本文選題：bFGF + 拮抗肽��； 參考：《暨南大學》2012年碩士論文

【摘要】：目的：研究bFGF新型拮抗肽P7對B16-F10細胞體內(nèi)外抗腫瘤活性及P7對bFGF誘導的結(jié)腸癌細胞對CPT-11耐藥的影響，并探討其相關(guān)作用機制。方法：1.選用黑色素瘤細胞株B16-F10，，用WST-1法檢測P7對細胞增殖的影響；PI染色結(jié)合流式細胞術(shù)分析P7對細胞周期的影響；westemblotting檢測信號分子Erk1/2、P38、Akt、MEK的活化水平以及凋亡相關(guān)蛋白的表達水平；胞質(zhì)胞核分離結(jié)合Western blotting檢測P7對B16-F10細胞胞外bFGF的內(nèi)化的影響；C57BL/6小鼠實體瘤模型評估P7在體內(nèi)的抗癌活性。 2.選用結(jié)腸癌細細胞株，用MTT法檢測P7對bFGF誘導的結(jié)腸癌細胞HT-29對化療藥物CPT-11耐藥的影響；Alexa Fluor488annexin V/PI雙染結(jié)合流式細胞術(shù)，檢測細胞凋亡；Western blotting分析P7對bFGF和CPT-11刺激的HT-29細胞胞內(nèi)凋亡相關(guān)信號分子活化以及細胞凋亡相關(guān)蛋白表達的影響；激光共聚焦、胞質(zhì)胞核分離結(jié)合Western blotting檢測P7對HT-29細胞胞外bFGF的內(nèi)化的影響。結(jié)果：1. P7減少bFGF刺激下處于S期的B16-F10細胞比率，使細胞周期阻滯在G0/G1期、下調(diào)Erkl/2、P38和Akt信號分子的磷酸化水平、抑制外源bFGF的內(nèi)化，可顯著抑制bFGF誘導的B16-F10細胞增殖；體內(nèi)實驗P7通過抑制MAPK信號通路、下調(diào)抗凋亡蛋白Bcl-2和促血管生長因子的表達水平，可顯著抑制荷瘤小鼠的腫瘤生長和血管生成。 2. P7可下調(diào)CPT-11作用下結(jié)腸癌細胞HT-29由bFGF誘導增加的細胞存活率、拮抗bFGF對CPT-11誘導的HT-29細胞凋亡的抑制作用、下調(diào)bFGF刺激的Akt活化水平、阻滯外源bFGF的內(nèi)化、反向調(diào)節(jié)bFGF上調(diào)抗凋亡蛋白Bcl-2和下調(diào)促凋亡蛋白Bax表達的作用，逆轉(zhuǎn)bFGF誘導的腫瘤耐藥。結(jié)論：P7可通過以bFGF為靶標，拮抗bFGF相關(guān)生物學活性，抑制細胞增殖和血管生成，逆轉(zhuǎn)化療耐藥，在黑色素瘤和結(jié)腸癌的治療上可能具有一定潛能。
[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.
【學位授予單位】：暨南大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R739.5

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