【摘要】：宮頸癌是女性常見的惡性腫瘤,對于中晚期患者通常放化療和手術(shù)治療的效果并不理想。自從發(fā)現(xiàn)宮頸癌與HPV感染密切相關(guān)后,HPV治療性疫苗成為一個重要的研究方向。本課題組前期構(gòu)建了針對HPV16的mdNCRT/E7/hsp重組融合蛋白疫苗,并通過體外實驗證明了該疫苗可誘導(dǎo)到較高水平的CTL活性及抑制血管生長作用。本研究進(jìn)一步通過體內(nèi)試驗研究其抗腫瘤作用及其分子機(jī)制。 我們建立了C57BL/6小鼠TC-1腫瘤模型,體內(nèi)實驗結(jié)果表明mdNCRT/E7/hsp重組融合蛋白疫苗能夠有效的抗腫瘤生長,誘發(fā)特異的腫瘤排斥反應(yīng),對未形成實體瘤的微小腫瘤病灶具有治療作用。將兩組腫瘤組織切片進(jìn)行免疫組化分析,發(fā)現(xiàn)mdNCRT/E7/hsp重組融合蛋白能夠減少腫瘤新生血管的形成,降低腫瘤的生長速度,減少發(fā)生轉(zhuǎn)移浸潤的機(jī)會。 將兩組腫瘤細(xì)胞進(jìn)行Western blot來初步研究mdNCRT/E7/hsp重組融合蛋白疫苗抗腫瘤生長的分子機(jī)制。(1)IL-6/pStat3信號傳導(dǎo)通路：結(jié)果顯示疫苗組的IL-6、p-Stat3、VEGF、HIF-1α、Snail蛋白表達(dá)水平明顯下降,活化的Caspase-3蛋白水平明顯提高。說明mdNCRT/E7/hsp重組融合蛋白疫苗能夠通過抑制IL-6和pStat3以及其下游相關(guān)基因的表達(dá)來抑制血管生成及促進(jìn)了腫瘤細(xì)胞的凋亡。(2)MAPK-ERK信號傳導(dǎo)通路：結(jié)果顯示疫苗組細(xì)胞中B-raf、p-MEK、p-ERK的表達(dá)降低,說明mdNCRT/E7/hsp重組融合蛋白疫苗能夠通過抑制MAPK-ERK信號通路鏈中相關(guān)MAPK激酶活性,抑制腫瘤細(xì)胞的生長。 本研究結(jié)果表明：mdNCRT/E7/hsp重組融合蛋白疫苗可能通過抑制IL-6/pStat3信號傳導(dǎo)通路和MAPK-ERK信號傳導(dǎo)通路來抑制腫瘤新生血管的形成和腫瘤浸潤轉(zhuǎn)移,最終產(chǎn)生有效的抗腫瘤作用。
[Abstract]:Cervical cancer is a common malignant tumor in women. Since the discovery of close association between cervical cancer and HPV infection, HPV therapeutic vaccine has become an important research direction. The recombinant mdNCRT/E7/hsp fusion protein vaccine against HPV16 was constructed in our research group, and it was proved in vitro that the vaccine could induce higher CTL activity and inhibit angiogenesis. In this study, the anti-tumor effect and its molecular mechanism were studied in vivo. The TC-1 tumor model of C57BL/6 mice was established. The results of in vivo experiments showed that the recombinant mdNCRT/E7/hsp fusion protein vaccine could effectively inhibit tumor growth, induce specific tumor rejection, and have therapeutic effect on small tumor lesions without solid tumors. Immunohistochemical analysis of the two groups showed that the recombinant mdNCRT/E7/hsp fusion protein could reduce the angiogenesis of tumor, decrease the growth rate of tumor and reduce the chance of metastasis and infiltration. Two groups of tumor cells were treated with Western blot to study the molecular mechanism of mdNCRT/E7/hsp recombinant fusion protein vaccine against tumor growth. (1) IL-6/pStat3 signal transduction pathway: the results showed that the expression of IL-6,p-Stat3,VEGF,HIF-1 偽 and Snail protein decreased significantly and the activated Caspase-3 protein level increased significantly in the vaccine group. The results showed that mdNCRT/E7/hsp recombinant fusion protein vaccine could inhibit angiogenesis and promote apoptosis of tumor cells by inhibiting the expression of IL-6, pStat3 and its downstream genes. (2) MAPK-ERK signaling pathway: the results showed that the expression of B-raftip-MEKp-ERK was decreased in the cells of vaccine group. The results showed that the recombinant mdNCRT/E7/hsp fusion protein vaccine could inhibit the growth of tumor cells by inhibiting the activity of MAPK kinase in the MAPK-ERK signaling pathway chain. Our results suggest that the recombinant mdNCRT/E7/hsp fusion protein vaccine may inhibit angiogenesis, tumor invasion and metastasis by inhibiting IL-6/pStat3 signal transduction pathway and MAPK-ERK signal transduction pathway, resulting in an effective anti-tumor effect.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R392

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