【摘要】： 目的:在過去的幾十年,有關(guān)疫苗的研發(fā)取得了很大的成績(jī),如經(jīng)典的疫苗開發(fā)戰(zhàn)略:減毒活疫苗、滅活疫苗、類毒素和亞單位疫苗。盡管這些戰(zhàn)略已在多種細(xì)菌和病毒疫苗開發(fā)方面取得了很大成功,但在許多胞內(nèi)寄生病原的疫苗開發(fā)上這些途徑表現(xiàn)了很大的限制性。核酸疫苗的問世,以其多方面的優(yōu)點(diǎn)如既可誘導(dǎo)體液免疫應(yīng)答又能誘導(dǎo)細(xì)胞免疫應(yīng)答;可編碼多種抗原,從而構(gòu)成多價(jià)疫苗;沒有死疫苗滅活不全和活疫苗毒力恢復(fù)的風(fēng)險(xiǎn);沒有亞單位疫苗因蛋白純化不夠誘發(fā)異源性免疫應(yīng)答的風(fēng)險(xiǎn);設(shè)計(jì)簡(jiǎn)單,制備容易,花費(fèi)較低;相對(duì)穩(wěn)定,能耐受50℃溫度,分送保存過程中不需冷凍系統(tǒng)等優(yōu)點(diǎn)為疫苗的開發(fā)提供了一個(gè)新的途徑。但是,經(jīng)過10余年的研究,至今尚無臨床可用的DNA疫苗可用于疾病的防治,重要原因在于DNA疫苗存在免疫保護(hù)作用不強(qiáng)的弱點(diǎn)。為克服DNA疫苗免疫保護(hù)差的弱點(diǎn),我室和國(guó)內(nèi)外眾多研究者探索了多種措施包括采用不同的接種方式以提高宿主細(xì)胞的轉(zhuǎn)染率;通過優(yōu)化遺傳密碼或質(zhì)粒的轉(zhuǎn)錄調(diào)節(jié)元件提高抗原的表達(dá);與DNA疫苗接種同時(shí)應(yīng)用編碼樹突狀細(xì)胞特異性生長(zhǎng)因子FLT3l、GM-CSF、MIP-1α的質(zhì)粒以提高抗原提呈作用;同時(shí)應(yīng)用編碼IL-2、IL-12、IFN-γ等細(xì)胞因子增加T淋巴細(xì)胞的擴(kuò)增以及應(yīng)用不同的載體等以期提高其免疫保護(hù)作用,但均未達(dá)到預(yù)期效果。 DNA疫苗誘導(dǎo)機(jī)體發(fā)生免疫應(yīng)答的機(jī)制尚未完全明了。一般認(rèn)為,DNA疫苗接種后,樹突狀細(xì)胞等專職抗原提呈細(xì)胞由于自身被轉(zhuǎn)染,或通過其攝取體細(xì)胞表達(dá)的抗原及死亡的抗原表達(dá)細(xì)胞,產(chǎn)生、處理、加工抗原,并經(jīng)MHC-I類、MHC-II類及交叉抗原提呈途徑將抗原肽提呈給免疫活性細(xì)胞是其誘導(dǎo)免疫應(yīng)答的主要機(jī)制。肌肉組織作為常用的接種部位,在DNA疫苗接種后可高效表達(dá)靶抗原,但由于作為非專職抗原提呈細(xì)胞的肌細(xì)胞缺乏B7.1、B7.2等共刺激分子的表達(dá),其在DNA疫苗免疫中的作用眾說紛紜。有報(bào)道認(rèn)為,肌細(xì)胞作為非專職抗原提呈細(xì)胞在DNA疫苗免疫過程中可誘導(dǎo)免疫應(yīng)答的產(chǎn)生,但更多的研究表明,肌細(xì)胞在此過程中導(dǎo)致的是免疫耐受而非免疫刺激,其機(jī)理可能涉及PD-L1/PD-1負(fù)性免疫調(diào)節(jié)通路的活化。鑒于上述以提高DNA疫苗免疫原性為目標(biāo)的嘗試未能取得滿意的免疫效果,我們認(rèn)為,換一個(gè)角度思考問題,阻斷負(fù)性調(diào)節(jié)信號(hào)轉(zhuǎn)導(dǎo)通路,有可能是解決DNA疫苗免疫效果不高的可行途徑。 基于上述考量,本研究以小鼠NOR-10細(xì)胞為研究對(duì)象,用不同的細(xì)胞因子處理細(xì)胞以模擬DNA疫苗接種后活化的T細(xì)胞與肌細(xì)胞相互作用的微環(huán)境,通過RT-PCR和流式細(xì)胞術(shù)檢測(cè)細(xì)胞免疫相關(guān)的MHC-I、共刺激分子B7及負(fù)性免疫調(diào)節(jié)分子PD-L1的表達(dá)。探討DNA疫苗肌肉注射接種后,肌細(xì)胞在免疫效應(yīng)誘導(dǎo)過程中的可能作用及通過阻斷負(fù)性調(diào)節(jié)信號(hào)PD-L1/PD-1通路提高DNA疫苗免疫效果的可能性。 方法:⑴體外刺激細(xì)胞:NOR-10細(xì)胞以5×105的密度傳到新的100ml培養(yǎng)瓶,24小時(shí)后,分別以干擾素-γ(IFN-γ,500U/ml)、白介素-4 (IL-4,50ng/ml),干擾素-β(IFN-β,500U/ml)刺激細(xì)胞,未處理細(xì)胞加新鮮培養(yǎng)基作為正常對(duì)照組。⑵Trizol提取細(xì)胞總RNA:不同的細(xì)胞因子處理細(xì)胞后,分別在3小時(shí)、6小時(shí)、12小時(shí)、24小時(shí),48小時(shí)后提取細(xì)胞總RNA,未處理組作為正常細(xì)胞對(duì)照組,進(jìn)行RT-PCR。⑶流式細(xì)胞術(shù)檢測(cè)細(xì)胞表面蛋白:細(xì)胞用不同的細(xì)胞因子處理后,分別在12小時(shí)、24小時(shí)、48小時(shí)后收集細(xì)胞,未處理組細(xì)胞作為正常對(duì)照。收集1×106個(gè)細(xì)胞/每個(gè)樣本。按流式細(xì)胞儀樣本準(zhǔn)備操作規(guī)程進(jìn)行,然后上機(jī)檢測(cè)。 結(jié)果:⑴共抑制分子PD-L1的表達(dá):證實(shí)正常小鼠NOR-10有少量的PD-L1mRNA但無PD-L1蛋白的表達(dá)。