以BAP31为靶点的新型肿瘤基因疫苗的研制及体内外免疫效果评价

发布时间：2017-12-31 18:35

本文关键词：以BAP31为靶点的新型肿瘤基因疫苗的研制及体内外免疫效果评价　出处：《第四军医大学》2011年硕士论文　论文类型：学位论文

更多相关文章： BAP31 LAMP 肿瘤 基因疫苗 免疫治疗

【摘要】：人口老龄化和人们长期不良生活习惯导致全球肿瘤患者增多,据国际肿瘤研究机构(international agency for research on cancer)统计,2008年全球肿瘤患者新增将近1270万,当年死于肿瘤的患者有760万,其中56%的新增患者和64%的死亡患者发生在发展中国家。目前肿瘤治疗的常规方法有外科手术切除、放疗和化疗,但这些方法都不能很有效地阻止肿瘤的转移、复发和侵袭,这就迫切需要有新的肿瘤治疗策略。 Wolff研究小组首先在小鼠上发现,通过直接将外源性DNA质粒肌肉注射至小鼠体内,该外源DNA编码的蛋白可以长期在骨骼肌表达,这就为DNA疫苗的发展提供了实际依据。严格意义上说,第一个DNA疫苗是用于对抗流感病毒和人类免疫缺陷病毒-1(HIV-1),在这次应用中发现,通过皮下或肌肉注射质粒DNA可以有效刺激机体的免疫应答,由此提示DNA疫苗能刺激机体免疫系统达到预防疾病作用,这就使大规模的预防接种变得更加有意义,特别是针对一些难以大量生产的疫苗,如重组蛋白或全肿瘤细胞疫苗。众所周知,肿瘤细胞之所以不容易被机体免疫系统清除的一个重要原因就是肿瘤抗原的低免疫原性,而DNA疫苗则为克服这一弊端提供了可能,从而成为肿瘤免疫治疗的一种有效策略。在DNA疫苗的动物实验中发现,虽然疫苗能诱导机体产生针对目的蛋白的特异性CTL免疫应答,但在动物体内却没有产生高滴度的特异性抗体,考虑原因为DNA疫苗进入机体,被细胞摄取后所表达的蛋白相对机体是内源性抗原,通过MHC I类提呈途径;而目的抗原通过MHCⅡ类提呈途径呈递给CD4~+T细胞是诱导机体既产生对目的抗原的细胞免疫反应又产生高滴度特异性抗体的必要条件。由于DNA疫苗进入细胞所表达的内源性蛋白无法进入MHCⅡ类提呈途径,因此只能通过引入定位分子将其靶向到MHCⅡ类提呈途径中,从而有效活化CD4~+T细胞。 1985年,通过单克隆抗体发现了定位于溶酶体外膜的溶酶体相关膜蛋白(lysosome associated membrane protein, LAMP),随后在树突状细胞和其他抗原提呈细胞(antigen presenting cells, APC)细胞中又发现LAMP与MHCⅡ类分子共定位于MHCⅡ类分子器室(MⅡC),MⅡC是抗原呈递细胞中降解加工抗原及组装抗原肽与MHCⅡ类分子的重要场所。LAMP分子可通过C末端短胞浆尾中的识别序列Tyr-X-X-φ(φ代表任意一亲水性氨基酸)将特定蛋白从细胞浆靶向输送至溶酶体。因此如果将肿瘤抗原的基因插入LAMP分子溶酶体内腔与穿膜区之间,构建DNA疫苗,可望通过LAMP分子的作用将肿瘤抗原靶向到MHC-Ⅱ类提呈途径中,诱导强的细胞和体液免疫应答。肿瘤/睾丸抗原(cancer/testis antigen, CTA)是一类可在多种肿瘤组织中表达,在除睾丸以外的正常组织中几乎不表达的抗原。由于CTA在肿瘤患者体内具有免疫原性,可以诱导高效的体液免疫和/或细胞免疫,因此以CTA为靶分子的肿瘤疫苗为治疗肿瘤提供了新的策略,如NY-ESO-1作为黑色素瘤治疗性疫苗的靶分子。我室发现BAP31分子高表达于睾丸组织,阳性产物主要定位于精原细胞和初级精母细胞,同时发现其在宫颈癌、卵巢癌、乳腺癌、食道癌、肝癌、结肠癌、直肠癌和肺癌组织中高表达,BAP31蛋白的基因定位与MAGE-A、NY-ESO-1、LAGE-1、SAGE等CTA编码基因毗邻。根据BAP31的限制性表达特点和基因定位,推测BAP31或许为CTA家族中的一种,可作为肿瘤基因疫苗的靶点。本研究将BAP31羧基端366个核苷酸作为目的基因(?BAP31)插入编码LAMP分子luminal domain和transmembrane/cytoplasmic tail基因之间,组成嵌合体构建新型肿瘤基因疫苗。设置DNA疫苗p-LAMP-?BAP31为实验组,p -?BAP31、p-LAMP、PBS为对照组,免疫6-8周龄雌性C57BL/6J小鼠。采取尾根部皮下注射的方式,每次50μg/只,按间隔3周的周期共免疫3次,第5、8周尾静脉采血分离血清用作ELISA检测抗BAP31抗体产生,第2次和最后一次免疫后2周行ELISPOT及杀伤试验。ELISPOT结果表明,?BAP31/GST融合蛋白可刺激实验组脾细胞分泌特异性IFN-γ,且水平显著高于对照组(P0.05)。杀伤试验中,实验组分离的脾细胞对B16细胞(小鼠黑素瘤细胞系)的杀伤率显著高于对照组(P0.05)。间接ELISA结果显示,p-LAMP-?BAP31和p -△BAP31组能产生针对?BAP31的特异性抗体,且随着免疫次数的增加,抗体滴度逐渐增加,至第3次免疫后达到峰值。体内实验证实,p-LAMP-△BAP31和p -△BAP31组能够对肿瘤在小鼠体内的生长产生明显的抑制。总之,以BAP31与LAMP构成的嵌合基因疫苗可通过LAMP分子的靶向作用将BAP31分子运输到MⅡC,并翻转入其中,从而实现DNA疫苗所表达蛋白从内源性抗原的MHC I类加工途径向外源性抗原的MHCⅡ类加工途径的转化,从而有效活化了CD4~+T细胞,诱导高效的细胞和体液免疫应答,为肿瘤治疗性疫苗的研制奠定了坚实的基础。
