芹菜素影响非小细胞肺癌A549细胞顺铂敏感性的RAD51基因调控机制研究  

Study on RAD51 Gene Regulatory Mechanism of Apigenin Affecting Cisplatin Sensitivity to NSCLC A549 Cells

作　　者：莫琳[1,2] 刘馨[1,2] 杨慧敏[1,2] 何欣蓉[1,2] 王小林[1,2] 唐春红[2] MO Lin;LIU Xin;YANG Huimin;HE Xinrong;WANG Xiaolin;TANG Chunhong(Pathological Teaching and Research Office,North Sichuan Medical College,Sichuan Nanchong 637000,China;Dept.of Pathology,the Affiliated Hospital of North Sichuan Medical College,Sichuan Nanchong 637000,China)

机构地区：[1]川北医学院病理教研室,四川南充637000 [2]川北医学院附属医院病理科,四川南充637000

出　　处：《中国药房》2020年第6期708-714,共7页China Pharmacy

基　　金：四川省教育厅(自筹经费)科研项目(No.12ZB227)。

摘　　要：目的:从RAD51基因途径研究芹菜素对非小细胞肺癌(NSCLC)A549细胞顺铂敏感性的影响及其机制。方法:取人肺癌顺铂耐药细胞A549/DDP,分为对照组(空白培养基)、顺铂组(5 g/L)和芹菜素低、高剂量组(10、20μmol/L),采用MTT法检测A549/DDP细胞生长,采用AnnexinⅤ/PI双染色法结合流式细胞术检测其凋亡情况。取A549/DDP细胞,分为顺铂单用组(1、2、4、8、16μg/mL)和芹菜素联用组(10μmol/L,在顺铂基础上加用),采用MTT法测定并计算细胞增殖抑制率;采用回归分析模型计算药物的半数抑制浓度(IC50),并以此计算芹菜素的逆转指数。将18只裸鼠随机分为对照组、顺铂单用组和芹菜素联用组,每组6只,接种A549/DDP细胞使形成移植瘤后,分别腹腔注射生理盐水、顺铂(2 mg/kg,隔天给药1次)、顺铂(剂量、用法同前)+芹菜素药液(30 mg/kg,每天给药1次),连续给药18 d,测量小鼠的体质量和移植瘤质量并计算抑瘤率。取人肺癌细胞A549和A549/DDP,分为正常组(A549细胞)、对照组(A549/DDP细胞)、顺铂组(5 g/L,A549/DDP细胞)和芹菜素低、高剂量组(10、20μmol/L,A549/DDP细胞),分别采用实时荧光定量聚合酶链反应和Western blotting法检测细胞中RAD51的mRNA及其蛋白表达情况。结果:与对照组比较,芹菜素低、高剂量组细胞增长数均显著降低,凋亡率均显著升高且显著高于顺铂组(P<0.05或P<0.01)。联用芹菜素后,A549/DDP细胞的增殖抑制率均较相应质量浓度的顺铂单用组显著升高(P<0.05);芹菜素联用组的IC50为(5.81±0.47)μg/mL,显著低于顺铂单用组的IC50(14.44±0.52)μg/mL(P<0.05);逆转指数为2.49。裸鼠抑瘤实验结果显示,联用芹菜素后,A549/DDP荷瘤裸鼠抑瘤率为68.72%,显著低于顺铂单用组的33.82%(P<0.05)。与正常组A549细胞比较,对照组A549/DDP细胞中RAD51mRNA及其蛋白的相对表达量均显著升高(P<0.05);与对照组A549/DDP细胞比较,芹菜素低、高剂量组A549/DDP细胞中RAD51 OBJECTIVE:To study the effects and mechanism of apigenin on cisplatin sensitivity of NSCLC A549 cells by regulating RAD51 gene. METHODS:Human lung cancer cisplatin-resistant cells A549/DDP were selected and divided into control group(blank culture medium),cisplatin group(5 g/L),apigenin low-dose and high-dose groups(10,20 μmol/L). MTT assay was used to detect the growth of A549/DDP cells,while the Annexin Ⅴ/PI double staining combined with flow cytometry were used to detect the apoptosis. A549/DDP cells were collected and divided into cisplatin alone group(1,2,4,8,16 μ g/mL),apigenin combination group(10 μmol/L,based on cisplatin). MTT method was used to determine and calculate inhibitory rate of cell proliferation. IC50 values of drugs were calculated by regression model,and reversion index of apigenin was calculated. 18 nude mice were randomly divided into control group,cisplatin alone group and apigenin combination group,with 6 mice in each group.After A549/DDP cells were inoculated to form the transplanted tumor,normal saline,cisplatin(2 mg/kg,once every other day),cisplatin(the same dosage and usage)+apigenin solution(30 mg/kg,once a day)were injected intraperitoneally respectively. After18 days of continuous administration,the body weight of mice and the mass of the transplanted tumor were detected and the tumor inhibition rate was calculated. Human lung cancer cells A549 and A549/DDP were collected and divided into normal group(A549 cells),control group(A549/DDP cells),cisplatin group(5 g/L,A549/DDP cells)and apigenin low-dose and high-dose groups(10,20 μmol/L,A549/DDP cells),respectively. m RNA and protein expression of RAD51 were detected by real-time fluorescence quantitative PCR and Western blotting assay. RESULTS:Compared with control group, the cell growth of apigenin low-dose and high-dose groups were decreased significantly,apoptosis rates of them were increased significantly and higher than those of cisplatin group(P<0.05 or P<0.01). After combined with apigenin,proliferation inhibition rate

关 键 词：芹菜素 RAD51 非小细胞肺癌 顺铂 耐药 敏感性 

分 类 号：R734.2[医药卫生—肿瘤] R963[医药卫生—临床医学]