective To investigate the effect of the antibody neutralization of astacin-like metalloendopeptidase (ASTL) on the radiosensitivity of human cervical carcinoma ME-180 cells. Methods ME-180 cells and HeLa cells were grouped as follows in accordance with different experimental treatments: (1) ME-180 cells were divided into the control group, irradiation (4 Gy) group, ASTL antibody group, and ASTL antibody + irradiation (4 Gy) group. The number and proliferative capacity of viable cells were detected via 5-ethynyl-2'deoxyuridine nucleoside (EdU) staining and 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) assay. Immunofluorescence and Western blot analyses were used to detect the localization of ASTL in the cells at 0, 24, 48, and 72 h after irradiation. (2) ME-180 cells were divided into two groups: the irradiation group and the ASTL antibody + irradiation group irradiated with 0, 2, 4, and 8 Gy γ-rays. The proliferative capacity of the cells was detected through the clone formation assay. (3) HeLa cells were divided into two groups: the irradiation group and the ASTL antibody + irradiation group irradiated with 0, 2, 4, and 8 Gy γ-rays. MTT assay was used to detect the proliferative capacity of the cells. (4) ME-180 cells were divided into two groups: the sh-control group and the sh-ASTL transfection group. Protein kinase B expression levels were analyzed through quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analyses; at the same time, ME-180 cells were treated with 0, 5, 10 nmoL/L antibodies respectively, and the effect of antibody neutralization on target gene expression was detected by western blot test. (5) ME-180 cells were divided into two groups: the control group and the irradiation (4 Gy) group. Western blot analysis was used to detect ASTL and AKT expression.For the relative experiments, the cells were treated with 137Cs γ-ray irradiation at the dose rate of 1 Gy/min. Independent samples t-test was used for comparison between two groups. Results (1) The results of the EdU staining experiments showed that neutralizing ASTL significantly inhibited the proliferation of ME-180 cells after irradiation (t=9.25, P<0.05). The results of the MTT assay validated that the proliferative capacity of the ASTL antibody + irradiation group (4 Gy) was significantly (t=5.17, 10.32, 14.27; all P<0.05) reduced compared with that of the irradiation group after 24, 48, and 72 h of irradiation. The results of immunofluorescence and Western blot analyses revealed that the cytoplasmic localization of ASTL in ME-180 cells was significantly enhanced after 24 h of 4 Gy irradiation, and ASTL was relocalized on the cell membrane after 48–72 h of irradiation. (2) The results of the clone formation assay demonstrated that ASTL antibodies could significantly reduce the survival rate of ME-180 cells after 8 Gy irradiation (t=7.63, P<0.05). (3) The results of the MTT assay showed that ASTL antibodies could significantly reduce the survival rates of HeLa cells after 2, 4, and 8 Gy irradiation (t=4.27, 9.66, 15.71; all P<0.05). (4) qRT-PCR analysis revealed that the expression level of AKT was significantly (t=13.94, P<0.05) down-regulated after ASTL interference, whereas the results of Western blot analysis showed that ASTL interference or ASTL antibody addition could reduce the protein expression levels of AKT and its target genes. (5) The results of Western blot analysis demonstrated that in ME-180 cells, the levels of the ASTL protein and AKT and its target genes significantly increased after 4 Gy irradiation. Conclusion The radiosensitivity of human cervical cancer ME-180 cells was correlated with the level of ASTL, and ASTL antibody or sh-ASTL could enhance the radiosensitivity of ME-180 cells after irradiation.