健脾中药诱导人胃癌细胞裸小鼠移植瘤细胞凋亡的实验研究

Chinese Jianpi herbs induce apoptosis of human gastric cancer grafted onto nude mice

目的研究健脾类中药复方四君子汤及以四君子汤等健脾药物为基础结合清热解毒、软坚散结中药组成的复方 SRRS 对人胃癌细胞裸小鼠移植瘤的抑瘤作用与诱导肿瘤细胞凋亡的关系.方法采用人胃癌细胞 SGC-7901裸小鼠皮下移植瘤模型.实验动物共30只,造模后随机分为3组,每组10只:SRRS 方剂组,予240 g/L SRRS 煎剂0.5mL/d 灌胃;四君子汤组,予160 g/L 四君子汤煎剂0.5 mL/d 灌胃;对照组,予生理盐水0.5 mL/d 灌胃,共40 d.应用末端脱氧核苷酸转移酶介导的dUTP 缺口末端标记法(TUNEL 法)、电镜,结合流式细胞仪分析技术,观察 SRRS 方剂及四君子汤的抑瘤作用与肿瘤细胞凋亡率及形态学改变,结果四君子汤、SRRS 方剂对人胃癌细胞裸小鼠移植瘤抑瘤率分别为34%,47%,TUNEL 法检测肿瘤细胞凋亡指数(AI)分别为:16%±3%,13%±3%,与对照组 AI:3%±1%比较,显著提高(四君子组 P<0.01;SRRS 方剂组 P<0.05,均为 t 检验);流式细胞分析示四君子汤组凋亡率:11%±6%,SRRS 方剂组:12%±6%,均较对照组:7%±1%提高(t 检验,P<0.05);但四君子汤和 SRRS 方剂组之间的凋亡率 TUNEL法和流式细胞分析均无差异.电镜下二中药治疗组见较多凋亡细胞和凋亡小体;对照组凋亡细胞和凋亡小体极少.结论中药四君子汤、SRRS 方剂在体内的抑瘤作用与诱导胃癌细胞凋亡有关,其诱导细胞凋亡作用可能主要与健脾药物有关.诱导肿瘤细胞凋亡可能是健脾药物抗癌作用的机制之一.

AIM To examine whether the Sijunzi decoction and Chinese herbal recipe (SRRS) of deheat-toxin,softening hard lumps and dissolving phlegm enhance apoptosis of human gastric cancer xenografts in nude mice. METHODS A human gastric adenocarcinoma cell line SGC-7901 was grafted onto 30 nude mice.The mice were divided into 3 groups,one control and the two experimental groups.Animals in the two experimental groups received either Sijunzi decoction or SRRS over a 40-day period beginning 2 days after grafting.Control animals received saline on an identical schedule.Animals were killed 41 days after being grafted.The effect of therapy was assessed by two ways:① tumor size was periodically measured during the life of the animals;② tumor weight was measured immediately by electron balance after the animals were killed.For detection of apoptotic cells,apoptotic indices were examined by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate fluorescence nick end labeling (TUNEL) method and flow cytometry analysis. Morphological variations were observed under electron microscopy. RESULTS When compared with controls,tumor growth (size and weight) was significantly inhibited by treatment with the Sijunzi decoction (t test,P<0.05) or SRRS (t test,P<0.01).The tumor inhibiting rate of the Sijunzi decoction group was 34% and of the SRRS group was 47%.Apoptotic index (AI) of human gastric cancer xenografts in nude mice was significantly increased to 15%±3% using TUNEL method and 11%±6% FACScan in the Sijunzi decoction treatment group compared with the controls (TUNEL:3%±1%,P<0.01,t test;FACScan: 7%±1%,P<0.05,t test).SRRS group had a significantly increased AI found by TUNEL method and flow cytometry analysis compared with the controls (TUNEL:13%±3%,P<0.05,t test;FACScan:12%± 6%,P<0.05,t test).But there was no significant difference between Sijunzi decoction group and SRRS treatment group using either TUNEL method or flow cytometry analysis.Under electron microscopy cell shrinkage,nuclear chromatin condensation,formation of membrane blebs and apoptotic bodies were frequently observed in Sijunzi decoction group and SRRS group. CONCLUSION Apoptosis was enhanced in human gastric cancer xenografts in nude mice after Sijunzi decoction and SRRS treatment.Apoptosis is one of the mechanisms of Jianpi herbs for gastric cancer therapies.