發(fā)布時(shí)間：2017-12-31 18:35

  本文關(guān)鍵詞：以BAP31為靶點(diǎn)的新型腫瘤基因疫苗的研制及體內(nèi)外免疫效果評(píng)價(jià)　出處：《第四軍醫(yī)大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： BAP31 LAMP 腫瘤 基因疫苗 免疫治療

【摘要】：人口老齡化和人們長期不良生活習(xí)慣導(dǎo)致全球腫瘤患者增多,據(jù)國際腫瘤研究機(jī)構(gòu)(international agency for research on cancer)統(tǒng)計(jì),2008年全球腫瘤患者新增將近1270萬,當(dāng)年死于腫瘤的患者有760萬,其中56%的新增患者和64%的死亡患者發(fā)生在發(fā)展中國家。目前腫瘤治療的常規(guī)方法有外科手術(shù)切除、放療和化療,但這些方法都不能很有效地阻止腫瘤的轉(zhuǎn)移、復(fù)發(fā)和侵襲,這就迫切需要有新的腫瘤治療策略。 Wolff研究小組首先在小鼠上發(fā)現(xiàn),通過直接將外源性DNA質(zhì)粒肌肉注射至小鼠體內(nèi),該外源DNA編碼的蛋白可以長期在骨骼肌表達(dá),這就為DNA疫苗的發(fā)展提供了實(shí)際依據(jù)。嚴(yán)格意義上說,第一個(gè)DNA疫苗是用于對(duì)抗流感病毒和人類免疫缺陷病毒-1(HIV-1),在這次應(yīng)用中發(fā)現(xiàn),通過皮下或肌肉注射質(zhì)粒DNA可以有效刺激機(jī)體的免疫應(yīng)答,由此提示DNA疫苗能刺激機(jī)體免疫系統(tǒng)達(dá)到預(yù)防疾病作用,這就使大規(guī)模的預(yù)防接種變得更加有意義,特別是針對(duì)一些難以大量生產(chǎn)的疫苗,如重組蛋白或全腫瘤細(xì)胞疫苗。眾所周知,腫瘤細(xì)胞之所以不容易被機(jī)體免疫系統(tǒng)清除的一個(gè)重要原因就是腫瘤抗原的低免疫原性,而DNA疫苗則為克服這一弊端提供了可能,從而成為腫瘤免疫治療的一種有效策略。 在DNA疫苗的動(dòng)物實(shí)驗(yàn)中發(fā)現(xiàn),雖然疫苗能誘導(dǎo)機(jī)體產(chǎn)生針對(duì)目的蛋白的特異性CTL免疫應(yīng)答,但在動(dòng)物體內(nèi)卻沒有產(chǎn)生高滴度的特異性抗體,考慮原因?yàn)镈NA疫苗進(jìn)入機(jī)體,被細(xì)胞攝取后所表達(dá)的蛋白相對(duì)機(jī)體是內(nèi)源性抗原,通過MHC I類提呈途徑;而目的抗原通過MHCⅡ類提呈途徑呈遞給CD4~+T細(xì)胞是誘導(dǎo)機(jī)體既產(chǎn)生對(duì)目的抗原的細(xì)胞免疫反應(yīng)又產(chǎn)生高滴度特異性抗體的必要條件。由于DNA疫苗進(jìn)入細(xì)胞所表達(dá)的內(nèi)源性蛋白無法進(jìn)入MHCⅡ類提呈途徑,因此只能通過引入定位分子將其靶向到MHCⅡ類提呈途徑中,從而有效活化CD4~+T細(xì)胞。 1985年,通過單克隆抗體發(fā)現(xiàn)了定位于溶酶體外膜的溶酶體相關(guān)膜蛋白(lysosome associated membrane protein, LAMP),隨后在樹突狀細(xì)胞和其他抗原提呈細(xì)胞(antigen presenting cells, APC)細(xì)胞中又發(fā)現(xiàn)LAMP與MHCⅡ類分子共定位于MHCⅡ類分子器室(MⅡC),MⅡC是抗原呈遞細(xì)胞中降解加工抗原及組裝抗原肽與MHCⅡ類分子的重要場所。LAMP分子可通過C末端短胞漿尾中的識(shí)別序列Tyr-X-X-φ(φ代表任意一親水性氨基酸)將特定蛋白從細(xì)胞漿靶向輸送至溶酶體。