發(fā)布時(shí)間：2018-05-14 10:16

  本文選題：重組免疫毒素 + CTLA-4��； 參考：《中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院》2012年博士論文

【摘要】：器官移植是治療終末期器官功能衰竭的有效手段,免疫抑制劑的應(yīng)用可以減輕移植排斥反應(yīng),但化學(xué)類免疫抑制劑毒副作用明顯,影響了其臨床應(yīng)用。生物制劑類免疫抑制劑尤其是免疫毒素可以通過(guò)其特異性的結(jié)合作用,清除抗原特異性T細(xì)胞,且毒副作用較小,有更好的應(yīng)用前景。 免疫毒素是具有導(dǎo)向能力的分子(載體)和具有細(xì)胞毒性的分子(毒素)偶聯(lián)而成的具有特異性細(xì)胞殺傷能力的雜合分子,在治療惡性腫瘤、移植排斥等方面有良好的應(yīng)用前景。 CTLA-4是一種僅表達(dá)于活化T細(xì)胞而靜止T細(xì)胞不表達(dá)的分子,因此CTLA-4的抗原結(jié)合分子是一種理想的免疫毒素導(dǎo)向分子,由之產(chǎn)生的免疫毒素只對(duì)活化的T細(xì)胞或腫瘤細(xì)胞有殺傷作用,避免了對(duì)正常靜息T細(xì)胞的非特異性損傷。 蜂毒肽(Melittin, Mel)是蜜蜂蜂毒的主要成分,是一種重要的抗菌肽,具有抗菌、抗輻射、抗腫瘤等作用。它由26個(gè)氨基酸組成,分子量小,免疫原性低,不易產(chǎn)生過(guò)敏反應(yīng)；具有膜活性,直接對(duì)細(xì)胞的磷脂膜起溶解作用,抑制細(xì)胞發(fā)育,是一種較理想毒素片段。 本研究以人源化抗CTLA-4單鏈抗體為靶向片段,特異性結(jié)合在表達(dá)CTLA-4的活化T細(xì)胞表面,以蜂毒肽類似物為毒素片段,特異性殺傷活化T細(xì)胞,設(shè)計(jì)構(gòu)建出了高效低毒的免疫毒素分子,主要用于抗移植排斥反應(yīng)。 利用重疊延伸PCR技術(shù),通過(guò)兩步PCR獲得了重組免疫毒素全長(zhǎng)基因,將其與表達(dá)載體pBV220連接,轉(zhuǎn)化大腸桿菌,重組基因在大腸桿菌表達(dá)系統(tǒng)中以包涵體形式表達(dá)。包涵體用8M脲變性,采用稀釋復(fù)性,然后用SP-Sepharose-Fast-Flow陽(yáng)離子交換柱分離純化,Sephacryl S-200凝膠排阻層析柱精制,制備得到目的蛋白CTLA4-ScFv-Mel. 在體外活性研究中,2μmo1/L的CTLA4-ScFv-Mel分別作用于非活化的T淋巴細(xì)胞、陰性對(duì)照細(xì)胞ECV-304、ConA激活的T淋巴細(xì)胞、CTLA-4陽(yáng)性細(xì)胞Raji知Jurkat細(xì)胞。結(jié)果顯示,給藥24h后各組細(xì)胞存活率下降,其中前兩組細(xì)胞的存活率分別為78.9%和77.3%；而后三組分別為31.2%、32.7%和30.8%。說(shuō)明CTLA4-ScFv-Mel對(duì)CTLA-4陽(yáng)性細(xì)胞的殺傷率高于正常細(xì)胞,表現(xiàn)出細(xì)胞殺傷的選擇性和高效率。 體內(nèi)活性研究以BEL-7402肝癌移植瘤模型和S180小鼠移植瘤模型為對(duì)象,分低、中、高三個(gè)劑量給藥,比較試驗(yàn)組和陰性對(duì)照組移植瘤相對(duì)體積等藥效學(xué)指標(biāo)。結(jié)果顯示,CTLA4-ScFv-Mel能夠發(fā)揮促進(jìn)兩個(gè)試驗(yàn)組模型中移植瘤的存活和生長(zhǎng)的作用,且呈現(xiàn)一定的劑量效應(yīng)關(guān)系。推測(cè)該現(xiàn)象的出現(xiàn)與抑制T細(xì)胞免疫有關(guān)。 本研究成功制備了一種全新的免疫毒素融合蛋白,并進(jìn)行了初步活性研究和藥效學(xué)評(píng)價(jià),為器官移植提供了潛在的選擇藥物,具有一定的科學(xué)價(jià)值和開發(fā)前景。
[Abstract]:Organ transplantation is an effective method for the treatment of end-stage organ failure. The application of immunosuppressant can alleviate the rejection of transplantation, but the toxicity and side effect of chemical immunosuppressant is obvious, which affects its clinical application. The immunosuppressants, especially the immunotoxins, can remove antigen-specific T cells through their specific binding, and the toxicity and side effects are relatively small, so it has a better application prospect. Immunotoxins are conjugated molecules (vectors) that have the ability to direct and have cytotoxic molecules (toxins) to form hybrids with specific cytotoxic abilities in the treatment of malignant tumors. Transplantation rejection and other aspects have a good application prospects. CTLA-4 is a molecule expressed only in activated T cells but not expressed in static T cells. Therefore, antigen-binding molecules of CTLA-4 are ideal immunotoxin oriented molecules. The resulting immunotoxins can only kill activated T cells or tumor cells and avoid nonspecific damage to normal resting T cells. Melittin (melittin) is