腺病毒介导CTLA4Ig和CD40Ig基因转染对人皮肤成纤维细胞免疫性能影响的体外研究

发布时间：2018-03-13 17:43

本文选题：腺病毒　切入点：CTLA4Ig　出处：《西南大学》2008年硕士论文　论文类型：学位论文

【摘要】： 异基因(异体、异种)皮肤移植后的排斥反应主要是由活化T细胞主导的急性细胞性排斥反应,此过程中T细胞共刺激信号的参与对于T细胞活化和免疫排斥的发生是必需的,阻断共刺激信号,可导致T细胞无反应、细胞凋亡或克隆丢失,从而诱导免疫耐受。在已发现的多种T细胞共刺激信号中,公认最为重要的是B7-CD28信号和CD40-CD40L信号。利用蛋白或抗体可有效对其阻断,诱导免疫耐受,但存在制备复杂、成本较高、半衰期短和需要长期施用等弊端。腺病毒载体安全性高、制备容易以其介导基因治疗的方式将共刺激分子基因导入靶细胞中,使治疗性蛋白在特定区域表达,阻断共刺激信号,有望降低成本、延长效应时间。皮肤成纤维细胞易获取、培养及大量扩增,也是常用的组织工程皮肤种子细胞和皮肤基因治疗理想的靶细胞。因此本研究以体外培养的人皮肤成纤维细胞为靶细胞,用腺病毒介导CTLA4Ig和CD40Ig基因转染阻断公认最为重要的B7/CD28和CD40/CD40L信号,通过对Adv-CTLA4Ig、Adv-CD40Ig感染人皮肤成纤维细胞的效率检测、感染后细胞形态和增殖能力检测、目的蛋白表达检测及对其免疫性能影响检测,研究、评价腺病毒基因治疗方式介导CTLA4Ig和CD40Ig阻断双共刺激信号应用于人皮肤成纤维细胞修饰和治疗的效果和可行性,为其应用提供基础资料。其主要内容如下: 1.采用流式细胞分析技术检测腺病毒对体外培养人皮肤成纤维细胞的转染效率。结果表明:Adv-EGFP在感染复数(MOI)为50,100时对人皮肤成纤维细胞转染效率约为51.7±7.24%、69.99±3.26%,同时对细胞形态和增殖能力无明显影响。因此本研究选用100为腺病毒感染人皮肤成纤维细胞的MOI。 2.采用形态学观察和MTT法细胞活力检测重组腺病毒转染对体外培养人皮肤成纤维细胞生长形态和增殖能力的影响。结果表明:重组病毒Adv-CTLA4Ig、Adv-CD40Ig在MOI为100时单独和混合转染人皮肤成纤维细胞后,实验组与对照组细胞形态均呈长梭形、不规则三角形,平行、放射状或漩涡状分布,有较长胞质突起,轮廓清晰,并且各组细胞增殖能力无显著差异(P＞0.05)。 3.采用酶联免疫吸附试验(ELISA)检测重组腺病毒介导CTLA4Ig和CD40Ig在体外培养人皮肤成纤维细胞培养上清中的表达情况。结果显示:重组病毒Adv-CTLA4Ig、Adv-CD40Ig在MOI为100单独和混合转染人皮肤成纤维细胞后,在培养上清中均检测到相应目的蛋白的表达,其表达量分别是:Adv-CTLA4Ig转染组:CTLA4Ig 2.79±0.08ug/ml;Adv-CD40Ig转染组:CD40Ig 1.25±0.11ug/ml;Adv-CTLA4Ig+Adv-CD40Ig转染组:CTLA4Ig2.14±0.21ug/ml,CD40Ig 1.00±0.04ug/ml。 4.采用重组腺病毒Adv-CTLA4Ig、Adv-CD40Ig转染后的体外培养人皮肤成纤维细胞与人外周血单个核细胞混合培养检测对人皮肤成纤维细胞免疫性能的影响。结果显示:重组病毒Adv-CTLA4Ig、Adv-CD40Ig以MOI为100单独和混合转染的体外培养人皮肤成纤维细胞对人外周血单个核细胞增殖抑制均优于对照组,有显著性差异(单处理组P＜0.05,混合组P＜0.01),Adv-CTLA4Ig和Adv-CD40Ig混合处理组抑制效果优于各单处理组(P＜0.05)达到31.67±2.25%,Adv-CTLA4Ig处理组(23.41±1.23%)优于Adv-CD40Ig(19.83±4.71%)(P＜0.05)。综上所述,重组腺病毒Adv-CTLA4Ig、Adv-CD40Ig可以有效转染体外培养人皮肤成纤维细胞;介导CTLA4Ig和CD40Ig表达;对细胞生长形态和增殖能力无明显影响;通过腺病毒介导方式能有效阻断B7/CD28、CD40/CD40L共刺激信号,抑制体外培养人外周血单个核细胞增殖,阻断B7/CD28信号诱导抑制效果优于CD40/CD40L信号,阻断双信号可更大程度诱导免疫抑制,因共刺激信号多样、免疫反应复杂性及施用量、处理时间的原因,并不能完全抑制。结论:腺病毒基因治疗方式介导CTLA4Ig和CD40Ig阻断B7/CD28、CD40/CD40L共刺激信号可应用于人皮肤成纤维细胞修饰和治疗。
[Abstract]:Allogeneic (allogeneic, xenogeneic) skin graft rejection after rejection is mainly by acute cellular activation of T cells, this process involved in T cell costimulatory signal for T cell activation and immune rejection is required, blocking costimulatory signals can lead to T cell reaction, cell cloning of apoptosis or loss, thereby inducing immune tolerance. T cells have been found in a variety of costimulatory signals, recognized as the most important is the B7-CD28 and CD40-CD40L signals. To be effective on the block by proteins or antibodies, induced immune tolerance, but in the preparation of complex, high cost, short half-life and need long-term application other drawbacks. Adenovirus vector has high safety, easy preparation for its mediated gene therapy to costimulatory molecule gene into the target cells, the expression of therapeutic proteins in specific areas, blocking costimulatory signals, is expected to drop Low cost, prolong the effect time. Skin fibroblasts were easy to obtain, culture and amplification of target cells, is also commonly used as seed cells for skin tissue engineering skin and ideal gene therapy. Therefore in this study, human skin fibroblasts cultured in vitro as target cells with adenovirus mediated CTLA4Ig gene transfection and CD40Ig blocking recognized the most important B7/CD28 and CD40/CD40L signal, through the Adv-CTLA4Ig, Adv-CD40Ig infection of human skin fibroblasts detection efficiency, after infection, cell morphology and proliferation ability of detection, protein expression and detection of its immune effect, performance detection, evaluation of adenovirus mediated CTLA4Ig gene therapy and CD40Ig double blocking costimulatory signal applied to human skin fibroblasts and the effect and feasibility of modified treatment, provide the basis for its application. The main contents are as follows:
1. by flow cytometry analysis technique for detection of adenovirus in human skin fibroblasts transfection efficiency in vitro. The results showed that Adv-EGFP infection in the complex (MOI) 50100 on human skin fibroblasts transfection efficiency is about 51.7 + 7.24%, 69.99 + 3.26%, but has no obvious effect on the morphology and proliferation of cells. Therefore, this study selected 100 adenovirus infection of human skin fibroblasts MOI.
2. by morphological observation and MTT method to detect the cell viability of recombinant adenovirus transfected into cultured human skin fibroblasts influence cell morphology and proliferation in vitro. The results showed that the recombinant virus Adv-CTLA4Ig, Adv-CD40Ig at MOI 100 alone and mixed transfected human skin fibroblast cells, the experimental group and the control group showed cell morphology long fusiform, irregular triangle, parallel, radial or whorled distribution, long cytoplasmic processes, clear outline, and no significant difference between the groups of cell proliferation (P > 0.05).
3. by enzyme-linked immunosorbent assay (ELISA) detection of recombinant adenovirus mediated CTLA4Ig and CD40Ig expression in the supernatant of cultured human cultured skin fibroblasts in vitro. The results showed that the recombinant virus Adv-CTLA4Ig, Adv-CD40Ig in MOI 100 alone and mixed into human skin fibroblasts, in culture supernatant were detected to express the corresponding protein and its expression are: Adv-CTLA4Ig transfection group: CTLA4Ig 2.79 + 0.08ug/ml; Adv-CD40Ig transfection group: CD40Ig 1.25 + 0.11ug/ml; Adv-CTLA4Ig+Adv-CD40Ig group: CTLA4Ig2.14 + 0.21ug/ml, CD40Ig + 0.04ug/ml. 1
4. using the recombinant adenovirus Adv-CTLA4Ig in cultured human skin fibroblasts and mixed peripheral blood mononuclear cells cultured fibroblasts cell immunity detection on human skin Adv-CD40Ig after transfection in vitro. The results showed that the recombinant virus Adv-CTLA4Ig, cultured human skin fibroblasts on human peripheral blood mononuclear cell proliferation inhibition better than the control group Adv-CD40Ig with MOI as 100 separate and mixed transfection in vitro, there was significant difference (P < 0.05 single treatment group, mixed group P < 0.01), Adv-CTLA4Ig and Adv-CD40Ig mixed treatment group the inhibitory effect is better than the single treatment group (P < 0.05) to 31.67 + 2.25%, 23.41 + 1.23% (Adv-CTLA4Ig treatment group) is better than that of Adv-CD40Ig (19.83 + 4.71%) (P < 0.05).
In summary, the recombinant adenovirus Adv-CTLA4Ig Adv-CD40Ig skin fibroblasts were cultured in vitro mediated transfection effectively; the expression of CD40Ig and CTLA4Ig; the cell growth morphology and proliferation ability has no effect; can effectively block B7/CD28 mediated by adenovirus, CD40/ CD40L costimulatory signal suppression in cultured human peripheral blood mononuclear cell proliferation cells, B7/CD28 signaling induced inhibitory effect than CD40/CD40L signal blocking, blocking double signal can be a greater degree of immunosuppression induced by costimulatory signals, because of diversity, complexity and application amount of the immune response causes the processing time, and can not be completely inhibited. Conclusion: adenovirus mediated CTLA4Ig gene therapy and CD40Ig blocking B7/CD28 costimulatory CD40/CD40L the signal can be applied to human skin fibroblasts and modification treatment.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392;Q78

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