[Abstract]:The aging of the population and the people long-term bad habits lead to increased cancer patients worldwide, according to the international agency for research on cancer (International Agency for research on cancer) statistics, in 2008 the global nearly 12 million 700 thousand new cancer patients, 7 million 600 thousand patients had died of cancer, which in 56% NEW patients and 64% deaths occur in developing countries. The current conventional method cancer treatment including surgery, radiotherapy and chemotherapy, but these methods are not very effective in preventing tumor metastasis, invasion and recurrence of the tumor, there is an urgent need for new therapeutic strategies.
The Wolff research team first discovered in mice, the direct intramuscular injection of exogenous DNA plasmid into mice, the exogenous DNA encoding the protein can be expressed in skeletal muscle, which provides the practical basis for the development of DNA vaccine. Strictly speaking, the first is for DNA vaccine against influenza virus and human immunodeficiency -1 virus (HIV-1), found in this application, by subcutaneous or intramuscular injection of plasmid DNA can effectively stimulate the body's immune response, suggesting that DNA vaccine can stimulate the immune system to achieve the effect of preventing disease, which makes mass vaccination becomes more meaningful, especially for some difficult to mass production of vaccines, such as recombinant protein or whole tumor cell vaccine. As everyone knows, an important reason for tumor cell is not easy to remove the immune system is the original low free anti tumor The pestilence, and the DNA vaccine is a possibility to overcome this disadvantage, and thus become an effective strategy for cancer immunotherapy.
