【摘要】：編碼腫瘤特異性抗原蛋白的DNA疫苗不僅能誘導(dǎo)機(jī)體產(chǎn)生特異性抗腫瘤的CD8+T細(xì)胞的免疫應(yīng)答,而且還能誘導(dǎo)機(jī)體產(chǎn)生體液免疫,從而顯示出抗腫瘤免疫的優(yōu)勢(shì)。對(duì)于顱內(nèi)腫瘤來(lái)說(shuō)編碼腫瘤特異性抗原蛋白的DNA免疫不僅可以減少自身免疫的風(fēng)險(xiǎn),還能精確地消除表達(dá)特異性抗原的腫瘤細(xì)胞。然而,裸DNA的免疫誘導(dǎo)能力極差,需要借助其他的載體或物理輸送方法來(lái)提高其免疫效果。電脈沖是目前最為有效的質(zhì)粒DNA物理輸送方法,而且還能提高DNA疫苗的免疫應(yīng)答效果。腫瘤特異性抗原蛋白,表皮生長(zhǎng)因子受體突變體Ⅲ(EGFRvⅢ)是腦膠質(zhì)瘤最常見(jiàn)的突變體抗原之一。本文中以EGFRvIII為抗原蛋白構(gòu)建DNA疫苗,并利用電脈沖介導(dǎo)DNA疫苗免疫小鼠,驗(yàn)證該DNA疫苗的免疫誘導(dǎo)能力及抑制EGFRvIII陽(yáng)性腫瘤細(xì)胞在顱內(nèi)成瘤和生長(zhǎng)的效果。我們首先克隆并構(gòu)建了EGFRvIII的表達(dá)質(zhì)粒;經(jīng)脂質(zhì)體轉(zhuǎn)染B16F10細(xì)胞后篩選穩(wěn)定表達(dá)該蛋白的細(xì)胞株(B16F10-EGFRvIII),并用蛋白印跡及細(xì)胞免疫化學(xué)方法進(jìn)行驗(yàn)證;將DNA疫苗用電脈沖方法免疫小鼠并用CTL檢測(cè)DNA疫苗的免疫效果;免疫小鼠皮下和顱內(nèi)接種腫瘤細(xì)胞,驗(yàn)證電脈沖DNA免疫對(duì)腫瘤形成和生長(zhǎng)的抑制效果。研究結(jié)果表明我們構(gòu)建的重組質(zhì)粒能高效表達(dá)EGFRvIII;篩選所得穩(wěn)轉(zhuǎn)細(xì)胞株,EGFRvIII能正確的表達(dá)和定位;與未免疫組相比,電脈沖介導(dǎo)DNA疫苗免疫能誘導(dǎo)更顯著的細(xì)胞免疫應(yīng)答(p0.05),并顯示對(duì)皮下和顱內(nèi)腫瘤的形成和生長(zhǎng)有明顯的抑制效果(s.c.,p=0.002,i.c.,p=0.02)。因此,電脈沖介導(dǎo)EGFRvIII DNA免疫有望用于顱內(nèi)腫瘤的免疫治療,并顯著的抑制顱內(nèi)腫瘤的生長(zhǎng)。
[Abstract]:The DNA vaccine encoding tumor specific antigen protein can not only induce the immune response of specific anti-tumor CD8 T cells, but also induce humoral immunity, thus showing the advantages of anti-tumor immunity. For intracranial tumors, DNA immunization encoding tumor specific antigen proteins can not only reduce the risk of autoimmunity, but also accurately eliminate tumor cells expressing specific antigens. However, the immune induction ability of naked DNA is very poor, so it is necessary to use other carriers or physical delivery methods to improve its immune effect. Electric pulse is the most effective physical delivery method of plasmid DNA at present, and it can also improve the immune response of DNA vaccine. Tumor specific antigen protein and epidermis growth factor receptor mutant III (EGFRv III) are one of the most common mutant antigens in glioma. In this paper, DNA vaccine was constructed with EGFRvIII as antigen protein, and mice were immunized with DNA vaccine mediated by electric pulse to verify the immune induction ability of DNA vaccine and the inhibitory effect of EGFRvIII positive tumor cells on intracranial tumorigenesis and growth. Firstly, the expression plasmid of EGFRvIII was cloned and constructed. The cell line (B16F10-EGFRvIII) stably expressing the protein was screened after B16F10 cells were stably expressed by liposomes and verified by Western imprinting and immunochemical methods. Mice were immunized with DNA vaccine by electric pulse method and the immune effect of DNA vaccine was detected by CTL. The inhibitory effect of electric pulse DNA immunization on tumor formation and growth was verified by subcutaneous and intracranial inoculation of tumor cells in immunized mice. The results showed that the recombinant plasmid could highly express the stable cell lines screened by EGFRvIII;, and EGFRvIII could be expressed and located correctly. Compared with the control group, electric pulse mediated DNA vaccine immunization could induce more significant cellular immune response (p0.05), and showed obvious inhibitory effect on the formation and growth of subcutaneous and intracranial tumors (s.c., p 鈮，

本文編號(hào)：2504702