the main component of bee venom and is an important antimicrobial peptide. It has antibacterial, anti-radiation and anti-tumor effects. It is composed of 26 amino acids with low molecular weight and low immunogenicity, which is not easy to produce allergic reaction. It has membrane activity, which directly dissolves phospholipid membrane and inhibits cell development. It is an ideal toxin fragment. In this study, the humanized single chain antibody against CTLA-4 was used as the target fragment, specifically binding to the surface of activated T cells expressing CTLA-4, and the propolis analogue was used as the toxin fragment to specifically kill activated T cells. A highly effective and low-toxic immunotoxin molecule was designed and constructed, which was mainly used to resist transplant rejection. The full-length gene of recombinant immunotoxin was obtained by two-step PCR using overlapping extension PCR technique. The recombinant gene was ligated with the expression vector pBV220 and transformed into E. coli. The recombinant gene was expressed in the form of inclusion body in the expression system of Escherichia coli. The inclusion bodies were denatured with 8M-urea, then refolded by dilution, then purified by SP-Sepharose-Fast-Flow cationic exchange column, then purified by Sephacryl S-200 gel exclusion chromatography. The target protein CTLA4-ScFv-Melwas obtained. In vitro activity study, 2 渭 mo1/L CTLA4-ScFv-Mel acted on non-activated T lymphocytes, while negative control cells, ECV-304 Cona activated T lymphocytes, Raji positive cells known Jurkat cells. The results showed that the cell survival rate of the first two groups was 78.9% and 77.3%, respectively, while that of the latter three groups was 31.2% and 30.8%, respectively. The results showed that the killing rate of CTLA4-ScFv-Mel to CTLA-4 positive cells was higher than that of normal cells, which showed the selectivity and high efficiency of cell killing. In vivo activity study was carried out on BEL-7402 liver cancer transplanted tumor model and S180 mouse transplanted tumor model. The pharmacodynamic indexes were compared between the experimental group and the negative control group at three doses: low, middle and high doses. The results showed that CTLA4-ScFv-Mel could promote the survival and growth of transplanted tumor in two experimental groups, and showed a dose-effect relationship. It is speculated that the occurrence of this phenomenon is related to the suppression of T cell immunity. In this study, a novel fusion protein of immunotoxin was successfully prepared, and the preliminary activity and pharmacodynamics evaluation were carried out, which provided a potential drug selection for organ transplantation, which has certain scientific value and development prospect.
【學(xué)位授予單位】：中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392.4

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