中药WCA对人胃癌细胞SGC-7901体内作用基因的筛选与验证  

Difference of gene expression profile in human gastric cancer grafted onto nude mice treated with WCA

作　　者：赵爱光[1] 杨金坤[1] 尤圣富[1] 李婷[1] 赵海磊[1] 顾缨[1] 唐莱娣[1] 

机构地区：[1]上海中医药大学附属龙华医院肿瘤一科

出　　处：《中国中药杂志》2007年第19期2028-2036,共9页China Journal of Chinese Materia Medica

基　　金：上海市科委青年科技启明星计划(02QB14043);上海市教委科技发展基金(01C06);上海市科委基础研究重点课题(02DJI4058);上海市教委E研究院建设项目(E03008);上海市重点学科建设项目(Y0302)

摘　　要：目的:探讨以四君子汤等健脾中药为基础的复方胃肠安(WCA)对胃癌细胞的体内作用分子机制。方法:采用人胃癌细胞SGC-7901裸小鼠皮下移植瘤模型系统评价WCA对胃癌细胞的体内抑瘤作用;链霉素抗生物素-过氧化酶法(SP法)检测增殖细胞核抗原PCNA表达,末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(TUNEL法)检测凋亡指数以及SP法检测caspase-3的活化表达;cDNA array筛选出WCA组N.S.对照组差异表达2倍以上基因,Real-tim e Quantitative PCR验证与增殖、凋亡、分化及信号转导密切相关的19条基因的表达;SP法检测部分有差异表达的基因在细胞内的蛋白表达。结果:与对照组比较,WCA组对皮下移植瘤生长的平均抑制率为(44.32+5.67)%,3次实验WCA组的平均瘤重分别为(0.99±0.76),(1.02±0.16),(0.88±0.47)g,分别低于对照组的(1.93±0.58),(1.65±0.46),(1.63±0.52)g(P<0.05);WCA组移植瘤PCNA阳性表达率(35.73±6.01)%、强阳性表达率(3.39±1.48)%和总阳性率(39.03±7.37)%均分别低于对照组的(53.48±9.34)%,(8.78±5.43)%,(62.26±13.80)%(P<0.05);凋亡指数为(9.72±4.51)%高于对照组的(2.45%±1.37)%(TUNEL法);WCA组活化caspase-3表达阳性率为(5.20±2.26)%,较对照组的(2.82±1.84)%高(P=0.0367);与对照组比较,全基因表达谱芯片筛选出WCA组共45个2倍以上差异表达基因,其中上调表达24个,下调表达21个。35个已克隆基因,10个表达序列标签(expression sequence tag,EST);2-ΔΔCT法对19条目标基因的表达量进行评估,与对照组比较,表达量显著差异4倍以上的有Stat3(2-ΔΔCT=0.16),R IPX(2-ΔΔCT=0.18),ROD1(2-ΔΔCT=0.23),bc l-2(2-ΔΔCT=0.10),均为下调表达作用;WCA组P-Stat3阳性表达率(35.93±12.67)%、强阳性表达率(3.64%±1.72)%和总阳性率(39.57±13.31)%均分别低于对照组的(56.49±9.34)%,(13.16±7.06)%,(69.65±10.80)%,差异有显著性;bc l-2蛋白阳性表达率(1.62±0.82)%低于对照组的(7.53±3.73)%(P<0.05)。结论:健脾中药复方WCA在�Objective： Chinese jianpi herbal recipe Weichangan （WCA） could increase the survival rate of advanced gastric cancer. This study was designed to investigate the molecular mechanism of WCA in treatment of gastric cancer by cDNA array, realtime quantitative PCR and immunohistochemical technique. Method： A human gastric adenocarcinoma cell line SGC-7901 grafted onto nude mouse was used as the animal model. The mice were divided into 3 groups, one control and the two representing experimental conditions. Animals in the two experimental groups received either WCA over a 34-day period or 5-fluorottraeil （5-FU） over 6-day period starting at 8th day after grafting. Control animals received saline on an identical schedule. Animals were killed 41 days after being grafted. To assess the effect of therapy tumor weight was determined by a electron balance immediately after the animals killed. SP immunohistoehemieal method was used to detent the expression of proliferating cell nuclear antigen （PCNA） in xenografts. For detention of apoptotie cells, apoptotie indices （AI） were examined by the terminal deoxynueleotidyl transferase-mediated deoxyuridine triphosphate fluorescence nick end labeling （TUNEL） method. SP method was also used to detect the expression of cleaved Caspase-3. The expression profiles in paired WCA treated gastric cancer samples and the N. S. control samples were studied by using a cDNA array representing 14, 181 cDNA clusters. The alterations in gene expression levels were eonfirmed by real-time quantitative PCR. SP method was used to detect the expression of Phospho-Stat3 （TyrT05） and bel-2. Result： When compared with controls, tumor growth was significantly inhibited by treatment with the WCA or 5-FU （P 〈 0. 01, respectively）. The average of tumor inhibitory rate in WCA group was （44. 32±5. 67）% and 5-FU （47.04 ± 11. 33）%. The average labeling index （LI） for PCNA in WCA group and 5-FU group was significantly decreased compared with the control group respect

关 键 词：胃癌 健脾中药 基因表达 CDNA微阵列 实时定量聚合酶链反应 

分 类 号：R285.5[医药卫生—中药学]