中药胃肠安诱导裸鼠胃癌移植瘤细胞凋亡的途径及基因调控的研究

目的:观察以健脾类中药为基础的复方“胃肠安”(Weichang'an,WCA)对人胃癌细胞SGC-7901裸小鼠皮下移植瘤生长的抑制作用和体内诱导胃癌细胞凋亡的作用和途径。方法:用高效液相色谱法监测WCA的制备,保证研究制剂的稳定性;用人胃癌细胞SGC-7901裸小鼠皮下移植瘤模型评价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(streptavidin peroxidase,SP)检测增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)表达,末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate fluorescence nick end labeling,TUNEL)法检测凋亡指数以及SP法检测活化caspase-3、caspase-8和caspase-9的表达;SYBR green dye I实时定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-PCR)检测bcl-2等5条凋亡调控相关基因的表达;SP法比较有差异表达基因在细胞内的蛋白表达。结果:WCA组的平均瘤质量为(0.99±0.76)g,低于模型组的(1.93±0.58)g,差异有统计学意义(P=0.011 7),WCA组对皮下移植瘤生长的抑制率为48.70%;WCA组移植瘤PCNA阳性表达率为(35.73±6.01)%,强阳性表达率为(3.39±1.48)%,总阳性率为(39.03±7.37)%,均低于模型组的(53.48±9.34)%、(8.78±5.43)%和(62.26±13.80)%,差异有统计学意义;凋亡指数为(9.72±4.51)%,高于模型组的(2.45±1.37)%(TUNEL法);WCA组活化caspase-3表达阳性率为(5.20±2.26)%,较模型组的(2.82±1.84)%高(P=0.036 7);caspase-8表达阳性率为(4.44±2.67)%,而模型组为(3.94±2.10)%,差异无统计学意义(P=0.682 7);caspase-9表达阳性率为(8.87±6.08)%,较模型组的(2.25±0.79)%高(P=0.0144);与模型组相比,WCA组bcl-2等5条与凋亡调控相关基因的表达量差异有统计学意义的为Stat3(2-ΔΔCT=0.16)和bcl-2(2-ΔΔCT=0.10),均为下调表达作用;WCA组P-Stat3阳性表达率为(35.93±12.67)%,强阳性表达率为(3.64±1.72)%,总阳性率为(39.57±13.31)%,均分别低于模型组的(56.49±9.34)%、(13.16±7.06)%和(69.65±10.80)%,差异有统计学意义;bcl-2蛋白阳性表达率为(1.62±0.82)%,低于模型组的(7.53±3.73)%,差异有统计学意义。结论:中药复方WCA在体内可抑制胃癌细胞增殖,诱导凋亡;WCA在体内诱导胃癌细胞凋亡可通过caspase-9和caspase-3的活化实现。诱导胃癌细胞凋亡的机制与下调stat3和bcl-2的mRNA表达,抑制P-stat3和bcl-2蛋白在细胞内的表达有关。

Effects of Chinese herbal recipe Weichang'an in inducing apoptosis and related gene expression in human gastric cancer grafted onto nude mice

OBJECTIVE: To investigate the mechanism of Chinese herbal recipe Weichang'an (WCA) in inducing cell apoptosis of human gastric cancer grafted onto nude mice. METHODS: The high performance liquid chromatography was used for monitoring the stability of WCA. A human gastric cancer cell line SGC-7901 grafted in nude mouse was used as the animal model. The mice were divided into untreated group and two experimental groups. Animals in the two experimental groups received either WCA over a 34-day period or 5-fluorouracil (5-FU) over a 6-day period starting at the 8th day after grafting. Animals in the untreated group received normal saline on an identical schedule. Animals were killed 41 days after being grafted. To assess the effect of the treatment on tumor, the tumor weight was determined by the electron balance immediately after the animals were killed. SP immunohistochemical method was used to detect the expression of proliferating cell nuclear antigen (PCNA) in grafts. Apoptotic indices (AI) of the tumor cells were examined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate fluorescence nick end labeling (TUNEL) method. SP method was also used to detect the expressions of cleaved caspase-3, caspase-8 and caspase-9. SYBR green dye I real-time quantitative polymerase chain reaction (real-time quantitative corrected] PCR) was used to assess the related gene alterations in mRNA level. The expressions of phospho-Stat3 (Tyr705) and bcl-2 proteins were detected by using SP method. RESULTS: Compared with the untreated group, tumor growth was significantly inhibited by treatment of WCA or 5-FU (P<0.01, respectively). The tumor inhibition rate in the WCA-treated group was 48.70% and that in the 5-FU-treated group was 60.10%. The average labeling index (LI) for PCNA in the WCA-treated group and 5-FU-treated group was significantly decreased as compared with that in the untreated group, respectively. The AI of human gastric cancer grafted in the nude mice detected by using TUNEL method was significantly increased to (9.72+/-4.51)% in the WCA-treated group, while it was (2.45+/-1.37)% in the untreated group. 5-FU-treated group was also found a significantly increased AI compared with the untreated group. The expressions of cleaved caspase-3 and caspase-9 in the WCA-treated group and 5-FU-treated group were significantly increased as compared with those in the untreated group. But caspase-8 showed no significant alteration either in the WCA-treated group or in the 5-FU-treated group. The expression levels of Stat3 (2(-)delta delta C(T))=0.16) and bcl-2 (2(-)delta delta C(T))=0.10) detected by using real-time quantitative corrected] PCR were lower in the WCA-treated group than those in the untreated group. The expressions of phospho-Stat3 (Tyr705) and bcl-2 in the WCA-treated group were significantly decreased as compared with those in the untreated group. CONCLUSIONS: Chinese herbal recipe WCA can inhibit gastric cancer cell SGC-7901 growth in vivo, induce gastric cancer cell apoptosis and suppress the cell proliferation. WCA induces apoptosis through the caspase-9 and caspase-3 pathway in vivo. Its mechanism might be involved in the down-regulation of Stat3 and bcl-2 genes.