P7抑制肿瘤生长及逆转肿瘤细胞耐药的作用机制研究
发布时间:2018-04-20 14:00
本文选题:bFGF + 拮抗肽 ; 参考:《暨南大学》2012年硕士论文
【摘要】:目的:研究bFGF新型拮抗肽P7对B16-F10细胞体内外抗肿瘤活性及P7对bFGF诱导的结肠癌细胞对CPT-11耐药的影响,并探讨其相关作用机制。方法:1.选用黑色素瘤细胞株B16-F10,,用WST-1法检测P7对细胞增殖的影响;PI染色结合流式细胞术分析P7对细胞周期的影响;westemblotting检测信号分子Erk1/2、P38、Akt、MEK的活化水平以及凋亡相关蛋白的表达水平;胞质胞核分离结合Western blotting检测P7对B16-F10细胞胞外bFGF的内化的影响;C57BL/6小鼠实体瘤模型评估P7在体内的抗癌活性。 2.选用结肠癌细细胞株,用MTT法检测P7对bFGF诱导的结肠癌细胞HT-29对化疗药物CPT-11耐药的影响;Alexa Fluor488annexin V/PI双染结合流式细胞术,检测细胞凋亡;Western blotting分析P7对bFGF和CPT-11刺激的HT-29细胞胞内凋亡相关信号分子活化以及细胞凋亡相关蛋白表达的影响;激光共聚焦、胞质胞核分离结合Western blotting检测P7对HT-29细胞胞外bFGF的内化的影响。结果:1. P7减少bFGF刺激下处于S期的B16-F10细胞比率,使细胞周期阻滞在G0/G1期、下调Erkl/2、P38和Akt信号分子的磷酸化水平、抑制外源bFGF的内化,可显著抑制bFGF诱导的B16-F10细胞增殖;体内实验P7通过抑制MAPK信号通路、下调抗凋亡蛋白Bcl-2和促血管生长因子的表达水平,可显著抑制荷瘤小鼠的肿瘤生长和血管生成。 2. P7可下调CPT-11作用下结肠癌细胞HT-29由bFGF诱导增加的细胞存活率、拮抗bFGF对CPT-11诱导的HT-29细胞凋亡的抑制作用、下调bFGF刺激的Akt活化水平、阻滞外源bFGF的内化、反向调节bFGF上调抗凋亡蛋白Bcl-2和下调促凋亡蛋白Bax表达的作用,逆转bFGF诱导的肿瘤耐药。结论:P7可通过以bFGF为靶标,拮抗bFGF相关生物学活性,抑制细胞增殖和血管生成,逆转化疗耐药,在黑色素瘤和结肠癌的治疗上可能具有一定潜能。
[Abstract]:Aim: to study the antitumor activity of bFGF antagonist peptide P7 on B16-F10 cells in vitro and in vivo and the effect of P7 on CPT-11 resistance induced by bFGF in colon cancer cells. Method 1: 1. Melanoma cell line B16-F10 was used to detect the effect of P7 on cell proliferation by WST-1 staining and flow cytometry. Western emblotting was used to detect the activation level of signal molecule Erk1 / 2P38 and the expression of apoptosis-related protein. The effect of P7 on the internalization of extracellular bFGF in B16-F10 cells was determined by cytoplasmic nuclear isolation and Western blotting. The tumor model of C57BL / 6 mice was used to evaluate the anticancer activity of P7 in vivo. 2. The effects of P7 on the chemotherapeutic drug resistance of colon cancer cell line HT-29 induced by bFGF to CPT-11 were detected by MTT assay, and Alexa Fluor488annexin V/PI double staining combined with flow cytometry was used to detect the effect of P7 on the chemotherapeutic drug resistance of colon cancer cell line. The effects of P7 on the activation of apoptosis-related signaling molecules and the expression of apoptosis-related proteins in HT-29 cells stimulated by bFGF and CPT-11 were detected by Western blotting. The effect of P7 on the internalization of extracellular bFGF in HT-29 cells was detected by cytoplasmic nuclear isolation and Western blotting. The result is 1: 1. P7 decreased the ratio of B16-F10 cells in S phase stimulated by bFGF, blocked the cell cycle at G0/G1 stage, down-regulated the phosphorylation levels of Erklr _ (32) P _ (38) and Akt signaling molecules, inhibited the internalization of exogenous bFGF, and significantly inhibited the proliferation of B16-F10 cells induced by bFGF. P7 could significantly inhibit tumor growth and angiogenesis in tumor-bearing mice by inhibiting MAPK signaling pathway and down-regulating the expression of anti-apoptotic protein Bcl-2 and angiogenic factor. 2. P7 could down-regulate the survival rate of colon cancer cell line HT-29 induced by bFGF induced by CPT-11, antagonize the inhibitory effect of bFGF on HT-29 cell apoptosis induced by CPT-11, down-regulate the Akt activation level stimulated by bFGF, and block the internalization of exogenous bFGF. Reverse regulation of bFGF up-regulated the expression of anti-apoptotic protein Bcl-2 and down-regulated the expression of pro-apoptotic protein Bax, and reversed the drug resistance induced by bFGF. Conclusion: P7 can antagonize the biological activity of bFGF by targeting bFGF, inhibit cell proliferation and angiogenesis, and reverse chemotherapeutic resistance, which may have potential in the treatment of melanoma and colon cancer.
【学位授予单位】:暨南大学
【学位级别】:硕士
【学位授予年份】:2012
【分类号】:R739.5
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