IFN-γ(500U/ml)和IFN-β(500U/ml)刺激既可上調(diào)PD-L1的mRNA表達(dá),也可上調(diào)其蛋白的表達(dá)。⑵共刺激分子MHC-I的表達(dá):正常的NOR-10細(xì)胞有少量的MHC-I mRNA表達(dá),給予IFN-γ和IFN-β處理后,細(xì)胞MHC-I的mRNA均可被上調(diào)。正常細(xì)胞表面無MHC-I蛋白,IFN-β可誘導(dǎo)MHC-I分子表達(dá),但I(xiàn)FN-γ刺激對(duì)MHC-I分子的表達(dá)不起作用。⑶共刺激分子B7的表達(dá):正常情況下,培養(yǎng)的NOR-10細(xì)胞有少量B7.1 mRNA表達(dá),但無B7.2 mRNA的表達(dá),給予IFN-γ和IFN-β刺激后,B7.1mRNA表達(dá)量均沒有明顯變化,B7.2 mRNA仍然陰性。通過流式細(xì)胞術(shù)檢測(cè),我們發(fā)現(xiàn)細(xì)胞表面無B7.1和B7.2表達(dá),同樣給予IFN-γ,IFN-β和IL-4刺激后,仍檢測(cè)不到B7.1和B7.2的表達(dá)。⑷IL-4刺激細(xì)胞,對(duì)PD-L1與MHC-I和B7.1、B7.2均沒有影響。 結(jié)論:⑴正常小鼠NOR-10細(xì)胞構(gòu)成性表達(dá)PD-L1,MHC-I和B7.1 mRNA,但無B7.2 mRNA的表達(dá);(2)正常的NOR-10細(xì)胞表面無MHC-I,共刺激分子B7.1、B7.2蛋白的表達(dá),也無共抑制分子PD-L1蛋白的表達(dá);(3)IFN-γ可上調(diào)PD-L1的mRNA和蛋白的表達(dá)水平,但對(duì)和B7.1和B7.2無影響。(4)MHC-I的mRNA可被IFN-γ上調(diào),細(xì)胞表面蛋白不受影響。(5)IFN-β可使PD-L1、MHC-I的mRNA和蛋白表達(dá)均增高,對(duì)B7.1和B7.2沒影響。(6)IL-4對(duì)MHC-I、B7.1以及PD-L1均無影響。
[Abstract]:OBJECTIVE: In the past few decades, great achievements have been made in vaccine research and development, such as classical vaccine development strategies: live attenuated, inactivated, toxoid and subunit vaccines. Nucleic acid vaccines have many advantages, such as inducing humoral and cellular immune responses; encoding multiple antigens to form a multivalent vaccine; no risk of inactivation of a dead vaccine or virulence recovery of a live vaccine; no subunit vaccine due to inadequate protein purification The risk of inducing heterologous immune response, the simplicity of design, the ease of preparation and the low cost, the relative stability, the tolerance to temperature of 50 C and the need for refrigeration during distribution and preservation provide a new way for vaccine development. To overcome the weakness of DNA vaccines, many researchers in our laboratory and at home and abroad have explored a variety of measures, including different ways of inoculation to improve the transfection rate of host cells, optimization of genetic codes or transcriptional regulatory elements of plasmids to enhance antigens. Expressions of cytokines encoding dendritic cell-specific growth factors FLT3l, GM-CSF and MIP-1a were used simultaneously with DNA vaccination to enhance the antigen presenting effect, while cytokines encoding IL-2, IL-12, IFN-gamma were used to increase the T lymphocyte proliferation and different carriers were used to enhance the immunoprotective effect, but none of them reached the expectation. Effect.
