电脉冲介导表达EGFRv ⅢDNA免疫预防小鼠颅内肿瘤研究
发布时间：2019-06-22 15:18
【摘要】：编码肿瘤特异性抗原蛋白的DNA疫苗不仅能诱导机体产生特异性抗肿瘤的CD8+T细胞的免疫应答,而且还能诱导机体产生体液免疫,从而显示出抗肿瘤免疫的优势。对于颅内肿瘤来说编码肿瘤特异性抗原蛋白的DNA免疫不仅可以减少自身免疫的风险,还能精确地消除表达特异性抗原的肿瘤细胞。然而,裸DNA的免疫诱导能力极差,需要借助其他的载体或物理输送方法来提高其免疫效果。电脉冲是目前最为有效的质粒DNA物理输送方法,而且还能提高DNA疫苗的免疫应答效果。肿瘤特异性抗原蛋白,表皮生长因子受体突变体Ⅲ(EGFRvⅢ)是脑胶质瘤最常见的突变体抗原之一。本文中以EGFRvIII为抗原蛋白构建DNA疫苗,并利用电脉冲介导DNA疫苗免疫小鼠,验证该DNA疫苗的免疫诱导能力及抑制EGFRvIII阳性肿瘤细胞在颅内成瘤和生长的效果。我们首先克隆并构建了EGFRvIII的表达质粒;经脂质体转染B16F10细胞后筛选稳定表达该蛋白的细胞株(B16F10-EGFRvIII),并用蛋白印迹及细胞免疫化学方法进行验证;将DNA疫苗用电脉冲方法免疫小鼠并用CTL检测DNA疫苗的免疫效果;免疫小鼠皮下和颅内接种肿瘤细胞,验证电脉冲DNA免疫对肿瘤形成和生长的抑制效果。研究结果表明我们构建的重组质粒能高效表达EGFRvIII;筛选所得稳转细胞株,EGFRvIII能正确的表达和定位;与未免疫组相比,电脉冲介导DNA疫苗免疫能诱导更显著的细胞免疫应答(p0.05),并显示对皮下和颅内肿瘤的形成和生长有明显的抑制效果(s.c.,p=0.002,i.c.,p=0.02)。因此,电脉冲介导EGFRvIII DNA免疫有望用于颅内肿瘤的免疫治疗,并显著的抑制颅内肿瘤的生长。
[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，
本文编号：2504702
[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，
本文编号：2504702
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