植物提取十八碳二烯酸对人胃癌组织细胞生态毒理学作用机制研究

Ecotoxicological mechanism of the octadecadienoic acid extract on human gastric cancer cells

为了探索植物提取十八碳二烯酸对人胃癌组织细胞生态毒理学作用机制,取胃癌患者肿瘤组织移植到重症联合免疫缺陷(SCID)小鼠皮下,腹腔注射100、300、900 mg·kg^(-1)植物提取物十八碳二烯酸,分析对移植瘤生长抑制作用;用酶联免疫吸附法(ELISA)检测瘤组织cAMP、P53、PI3K水平。将瘤组织经酶解消化分离出细胞进行培养,给予胃癌瘤细胞2.0、4.0、8.0 mg·L^(-1)十八碳二烯酸,用MTT法检测十八碳二烯酸对胃癌瘤细胞生态毒性作用,用酶联免疫吸附法(ELISA)检测胃癌瘤细胞中P21、MDM2、PKB/Akt水平。结果发现,低中高剂量组和5-FU组胃癌移植瘤生长抑制率比模型组升高17.92%—45.75%和35.85%;胃癌移植瘤cAMP水平比模型组升高14.88%—40.70%和23.21%(P<0.05—0.01),P53水平比模型组降低37.94%—63.96%和58.54%(P<0.01),PI3K水平比模型组降低20.77%—52.94%和35.29%(P<0.01)。低中高剂量组和5-FU组胃癌活细胞数随给药浓度的增加和作用时间的延长而减少,细胞增殖抑制率为23.11%—59.66%和51.26%,半数抑制浓度(IC_(50))为5.811 mg·L^(-1);流式细胞仪检测,G_(0)G_(1)期细胞比对照组增加16.54%—27.51%和20.45%;G_(2)M期细胞比对照组减少36.40%—67.98%和41.67%;凋亡率为17.44%—23.37%和19.21%;胃癌瘤细胞P21水平比对照组升高13.33%—62.81%和52.63%,PKB/Akt水平比对照组降低19.39%—51.02%和46.94%,MDM2水平比对照组升高10.26%—76.92%和33.33%。结果提示,重症综合性免疫缺乏症(SCID)小鼠肿瘤模型的建立,为研究人类肿瘤治疗和康复提供了生态毒理学实验对象;十八碳二烯酸对胃癌移植瘤和瘤细胞增殖具有明显的生态毒性和抑制作用。其机制可能与升高移植瘤细胞cAMP、p21、MDM2水平,降低P13K、PKB/Akt、P53水平,抑制细胞分裂周期,降低PI3K-Akt信号转导通路活性有关。

In order to understand the ecotoxicological mechanism of the extracted octadecadienoic acid from plant on human gastric cancer,the tumor tissues from the gastric cancer patients were transplanted subcutaneously into severe combined immunodeficiency(SCID)mice.The mice in different groups were respectively treated with 100,300 and 900 mg·kg^(-1) extract intraperitoneally to evaluate the inhibitory effect on the growth of transplanted tumor cells and the levels of cAMP,P53 and PI3K with the enzyme-linked immunosorbent assay(ELISA).The tumor tissues were digested with enzyme for cell culture.The cultured tumor cells were administered 2.0,4.0 and 8.0 mg·L^(-1) octadecadienoic acid extract,and the ecotoxicity of extract on gastric cancer cells was detected by MTT assay and the levels of P21,MDM2 and PKB/Akt were measured by ELISA.Our results showed that,after treated with different extract doses and 5-FU,compared with the control group,the growth inhibition rates of the transplanted gastric cancer were significantly increased by 17.92%-45.75%and 35.85%and the cAMP level increased by 14.88%-40.70%and 23.21%(P<0.05,P<0.01),respectively;while the P53 levels were decreased by 37.94%-63.96%and 58.54%(P<0.01)and the PI3K level decreased by 20.77%-52.94%and 35.29%(P<0.01),respectively.In addition,the numbers of living gastric cancer cells in the extract treated groups and the 5-FU group were decreased with the increase of the extract concentration and the prolongation of action time and the IC_(50) was 5.811 mg·L^(-1) and the inhibitory rates of cells proliferation by 23.11%-59.66%and 51.26%,respectively.The results from the flow cytometry showed that,when the gastric cancer cells were treated with different extract doses and the 5-FU,the G_(0)G_(1) phase cells were increased by 16.54%-7.51%and 20.45%,respectively,while the G2M phase cells were decreased by 36.40%-67.98%and 41.67%,and the apoptosis rates were 17.44%-23.37%and 19.21%,respectively,compared with the control group.The results from the ELISA suggested that,when the gastric cancer cells were treated with different extract doses and the 5-FU,the levels of P21 and MDM2 in gastric cancer cells were increased by 13.33%-62.81%and 52.63%,and 10.26%-76.92%and 33.33%,respectively,while the levels of PKB/Akt were decreased by 19.39%-51.02%and 46.94%,respectively.Our findings indicate that the SCID mouse tumor model can be used as an ecotoxicological experimental model for human tumor treatment and rehabilitation.The octadecadienoic acid extract has significant ecotoxic and inhibitory effects on the proliferation of the transplanted gastric cancer and tumor cells.The potential mechanism may be related to the increases of the cAMP,P21 and MDM2 levels,the decrease of the PI3K,PKB/Akt and P53 levels,the inhibition of the cell division cycle,and the reduced activity of PI3K-Akt signal transduction pathway.