The mechanism by which DNA vaccines induce immune responses has not been fully understood. It is generally believed that after DNA vaccination, full-time antigen presenting cells, such as dendritic cells, are produced, processed, and transfected by themselves, or by their uptake of antigens expressed in somatic cells and dead antigen-expressing cells, and are subjected to MHC-I, MHC-II and cross-antibodies. The main mechanism of inducing immune response is presenting antigen peptides to immunocompetent cells by the original presenting pathway. Muscle tissue, as a common site of vaccination, can efficiently express target antigens after DNA vaccination. However, as a non-full-time antigen presenting cell, muscle cells lack the expression of costimulatory molecules such as B7.1 and B7.2, so they are immunized with DNA vaccines. It has been reported that myocytes, as non-professional antigen presenting cells, can induce immune responses during DNA vaccine immunization, but more studies have shown that myocytes induce immune tolerance rather than immune stimulation in this process. The mechanism may involve the activation of the negative immune regulatory pathway of PD-L1/PD-1. In view of the above attempts to improve the immunogenicity of DNA vaccines have failed to achieve satisfactory immune results, we believe that a change of perspective to block the negative regulatory signal transduction pathway may be a feasible way to solve the problem of low immune effect of DNA vaccines.
Based on the above considerations, NOR-10 cells were treated with different cytokines to simulate the microenvironment of interaction between activated T cells and myocytes after DNA vaccination. The expression of MHC-I, costimulatory molecule B7 and negative immunoregulatory molecule PD-L1 was detected by RT-PCR and flow cytometry. To investigate the possible role of muscle cells in the induction of immune response after intramuscular injection of DNA vaccine and the possibility of enhancing the immune response of DNA vaccine by blocking the negative regulatory signal PD-L1/PD-1 pathway.
METHODS: _NOR-10 cells were stimulated by interferon-gamma (IFN-gamma, 500U/ml), interleukin-4 (IL-4, 50ng/ml), interferon-beta (IFN-beta, 500U/ml) in vitro. The untreated cells were cultured with fresh medium as the normal control group. _Total RNA of cells was extracted by Trizol: different fines. After cytokine treatment, total RNA was extracted at 3, 6, 12, 24 and 48 hours respectively. The untreated cells were used as the control group and the cell surface proteins were detected by RT-PCR. _Flow cytometry: After treated with different cytokines, the cells were collected at 12, 24, 48 hours, respectively. Cells were taken as normal controls. 1 106 cells were collected per sample. The samples were prepared according to the procedure of flow cytometry, and then tested by computer.
Results: _Co-inhibitory molecule PD-L1 expression: NOR-10 in normal mice was confirmed to have a small amount of PD-L1 mRNA but no expression of PD-L1 protein. IFN-gamma (500U/ml) and IFN-beta (500U/ml) stimulation can both up-regulate the expression of PD-L1 mRNA and up-regulate the expression of PD-L1 protein. _Co-stimulatory molecule MHC-I expression: Normal NOR-10 cells have a small amount of MHC-I mRNA expression. After treatment with IFN-gamma and IFN-beta, the mRNA of MHC-I was up-regulated. There was no MHC-I protein on the surface of normal cells. IFN-beta could induce the expression of MHC-I molecule, but IFN-gamma did not affect the expression of MHC-I molecule. _Co-stimulatory molecule B7: Normally, a small amount of B7.1 mRNA was expressed in NOR-10 cells, but no B7.2 mRNA was expressed. After stimulation with IFN-gamma and IFN-beta, the expression of B7.1 mRNA did not change significantly, but the expression of B7.2 mRNA was still negative. Flow cytometry showed that there was no expression of B7.1 and B7.2 on the cell surface. After stimulation with IFN-gamma, IFN-beta and IL-4, the expression of B7.1 and B7.2 could not be detected. Influence.
CONCLUSION: _Normal NOR-10 cells express PD-L1, MHC-I and B7.1 mRNA constructively, but not B7.2 mRNA; (2) Normal NOR-10 cells do not express MHC-I, costimulatory molecule B7.1, B7.2 protein, nor co-inhibitory molecule PD-L1 protein; (3) IFN-gamma can up-regulate the expression of PD-L1 mRNA and protein, but has no effect on B7.1 and B7.2. (4) The mRNA of MHC-I was up-regulated by IFN-gamma, and the cell surface protein was not affected. (5) IFN-beta increased the mRNA and protein expression of PD-L1 and MHC-I, but had no effect on B7.1 and B7.2. (6) IL-4 had no effect on MHC-I, B7.1 and PD-L1.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392

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