因此如果將腫瘤抗原的基因插入LAMP分子溶酶體內(nèi)腔與穿膜區(qū)之間,構(gòu)建DNA疫苗,可望通過LAMP分子的作用將腫瘤抗原靶向到MHC-Ⅱ類提呈途徑中,誘導(dǎo)強(qiáng)的細(xì)胞和體液免疫應(yīng)答。 腫瘤/睪丸抗原(cancer/testis antigen, CTA)是一類可在多種腫瘤組織中表達(dá),在除睪丸以外的正常組織中幾乎不表達(dá)的抗原。由于CTA在腫瘤患者體內(nèi)具有免疫原性,可以誘導(dǎo)高效的體液免疫和/或細(xì)胞免疫,因此以CTA為靶分子的腫瘤疫苗為治療腫瘤提供了新的策略,如NY-ESO-1作為黑色素瘤治療性疫苗的靶分子。我室發(fā)現(xiàn)BAP31分子高表達(dá)于睪丸組織,陽性產(chǎn)物主要定位于精原細(xì)胞和初級(jí)精母細(xì)胞,同時(shí)發(fā)現(xiàn)其在宮頸癌、卵巢癌、乳腺癌、食道癌、肝癌、結(jié)腸癌、直腸癌和肺癌組織中高表達(dá),BAP31蛋白的基因定位與MAGE-A、NY-ESO-1、LAGE-1、SAGE等CTA編碼基因毗鄰。根據(jù)BAP31的限制性表達(dá)特點(diǎn)和基因定位,推測BAP31或許為CTA家族中的一種,可作為腫瘤基因疫苗的靶點(diǎn)。 本研究將BAP31羧基端366個(gè)核苷酸作為目的基因(?BAP31)插入編碼LAMP分子luminal domain和transmembrane/cytoplasmic tail基因之間,組成嵌合體構(gòu)建新型腫瘤基因疫苗。設(shè)置DNA疫苗p-LAMP-?BAP31為實(shí)驗(yàn)組,p -?BAP31、p-LAMP、PBS為對(duì)照組,免疫6-8周齡雌性C57BL/6J小鼠。采取尾根部皮下注射的方式,每次50μg/只,按間隔3周的周期共免疫3次,第5、8周尾靜脈采血分離血清用作ELISA檢測抗BAP31抗體產(chǎn)生,第2次和最后一次免疫后2周行ELISPOT及殺傷試驗(yàn)。ELISPOT結(jié)果表明,?BAP31/GST融合蛋白可刺激實(shí)驗(yàn)組脾細(xì)胞分泌特異性IFN-γ,且水平顯著高于對(duì)照組(P0.05)。殺傷試驗(yàn)中,實(shí)驗(yàn)組分離的脾細(xì)胞對(duì)B16細(xì)胞(小鼠黑素瘤細(xì)胞系)的殺傷率顯著高于對(duì)照組(P0.05)。間接ELISA結(jié)果顯示,p-LAMP-?BAP31和p -△BAP31組能產(chǎn)生針對(duì)?BAP31的特異性抗體,且隨著免疫次數(shù)的增加,抗體滴度逐漸增加,至第3次免疫后達(dá)到峰值。體內(nèi)實(shí)驗(yàn)證實(shí),p-LAMP-△BAP31和p -△BAP31組能夠?qū)δ[瘤在小鼠體內(nèi)的生長產(chǎn)生明顯的抑制。 總之,以BAP31與LAMP構(gòu)成的嵌合基因疫苗可通過LAMP分子的靶向作用將BAP31分子運(yùn)輸?shù)組ⅡC,并翻轉(zhuǎn)入其中,從而實(shí)現(xiàn)DNA疫苗所表達(dá)蛋白從內(nèi)源性抗原的MHC I類加工途徑向外源性抗原的MHCⅡ類加工途徑的轉(zhuǎn)化,從而有效活化了CD4~+T細(xì)胞,誘導(dǎo)高效的細(xì)胞和體液免疫應(yīng)答,為腫瘤治療性疫苗的研制奠定了堅(jiān)實(shí)的基礎(chǔ)。
[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.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392.1

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