细胞因子对小鼠肌细胞免疫调节相关分子表达的影响
发布时间：2018-08-24 09:57
【摘要】： 目的:在过去的几十年,有关疫苗的研发取得了很大的成绩,如经典的疫苗开发战略:减毒活疫苗、灭活疫苗、类毒素和亚单位疫苗。尽管这些战略已在多种细菌和病毒疫苗开发方面取得了很大成功,但在许多胞内寄生病原的疫苗开发上这些途径表现了很大的限制性。核酸疫苗的问世,以其多方面的优点如既可诱导体液免疫应答又能诱导细胞免疫应答;可编码多种抗原,从而构成多价疫苗;没有死疫苗灭活不全和活疫苗毒力恢复的风险;没有亚单位疫苗因蛋白纯化不够诱发异源性免疫应答的风险;设计简单,制备容易,花费较低;相对稳定,能耐受50℃温度,分送保存过程中不需冷冻系统等优点为疫苗的开发提供了一个新的途径。但是,经过10余年的研究,至今尚无临床可用的DNA疫苗可用于疾病的防治,重要原因在于DNA疫苗存在免疫保护作用不强的弱点。为克服DNA疫苗免疫保护差的弱点,我室和国内外众多研究者探索了多种措施包括采用不同的接种方式以提高宿主细胞的转染率;通过优化遗传密码或质粒的转录调节元件提高抗原的表达;与DNA疫苗接种同时应用编码树突状细胞特异性生长因子FLT3l、GM-CSF、MIP-1α的质粒以提高抗原提呈作用;同时应用编码IL-2、IL-12、IFN-γ等细胞因子增加T淋巴细胞的扩增以及应用不同的载体等以期提高其免疫保护作用,但均未达到预期效果。 DNA疫苗诱导机体发生免疫应答的机制尚未完全明了。一般认为,DNA疫苗接种后,树突状细胞等专职抗原提呈细胞由于自身被转染,或通过其摄取体细胞表达的抗原及死亡的抗原表达细胞,产生、处理、加工抗原,并经MHC-I类、MHC-II类及交叉抗原提呈途径将抗原肽提呈给免疫活性细胞是其诱导免疫应答的主要机制。肌肉组织作为常用的接种部位,在DNA疫苗接种后可高效表达靶抗原,但由于作为非专职抗原提呈细胞的肌细胞缺乏B7.1、B7.2等共刺激分子的表达,其在DNA疫苗免疫中的作用众说纷纭。有报道认为,肌细胞作为非专职抗原提呈细胞在DNA疫苗免疫过程中可诱导免疫应答的产生,但更多的研究表明,肌细胞在此过程中导致的是免疫耐受而非免疫刺激,其机理可能涉及PD-L1/PD-1负性免疫调节通路的活化。鉴于上述以提高DNA疫苗免疫原性为目标的尝试未能取得满意的免疫效果,我们认为,换一个角度思考问题,阻断负性调节信号转导通路,有可能是解决DNA疫苗免疫效果不高的可行途径。 基于上述考量,本研究以小鼠NOR-10细胞为研究对象,用不同的细胞因子处理细胞以模拟DNA疫苗接种后活化的T细胞与肌细胞相互作用的微环境,通过RT-PCR和流式细胞术检测细胞免疫相关的MHC-I、共刺激分子B7及负性免疫调节分子PD-L1的表达。探讨DNA疫苗肌肉注射接种后,肌细胞在免疫效应诱导过程中的可能作用及通过阻断负性调节信号PD-L1/PD-1通路提高DNA疫苗免疫效果的可能性。 方法:⑴体外刺激细胞:NOR-10细胞以5×105的密度传到新的100ml培养瓶,24小时后,分别以干扰素-γ(IFN-γ,500U/ml)、白介素-4 (IL-4,50ng/ml),干扰素-β(IFN-β,500U/ml)刺激细胞,未处理细胞加新鲜培养基作为正常对照组。⑵Trizol提取细胞总RNA:不同的细胞因子处理细胞后,分别在3小时、6小时、12小时、24小时,48小时后提取细胞总RNA,未处理组作为正常细胞对照组,进行RT-PCR。⑶流式细胞术检测细胞表面蛋白:细胞用不同的细胞因子处理后,分别在12小时、24小时、48小时后收集细胞,未处理组细胞作为正常对照。收集1×106个细胞/每个样本。按流式细胞仪样本准备操作规程进行,然后上机检测。 结果:⑴共抑制分子PD-L1的表达:证实正常小鼠NOR-10有少量的PD-L1mRNA但无PD-L1蛋白的表达。IFN-γ(500U/ml)和IFN-β(500U/ml)刺激既可上调PD-L1的mRNA表达,也可上调其蛋白的表达。⑵共刺激分子MHC-I的表达:正常的NOR-10细胞有少量的MHC-I mRNA表达,给予IFN-γ和IFN-β处理后,细胞MHC-I的mRNA均可被上调。正常细胞表面无MHC-I蛋白,IFN-β可诱导MHC-I分子表达,但IFN-γ刺激对MHC-I分子的表达不起作用。⑶共刺激分子B7的表达:正常情况下,培养的NOR-10细胞有少量B7.1 mRNA表达,但无B7.2 mRNA的表达,给予IFN-γ和IFN-β刺激后,B7.1mRNA表达量均没有明显变化,B7.2 mRNA仍然阴性。通过流式细胞术检测,我们发现细胞表面无B7.1和B7.2表达,同样给予IFN-γ,IFN-β和IL-4刺激后,仍检测不到B7.1和B7.2的表达。⑷IL-4刺激细胞,对PD-L1与MHC-I和B7.1、B7.2均没有影响。 结论:⑴正常小鼠NOR-10细胞构成性表达PD-L1,MHC-I和B7.1 mRNA,但无B7.2 mRNA的表达;(2)正常的NOR-10细胞表面无MHC-I,共刺激分子B7.1、B7.2蛋白的表达,也无共抑制分子PD-L1蛋白的表达;(3)IFN-γ可上调PD-L1的mRNA和蛋白的表达水平,但对和B7.1和B7.2无影响。(4)MHC-I的mRNA可被IFN-γ上调,细胞表面蛋白不受影响。(5)IFN-β可使PD-L1、MHC-I的mRNA和蛋白表达均增高,对B7.1和B7.2没影响。(6)IL-4对MHC-I、B7.1以及PD-L1均无影响。
[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.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
本文编号：2200464
[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.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
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