發(fā)布時間：2018-03-14 23:28

  本文選題：程序性死亡配體-1　切入點(diǎn)：嵌合抗原受體　出處：《浙江理工大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：繼手術(shù),放療,化療后,腫瘤的免疫療法已成為第四種行之有效的治療手段,近期因發(fā)展迅速,而廣受關(guān)注。其中,嵌合抗原受體T細(xì)胞技術(shù)(Chimeric Antigen Receptors-T cell,CAR-T)是一種新興的過繼細(xì)胞療法(Adoptive cell transfer therapy,ACT),該技術(shù)將患者的T細(xì)胞在體外修飾,活化和增殖后回輸?shù)交颊唧w內(nèi),通過T細(xì)胞表達(dá)的特異性受體,靶向性的識別腫瘤細(xì)胞,并顯示較強(qiáng)的殺傷活性和持久性。PD-L1(Programmed death ligand-1)是程序性死亡分子1(Programmed death-1,PD-1)的配體,主要誘導(dǎo)性的表達(dá)在上皮細(xì)胞(如腫瘤細(xì)胞)和免疫細(xì)胞(如腫瘤浸潤淋巴細(xì)胞)等,在免疫應(yīng)答負(fù)調(diào)控中發(fā)揮重要作用,PD-1/PD-L1信號通路的激活,有利于形成腫瘤微環(huán)境,使腫瘤細(xì)胞逃避免疫系統(tǒng)的免疫監(jiān)視和免疫殺傷,而阻斷該信號通路,在一定程度上,促進(jìn)可識別腫瘤抗原并產(chǎn)生特異性反應(yīng)的T細(xì)胞增殖,發(fā)揮其殺傷腫瘤細(xì)胞的作用。故本課題設(shè)計(jì)了PD-L1抗原特異CAR-T細(xì)胞,既可以阻斷PD-1/PD-L1信號通路,提高T細(xì)胞介導(dǎo)的腫瘤免疫治療,又可以特異性的殺死腫瘤細(xì)胞,并且構(gòu)建的第三代CAR基因,可以增強(qiáng)殺傷的活性和持久性。本研究構(gòu)建了PD-L1(73-739)基因的原核表達(dá)載體,分離純化His-PD-L1融合蛋白,免疫BALB/c小鼠,利用細(xì)胞融合技術(shù)和有限稀釋法,篩選獲得2株能夠穩(wěn)定分泌PD-L1單克隆抗體的雜交瘤細(xì)胞株,雜交瘤細(xì)胞腹腔接種BALB/c雌性小鼠,通過收集腹水制備并純化抗體,進(jìn)行Western Boltting驗(yàn)證,利用ELISA法檢測,優(yōu)選其中一株雜交瘤細(xì)胞株,提取雜交瘤細(xì)胞總RNA,逆轉(zhuǎn)錄合成第一鏈cDNA,PCR擴(kuò)增單克隆抗體單鏈可變區(qū)片段,克隆片段經(jīng)人源化替換與共刺激信號分子CD28、CD137和胞內(nèi)信號分子CD3-ζ鏈的基因組合,構(gòu)建并合成第三代CAR基因,將合成的CAR基因構(gòu)建于慢病毒載體pCDH-CMV-MCS-EF1-copGFP上,慢病毒載體與包膜載體pLP1、pLP2以及包被載體pLP-VSVG按照優(yōu)化比例,利用脂質(zhì)體3000共轉(zhuǎn)染到293T細(xì)胞完成慢病毒包裝,使用PEG濃縮法,獲得有活性的抗PD-L1慢病毒,使用CD8+磁珠分離外周血單個核細(xì)胞(peripheral blood mononuclear cell,PBMC)中的CD8+T細(xì)胞,經(jīng)過體外刺激,慢病毒感染CD8+T細(xì)胞,待其擴(kuò)增5 d左右,進(jìn)行CAR表達(dá)的測定,擴(kuò)增20倍左右,利用非放射性細(xì)胞毒性殺傷CytoTox 96和ELISA法檢測IFN-γ的分泌來測定CAR-T細(xì)胞的體外活性。本研究在成功制備了PD-L1單克隆抗體的基礎(chǔ)上,進(jìn)一步構(gòu)建了PD-L1特異性的嵌合抗原受體pCDH-CMV-MCS-EF1-copGFP-CAR,轉(zhuǎn)染293T細(xì)胞的效率可達(dá)到95.66%以上。包裝的慢病毒的滴度可達(dá)到測定滴度的結(jié)果為5×10~7 TU/mL,可成功感染CD8+T細(xì)胞,CAR的表達(dá)率可達(dá)到22%以上。經(jīng)過CAR-T細(xì)胞的體外活性測定,證明PD-L1特異性CAR-T細(xì)胞有一定的殺傷力,為進(jìn)一步的體內(nèi)試驗(yàn)研究和臨床試驗(yàn)提供了重要的技術(shù)支撐。
[Abstract]:After surgery, radiotherapy and chemotherapy, immunotherapy for tumors has become an effective treatment for 4th types of cancer. Recently, because of its rapid development, it has attracted much attention. Chimeric Antigen Receptors-T cell (CAR-T) is a novel adoptive cell transfer therapeutic technique that modifies, activates and proliferates T cells in vitro and infuses them into the body of patients. Targeted recognition of tumor cells and their strong cytotoxicity and persistence. PD-L1 programmatic death ligand-1 are the ligands of programmed death molecule 1hp death-1 (PD-1). The main inducible expression in epithelial cells (such as tumor cells) and immune cells (such as tumor infiltrating lymphocytes) plays an important role in the negative regulation of immune response and the activation of PD-1 / PD-L1 signaling pathway, which is beneficial to the formation of tumor microenvironment. To prevent tumor