【摘要】：Mucin 1(簡稱MUC1),是黏蛋白家族的跨膜糖蛋白,由多肽核芯和側(cè)枝糖鏈構(gòu)成,主要分布于正常腺管上皮細(xì)胞及其來源的癌細(xì)胞以及部分血液腫瘤細(xì)胞表面,作為癌基因在癌細(xì)胞的發(fā)生發(fā)展中發(fā)揮重要作用。由于MUC1在正常組織和癌組織中存在明顯的差別,使其成為腫瘤疫苗研究的有效靶點(diǎn)。目前,以MUC1為靶點(diǎn)研制的腫瘤疫苗主要包括糖疫苗、蛋白或多肽疫苗和DNA疫苗。其中重組蛋白疫苗因安全、成本低廉而倍受關(guān)注,并且反復(fù)動物實(shí)驗(yàn)和部分人體實(shí)驗(yàn)證實(shí)其安全有效。本研究組將編碼人類MUC1基因按正確閱讀框插入高效表達(dá)麥芽糖結(jié)合蛋白(maltose binding protein,MBP)的載體中,構(gòu)建了表達(dá)MUC1-MBP的重組質(zhì)粒,獲得穩(wěn)定表達(dá)重組MUC1-MBP融合蛋白的工程菌,建立了中試發(fā)酵和蛋白生產(chǎn)工藝。我們前期研究結(jié)果顯示,重組MUC1-MBP抗腫瘤疫苗可以誘導(dǎo)MUC1特異性Th1型免疫應(yīng)答,促進(jìn)MUC1特異性細(xì)胞毒性T細(xì)胞的殺傷活性,并且可以抑制MUC1+B16細(xì)胞在小鼠體內(nèi)的生長。為了促使重組MUC1-MBP抗腫瘤疫苗進(jìn)入I期臨床試驗(yàn),本課題對重組MUC1-MBP抗腫瘤疫苗的毒性及免疫原性進(jìn)行研究,包括小鼠單劑量急性毒性、大鼠反復(fù)劑量慢性毒性和免疫原性以及探索性食蟹猴毒性和免疫原性研究。結(jié)果顯示,除了大鼠中偶發(fā)BCG誘導(dǎo)的關(guān)節(jié)炎和局部結(jié)節(jié),在動物體內(nèi)重組MUC1-MBP抗腫瘤疫苗沒有導(dǎo)致明顯的器官毒性。另外,重組MUC1-MBP抗腫瘤疫苗在大鼠中顯著的誘導(dǎo)IFN-γ、TNF的分泌,揭示Th1細(xì)胞被激活。重組MUC1-MBP抗腫瘤疫苗在大鼠和食蟹猴中均誘導(dǎo)了MUC1特異性Ig G抗體。本研究提示重組MUC1-MBP抗腫瘤疫苗對大鼠和小鼠無明顯毒性,并且大鼠和食蟹猴體免疫后產(chǎn)生較強(qiáng)的免疫活性。本研究為推動重組MUC1-MBP進(jìn)入臨床試驗(yàn)奠定實(shí)驗(yàn)基礎(chǔ),為臨床研究方案的設(shè)計提供依據(jù)。
[Abstract]:Mucin 1 (MUC1), a transmembrane glycoprotein of mucin family, is composed of polypeptide core and side sugar chain. It mainly distributes on the surface of normal adenoepithelial cells, cancer cells derived from them and some blood tumor cells. As a oncogene, it plays an important role in the carcinogenesis and development of cancer cells. Because of the obvious difference between normal tissue and cancer tissue, MUC1 has become an effective target for tumor vaccine research. At present, tumor vaccines targeting MUC1 include sugar vaccine, protein or polypeptide vaccine and DNA vaccine. Among them, recombinant protein vaccine has attracted much attention because of its safety and low cost, and has been proved to be safe and effective by repeated animal experiments and some human experiments. In this study, the human MUC1 gene was inserted into the vector expressing maltose binding protein (maltose binding protein,MBP) according to the correct reading frame, and the recombinant plasmid expressing MUC1-MBP was constructed, and the engineering bacteria stably expressing the recombinant MUC1-MBP fusion protein were obtained. A pilot-scale fermentation and protein production process was established. Our previous studies showed that the recombinant MUC1-MBP antitumor vaccine could induce MUC1 specific Th1 type immune response, promote the cytotoxic T cell killing activity of MUC1 specific cells, and inhibit the growth of MUC1 B16 cells in mice. In order to make recombinant MUC1-MBP antitumor vaccine enter phase I clinical trial, the toxicity and immunogenicity of recombinant MUC1-MBP antitumor vaccine were studied, including single dose acute toxicity in mice. The chronic toxicity and immunogenicity of repeated doses of rats and the toxicity and immunogenicity of exploratory crab-eating monkeys were studied. The results showed that, except for the occasional BCG induced arthritis and local nodules in rats, the recombinant MUC1-MBP antitumor vaccine did not cause significant organ toxicity in animals. In addition, recombinant MUC1-MBP antitumor vaccine significantly induced the secretion of IFN- 緯 and TNF in rats, which revealed that Th1 cells were activated. The recombinant MUC1-MBP antitumor vaccine induced MUC1 specific Ig G antibody in both rat and monkey. This study suggested that the recombinant MUC1-MBP antitumor vaccine had no obvious toxicity to rats and mice, and the immune activity of rats and crab-eating monkeys was stronger. This study provides experimental basis for promoting recombinant MUC1-MBP into clinical trials and provides basis for the design of clinical research scheme.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R730.3

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