本文選題：人參皂甙　切入點(diǎn)：Rg3　出處：《大連醫(yī)科大學(xué)》2011年碩士論文

【摘要】：目的:人參皂甙Rg3是從紅參中提取的中藥單體,具有抑制多種腫瘤生長和轉(zhuǎn)移等作用。表皮生長因子受體(epidermal growth factor receptor, EGFR)屬于ErbB家族成員,是一種重要的跨膜受體。EGFR與配體結(jié)合后激活細(xì)胞內(nèi)信號轉(zhuǎn)導(dǎo),參與細(xì)胞的增殖、遷移、黏附、分化和凋亡等過程。IRESSA是一種EGFR的小分子酪氨酸激酶抑制劑,臨床上用于非小細(xì)胞肺癌等惡性腫瘤的晚期治療。根據(jù)文獻(xiàn)報道,在腫瘤的治療中,單純用藥的模式已經(jīng)不能滿足臨床需要,化療藥物的聯(lián)合應(yīng)用是最近治療的熱點(diǎn),具有應(yīng)用前景。本實(shí)驗采用高表達(dá)EGFR的人表皮鱗狀細(xì)胞癌細(xì)胞A431為研究對象,觀察Rg3與IRESSA聯(lián)合應(yīng)用對A431細(xì)胞生長的抑制作用,并初步探討其作用機(jī)制。 方法:體外培養(yǎng)人A431細(xì)胞。將實(shí)驗分為4組(對照組,Rg3處理組,IRESSA處理組和聯(lián)合用藥組)。MTT法檢測藥物對細(xì)胞增殖的影響;分析藥物對細(xì)胞增殖的抑制率;用倒置顯微鏡對藥物處理后第四天的細(xì)胞進(jìn)行形態(tài)學(xué)觀察;用Western Blot的方法檢測細(xì)胞核增殖抗原(PCNA)的表達(dá);用RT-PCR檢測FUT4的表達(dá),用Western Blot的方法檢測p-EGFR的表達(dá)。 結(jié)果: 1.MTT法檢測細(xì)胞的增殖。處理方法為對照組(加入等量培養(yǎng)基),Rg3組( 40μg/ml) , IRESSA組( 0.025μmol/l ),聯(lián)合用藥組Rg3 ( 40μg/ml )和IRESSA(0.025μmol/l)。聯(lián)合用藥組細(xì)胞的增殖速率明顯小于其他組,24小時后Rg3組抑制率為20.92%,IRESSA組抑制率為23.47%,聯(lián)合用藥組抑制率為35.71%;48小時后Rg3組抑制率25.00%,IRESSA組抑制率為32.98%,聯(lián)合用藥組抑制率為44.37%;72小時后Rg3組抑制率為42.10%,IRESSA組抑制率為48.58%,聯(lián)合用藥組抑制率為55.48%;96小時后Rg3組抑制率為57.55%,IRESSA組抑制率為65.86%,聯(lián)合用藥組抑制率為70.52%。聯(lián)合用藥組的抑制率高于單用其中任何一種藥物組(p0.05)。 2.用倒置顯微鏡對藥物處理后第四天的細(xì)胞進(jìn)行形態(tài)學(xué)觀察。對照組A431細(xì)胞貼壁良好,飽滿,呈不規(guī)則多邊形,有棱角;Rg3組則細(xì)胞略微變圓,細(xì)胞與細(xì)胞的間隙縮小;IRESSA組可見部分細(xì)胞凋亡,細(xì)胞萎縮;聯(lián)合用藥組則有大量細(xì)胞死亡,可見部分細(xì)胞殘片。 3.Western Blot方法檢測PCNA的表達(dá)。處理方法為對照組(加入等量培養(yǎng)基),Rg3組( 320μg/ml),IRESSA組(1μmol/l,聯(lián)合用藥組Rg3(320μg/ml)和IRESSA(1μmol/l)。單純用藥組和聯(lián)合用藥組PCNA的表達(dá)均下降,聯(lián)合用藥組下降的程度更高(p0.05)。 4. RT-PCR檢測FUT4基因的表達(dá)(處理方法同PCNA的檢測)。A431細(xì)胞的FUT4表達(dá)下降,聯(lián)合用藥組下降的程度更高(p0.05)。 5.Western Blot檢測p-EGFR的表達(dá)(處理方法同PCNA的檢測)。單純用藥組和聯(lián)合用藥組p-EGFR的表達(dá)下降,聯(lián)合用藥組下降的程度更高(p0.05)。 結(jié)論:人參皂甙Rg3和IRESSA均能抑制細(xì)胞的增殖,二者聯(lián)合應(yīng)用則可增加對腫瘤細(xì)胞生長的抑制作用,提示二者有協(xié)同作用。人參皂甙Rg3和IRESSA均能下調(diào)EGFR的激活,下調(diào)FUT4的表達(dá)有關(guān)。其機(jī)制有待于進(jìn)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嵂緇勭粏鑳?yōu)鐨勫娈栭�熺巼鏄庢樉灝忎簬鍏朵粬緇，

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