cells from immune surveillance and killing in the immune system, and to block the signaling pathway, to some extent, to promote the proliferation of T cells that recognize tumor antigens and produce specific responses. Therefore, we designed PD-L1 antigen-specific CAR-T cells, which can block PD-1/PD-L1 signaling pathway, improve tumor immunotherapy mediated by T cells, and specifically kill tumor cells. In this study, the prokaryotic expression vector of PD-L1O73-739) gene was constructed, His-PD-L1 fusion protein was isolated and purified, BALB/c mice were immunized, and cell fusion technique and limited dilution method were used to immunize BALB/c mice. Two hybridoma cell lines which could stably secrete monoclonal antibody to PD-L1 were obtained. The hybridoma cells were inoculated intraperitoneally with BALB/c female mice. The antibody was prepared and purified by collecting ascites, and the antibody was verified by Western Boltting, and detected by ELISA method. One of the hybridoma cell lines was selected, the total RNAs were extracted from the hybridoma cells, and the single-strand variable region fragment of monoclonal antibody was amplified by reverse transcription synthesis of the first strand cDNA-PCR. The third generation CAR gene was constructed and synthesized by the combination of human substitution and costimulatory signal molecule CD28 + CD137 and intracellular signal molecule CD3- 味 chain. The synthesized CAR gene was constructed on the lentivirus vector pCDH-CMV-MCS-EF1-copGFP. Lentivirus vector, pLP1pLP2 and pLP-VSVG were cotransfected into 293T cells by liposome 3000 to complete the packaging of lentivirus. The active anti-lentivirus was obtained by PEG concentration method. CD8 magnetic beads were used to isolate CD8 T cells from peripheral blood mononuclear cells (PBMC). After stimulation in vitro, lentivirus was infected with CD8 T cells. After 5 days of expansion, the expression of CAR was detected and the expression of CAR was increased by about 20 times. CytoTox 96 and ELISA were used to detect the activity of CAR-T cells in vitro. In this study, monoclonal antibodies against PD-L1 were successfully prepared. The chimeric antigen receptor pCDH-CMV-MCS-EF1-copGFP-CARwas further constructed. The efficiency of transfection into 293T cells was over 95.66%. The titer of packaged lentivirus was 5 脳 107TU / mL, and the expression rate of car in CD8 T cells could be successfully infected with pCDH-CMV-MCS-EF1-copGFP-CAR. More than 22%. The activity of CAR-T cells was determined in vitro. It was proved that PD-L1 specific CAR-T cells had a certain cytotoxicity, which provided important technical support for further in vivo experimental research and clinical trials.
【學(xué)位授予單位】：浙江理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R730.51

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本文編號：1613446