Found in the animal experiment of DNA vaccine, although the vaccine can induce specific immune responses against CTL protein, but in the animal did not produce high titer antibody, consider the reasons for the DNA vaccine into the body, is after cellular uptake expressed relative to body endogenous protein antigen by MHC. I classpresentation pathway; and the target antigen by MHC class II presentation pathway presented to CD4~+T cells is a necessary condition to induce the generation of cellular immune responses to both the target antigen and produce high titers of specific antibody. The endogenous protein DNA vaccine into cells by the expression of MHC can not enter the class II presentation pathway, therefore only through the introduction of targeted molecular targeting to MHC class II presentation pathway, thus effectively activate CD4~+T cells.
In 1985, the monoclonal antibody was found localized in the lysosome associated membrane protein lysosome membrane (lysosome associated membrane protein, LAMP), followed by dendritic cells and other antigen-presenting cells (antigen presenting cells, APC LAMP) and MHC class II molecules were located in MHC class II molecules is also found in cells (room M C, M II C II) is an important place for.LAMP molecular antigen-presenting cells in the degradation of processing and assembly of peptide antigen and MHC antigen class II molecules can be identified by Tyr-X-X- sequence with C terminal short cytoplasmic tail (the phi represents any a hydrophilic amino acid) specific proteins from the cytoplasm to the targeted delivery lysosomes. So if the tumor antigen genes into the LAMP lysosomal lumen and the transmembrane construct DNA vaccine is targeting the tumor antigen to the MHC- class II presentation pathway by LAMP molecules, induced strong fine Cellular and humoral immune responses.
Cancer / testis antigens (cancer/testis, antigen, CTA) is a kind of expression in various tumor tissues, almost no expression in normal tissues except for testis than in antigen. Because CTA has immunogenicity in cancer patients, can induce efficient humoral and / or cellular immunity, so CTA to provide a new strategy of tumor vaccine molecules for the treatment of tumors, such as NY-ESO-1 as a therapeutic melanoma vaccine target molecules. I found BAP31 molecule highly expressed in testis, was mainly expressed in spermatogonia and primary spermatocyte, also found in cervical cancer, ovarian cancer, breast cancer, esophageal cancer, liver cancer, colon cancer, rectal cancer and high expression in lung cancer tissues, BAP31 protein gene mapping and MAGE-A, NY-ESO-1, LAGE-1, SAGE and CTA. According to the BAP31 encoding gene adjacent to the restricted expression characteristics and gene mapping, suggesting that BAP3 1 may be one of the CTA family, which can be used as a target for tumor gene vaccine.
In this study, BAP31 carboxy terminal 366 nucleotides as target genes (? BAP31) is inserted between LAMP molecules and transmembrane/cytoplasmic domain encoding luminal tail gene, composed of chimeric construct new tumor gene vaccine. The vaccine set DNA p-LAMP-? BAP31 as the experimental group, BAP31, p-LAMP, P -? PBS as control group, immune 6-8 week old female C57BL/6J mice. Take tail root subcutaneous injection, each 50 g/, according to the interval 3 weeks were immunized 3 times in 5,8 weeks of tail vein blood serum was separated for ELISA detection of anti BAP31 antibody, the second and the last 2 weeks after immunization with ELISPOT and.ELISPOT cytotoxicity assay results show that? BAP31/GST fusion protein can stimulate the secretion of spleen cells of experimental group specific IFN- gamma, and was significantly higher than the control group (P0.05). The killing experiment, the experimental group of B16 cells isolated spleen cells (mouse melanoma cell line) killer The rate was significantly higher than the control group (P0.05). The results of indirect ELISA showed that p-LAMP-? BAP31 and P - a BAP31 group can produce for? BAP31 specific antibody, and with the increase of immunization times, antibody titer gradually increased to third after immunization and reached the peak. In vivo experiments confirmed that p-LAMP- BAP31 and P - a the BAP31 group can significantly inhibit the tumor growth in mice.
In short, the chimeric gene vaccine consisting of BAP31 and LAMP by LAMP molecular targeting of BAP31 molecular transport to M II C, and turned into one, so as to realize the DNA vaccine protein expression from the endogenous antigen processing pathway to MHC class I MHC class II processing way of exogenous antigens to the diameter of the transformation. The effective activation of CD4~+T cells, induce cellular and humoral immune responses, and developed as a therapeutic cancer vaccine has laid a solid foundation.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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