补肾疏肝方对肺腺癌A549细胞的凋亡作用及其机制研究  

Effects of Bushenshugan Formula on the Apoptosis of Human Lung Adenocarcinoma A549 Cells and Its Mechanism

作　　者：苗学红 王淑玲[2] 

机构地区：[1]郑州大学第一附属医院中西医结合科,河南省郑州市450052 [2]郑州大学基础医学院,河南省郑州市450001

出　　处：《中国全科医学》2015年第32期3964-3968,共5页Chinese General Practice

基　　金：国家自然科学基金资助项目(81473497);河南省科技厅科技攻关项目(112300410049)

摘　　要：目的探讨补肾疏肝方对肺腺癌A549细胞的凋亡作用,对肿瘤坏死因子（TNF）-α分泌、Survivin及半胱氨酸天冬氨酸蛋白酶（Caspase）-3 mRNA表达的影响。方法将补肾疏肝方制成低剂量（1.25 g/ml）、中剂量（2.50 g/ml）和高剂量（3.75 g/ml）药液。以随机数字表法将大鼠分为对照组、低剂量组、中剂量组、高剂量组、顺铂（DDP）组、联合组（中剂量药液联合DDP）,每组10只。对照组于第1～5天给予0.9%氯化钠溶液4 ml/d灌胃;低剂量组、中剂量组、高剂量组于第1～5天给予相应剂量药液4 ml/d灌胃;DDP组分别于第1、3、5天腹腔注射2 ml DDP（20%）;联合组于第1～5天给予中剂量灌胃药液灌胃4 ml/d及第1、3、5天腹腔注射2 ml DDP（20%）。末次给药2 h后采血,分离血清。A549细胞分别加入各组血清,培养72 h,倒置荧光显微镜下观察细胞凋亡情况。A549细胞分别加入各组血清,培养24、48、72 h后,采用酶联免疫吸附法（ELISA）检测TNF-α水平。A549细胞与各组血清共培养72 h后,采用荧光定量PCR检测Survivin mRNA、Caspase-3 mRNA的相对表达量。结果各组血清作用于A549细胞72 h后,凋亡细胞核固缩,且随药液浓度增加凋亡细胞增多。各组血清作用于A549细胞24、48、72h后TNF-α分泌水平比较,差异均有统计学意义（P〈0.05）。其中,低剂量组、中剂量组、高剂量组、DDP组和联合组作用于A549细胞24、48、72 h后TNF-α分泌水平高于对照组,差异均有统计学意义（P〈0.05）。各组血清作用于A549细胞72 h后Survivin mRNA相对表达量比较,差异有统计学意义（F=13.238,P〈0.05）。其中,高剂量组、DDP组和联合组血清作用于A549细胞72 h后Survivin mRNA相对表达量低于对照组,差异有统计学意义（P〈0.05）。各组血清作用于A549细胞72 h后Caspase-3 mRNA相对表达量比较,差异有统计学意义（F=10.814,P〈0.05）。其中,中剂量组、高剂量组、DDP组和联合组�Objective To explore the effects of Bushenshugan Formula（ BSSGF） on the apoptosis of human lung adenocarcinoma A549 cells,the level of TNF- α and the mRNA expression of Survivin and Caspase- 3. Methods BSSGF was used to make low- dose（ 1. 25 g / ml）, medium- dose（ 2. 50 g / ml） and high- dose（ 3. 75 g / ml） liquid. Using random number table method,we divided the rats into control group,low- dose group,medium- dose group,high- dose group,DDP group and combined group（ medium- dose liquid combined with DDP） with 10 rats in each group. Control group was given0. 9% sodium chloride solution from day 1 to day 5; low- dose,medium- dose and high- dose groups were given corresponding dose of liquid by 4 ml / d; DDP group was given 2 ml DDP（ 20%） on day 1,day 3 and day 5 by intraperitoneal infection;combined group was given medium- dose liquid by 4 ml / d from day 1 to day 5 and was also given 2 ml DDP（ 20%） on day 1,day 3 and day 5 by intraperitoneal infection. Two hours after the last administration,blood samples were taken and serum was separated. A549 cells were added into the serum of each group and were cultured for 72 hours,then observed cell apoptosis by inverted fluorescence microscope. ELISA method was used to determine TNF- α level at 24 h,48 h and 72 h after cultivatation.After 72 hours,fluorogenic quantitative PCR was employed to determine the relative expression of Survivin mRNA and Caspase-3 mRNA. Results After 72 hours＇ influence of serum on A549 cells, cell apoptosis and karyopyknosis occurred, and the apoptotic cells increased with liquid concentration increasing. At 24 h,48 h and 72 h,the 6 groups were significantly different in the expression of TNF- α（ P 〈0. 05）. At 24 h,48 h and 72 h,low- dose group,medium- dose group,high- dose group,DDP group and combined group were higher than control group in the expression of TNF- α（ P 〈0. 05）. The 6 groups were significantly different in the relative expression of Survivin mRNA at 72 h（ F = 13. 238,P 〈0. 05）. Hi

关 键 词：腺癌 细支气管肺泡 补肾疏肝方 A549细胞 细胞凋亡 SURVIVIN 肿瘤坏死因子α 半胱氨酸天冬氨酸蛋白酶3 

分 类 号：R730.261[医药卫生—肿瘤]