人参皂甙Rg3联合IRESSA对人A431细胞生长的抑制作用

发布时间：2018-04-03 11:50

  本文选题：人参皂甙　切入点：Rg3　出处：《大连医科大学》2011年硕士论文

【摘要】：目的:人参皂甙Rg3是从红参中提取的中药单体,具有抑制多种肿瘤生长和转移等作用。表皮生长因子受体(epidermal growth factor receptor, EGFR)属于ErbB家族成员,是一种重要的跨膜受体。EGFR与配体结合后激活细胞内信号转导,参与细胞的增殖、迁移、黏附、分化和凋亡等过程。IRESSA是一种EGFR的小分子酪氨酸激酶抑制剂,临床上用于非小细胞肺癌等恶性肿瘤的晚期治疗。根据文献报道,在肿瘤的治疗中,单纯用药的模式已经不能满足临床需要,化疗药物的联合应用是最近治疗的热点,具有应用前景。本实验采用高表达EGFR的人表皮鳞状细胞癌细胞A431为研究对象,观察Rg3与IRESSA联合应用对A431细胞生长的抑制作用,并初步探讨其作用机制。 方法:体外培养人A431细胞。将实验分为4组(对照组,Rg3处理组,IRESSA处理组和联合用药组)。MTT法检测药物对细胞增殖的影响;分析药物对细胞增殖的抑制率;用倒置显微镜对药物处理后第四天的细胞进行形态学观察;用Western Blot的方法检测细胞核增殖抗原(PCNA)的表达;用RT-PCR检测FUT4的表达,用Western Blot的方法检测p-EGFR的表达。 结果: 1.MTT法检测细胞的增殖。处理方法为对照组(加入等量培养基),Rg3组( 40μg/ml) , IRESSA组( 0.025μmol/l ),联合用药组Rg3 ( 40μg/ml )和IRESSA(0.025μmol/l)。联合用药组细胞的增殖速率明显小于其他组,24小时后Rg3组抑制率为20.92%,IRESSA组抑制率为23.47%,联合用药组抑制率为35.71%;48小时后Rg3组抑制率25.00%,IRESSA组抑制率为32.98%,联合用药组抑制率为44.37%;72小时后Rg3组抑制率为42.10%,IRESSA组抑制率为48.58%,联合用药组抑制率为55.48%;96小时后Rg3组抑制率为57.55%,IRESSA组抑制率为65.86%,联合用药组抑制率为70.52%。联合用药组的抑制率高于单用其中任何一种药物组(p0.05)。 2.用倒置显微镜对药物处理后第四天的细胞进行形态学观察。对照组A431细胞贴壁良好,饱满,呈不规则多边形,有棱角;Rg3组则细胞略微变圆,细胞与细胞的间隙缩小;IRESSA组可见部分细胞凋亡,细胞萎缩;联合用药组则有大量细胞死亡,可见部分细胞残片。 3.Western Blot方法检测PCNA的表达。处理方法为对照组(加入等量培养基),Rg3组( 320μg/ml),IRESSA组(1μmol/l,联合用药组Rg3(320μg/ml)和IRESSA(1μmol/l)。单纯用药组和联合用药组PCNA的表达均下降,联合用药组下降的程度更高(p0.05)。 4. RT-PCR检测FUT4基因的表达(处理方法同PCNA的检测)。A431细胞的FUT4表达下降,联合用药组下降的程度更高(p0.05)。 5.Western Blot检测p-EGFR的表达(处理方法同PCNA的检测)。单纯用药组和联合用药组p-EGFR的表达下降,联合用药组下降的程度更高(p0.05)。 结论:人参皂甙Rg3和IRESSA均能抑制细胞的增殖,二者联合应用则可增加对肿瘤细胞生长的抑制作用,提示二者有协同作用。人参皂甙Rg3和IRESSA均能下调EGFR的激活,下调FUT4的表达有关。其机制有待于进一步研究。
[Abstract]:Objective: ginsenoside Rg3 is a traditional Chinese medicine monomer extracted from Rhizoma Ginseng, which can inhibit the growth and metastasis of many kinds of tumors.Epidermal growth factor receptor (EGFR) is a member of the ErbB family. It is an important transmembrane receptor. EGFR binds to ligand and activates intracellular signal transduction and participates in cell proliferation, migration and adhesion.IRESSA is a small molecular tyrosine kinase inhibitor of EGFR, which is used in advanced treatment of malignant tumors such as non-small cell lung cancer (NSCLC).According to literature reports, in the treatment of cancer, the mode of drug use alone can no longer meet the clinical needs, the combined use of chemotherapeutic drugs is a hot spot of recent treatment, and has a prospect of application.In this study, human epidermal squamous cell carcinoma A431 with high expression of EGFR was used to observe the inhibitory effect of Rg3 and IRESSA on the growth of A431 cells, and to explore its mechanism.Methods: human A431 cells were cultured in vitro.The experiment was divided into 4 groups (control group, Rg3 treatment group, IRESSA treatment group and combined drug group, the effect of drug on cell proliferation was detected by MTT method, the inhibition rate of drug on cell proliferation was analyzed.The cell morphology was observed by inverted microscope on the fourth day after drug treatment, the expression of nuclear proliferating antigen (PCNA) was detected by Western Blot, the expression of FUT4 was detected by RT-PCR, and the expression of p-EGFR was detected by Western Blot.Results:Cell proliferation was detected by 1.MTT assay.鑱斿悎鐢ㄨ嵂缁勭粏鑳炵殑澧炴畺閫熺巼鏄庢樉灏忎簬鍏朵粬缁，

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