PTD-Foxp3融合蛋白体外功能的初步研究

发布时间：2019-03-12 15:57

【摘要】： Foxp3是FOX(forkhead box)家族转录因子中的一员,是调节性T细胞(regulatory T cells,Treg)的特异性标志,同时在Treg的发生、发育、功能的维持与发挥方面起着关键作用。研究发现表达外源性Foxp3可使非Treg细胞具有类似Treg细胞样的表型及免疫抑制功能。人类免疫缺陷病毒1型(human immunodeficiency virus type 1,HIV-1)基因编码的反式激活蛋白(Trans-activator transcription,TAT)中的蛋白转导结构域(protein transduction domain,PTD)能够高效、快速地携带外源性蛋白穿入几乎所有的真核细胞,而进入细胞浆。由于其具有核定位信号,因此也能进入细胞核内。同时PTD具有不影响其携带的目的蛋白功能的特点。目的:我们的研究是要利用生物大分子载体工具蛋白转导结构域(PTD)将Foxp3蛋白带到细胞内,以获取大量的免疫抑制细胞,从而突破Treg制约基础和临床器官移植的研究瓶颈,为最终应用于器官移植和自身免疫性疾病治疗奠定基础。方法:流式细胞术检测PTD融合蛋白穿过细胞膜进入EL-4细胞中的能力和量效关系;Western Blot分析和激光共聚焦显微镜观察确认融合蛋白进入细胞浆和细胞核;采用CCK-8法检测融合蛋白对DO11.10鼠的CD4~+CD25~- T细胞活化增殖的影响,以及蛋白转导的CD4~+CD25~- T细胞对效应细胞的增殖抑制能力;RT-PCR检测IL-2、INF-γ、IL-10、TGF-β和CTLA-4的mRNA表达水平;EHSA测定上清中IL-2、INF-γ、IL-10和TGF-β的分泌水平。结果:通过流式细胞术分析证实表达的PTD-eGFP-Foxp3融合蛋白能高效的转入细胞内;经Western Blot和激光共聚焦显微镜观察确认PTD-Foxp3和PTD-eGFP-Foxp3融合蛋白能够进入细胞浆和细胞核;同时经细胞增殖抑制实验证明PTD-Foxp3融合蛋白能明显抑制OVA特异性TCR转基因小鼠DO11.10小鼠来源的CD4~+CD25~- T细胞的活化增殖能力,并且PTD-Foxp3转导的DO11.10小鼠CD4~+CD25~- T细胞也有抑制细胞活化的能力;RT-PCR和ELISA结果可以看出PTD-Foxp3与Foxp3、PTD-eGFP相比可以明显的抑制效应细胞分泌的IL-2、IFN-γ,并促进抑制性因子IL-10的产生;RT-PCR检测PTD-Foxp3转导的D011.10小鼠CD4~+CD25~- T细胞,证实表面标志分子CTLA-4的表达有所升高。结论:PTD-Foxp3具有调节CD4~+CD25~- T细胞的作用,并且有可能将CD4~+CD25~- T细胞转化为Treg样细胞,希望能应用于自身免疫性疾病和抗器官移植排斥反应的治疗中。
[Abstract]:Foxp3 is a member of the FOX (forkhead box) family of transcription factors and a specific marker of regulatory T cell (regulatory T cells,Treg. At the same time, it plays a key role in the occurrence, development, maintenance and play of Treg. It was found that the expression of exogenous Foxp3 could induce Treg-like phenotype and immunosuppressive function in non-Treg cells. The protein transduction domain (protein transduction domain,PTD in transactivator protein (Trans-activator transcription,TAT) encoded by human immunodeficiency virus type 1 (human immunodeficiency virus type-1, HIV-1) gene is highly efficient. It quickly carries exogenous proteins through almost all eukaryotes and into the cytoplasm. Because of its nuclear localization signal, it can also enter the nucleus. At the same time, PTD has the characteristics of not affecting the function of the target protein it carries. Objective: our research is to use the biological macromolecule vector tool protein transduction domain (PTD) to carry the Foxp3 protein into the cell, in order to obtain a large number of immunosuppressive cells, so as to break through the bottleneck of Treg restriction foundation and clinical organ transplantation research. It lays a foundation for the final application in organ transplantation and autoimmune disease treatment. Methods: flow cytometry was used to detect the ability and dose-effect relationship of PTD fusion protein entering into EL-4 cells.; Western Blot analysis and laser confocal microscopy showed that the fusion protein entered into cytoplasm and nucleus. The effect of fusion protein on the activation and proliferation of CD4~ CD25~- T cells in DO11.10 mice and the inhibitory effect of CD4~ CD25~- T cells on the proliferation of effector cells were detected by CCK-8 assay. The mRNA expression of IL-2,INF- 纬, IL-10,TGF- 尾 and CTLA-4 was detected by RT-PCR, and the secretion of IL-2,INF- 纬, IL-10 and TGF- 尾 in supernatant was measured by EHSA. Results: the expressed PTD-eGFP-Foxp3 fusion protein could be efficiently transferred into the cell by flow cytometry, and the PTD-Foxp3 and PTD-eGFP-Foxp3 fusion protein could enter the cytoplasm and nucleus by Western Blot and laser confocal microscopy. The results of cell proliferation inhibition assay showed that PTD-Foxp3 fusion protein could significantly inhibit the activation and proliferation of CD4~ CD25~- T cells derived from OVA-specific TCR transgenic mice DO11.10 mice. In addition, CD4~ CD25~- T cells of DO11.10 mice transfected with PTD-Foxp3 also had the ability to inhibit the activation of cells. The results of RT-PCR and ELISA showed that compared with Foxp3,PTD-eGFP, PTD-Foxp3 could significantly inhibit the secretion of IL-2,IFN- 纬 by effector cells and promote the production of inhibitory factor IL-10. The expression of surface marker molecule CTLA-4 was detected by RT-PCR in D011.10 CD4~ CD25~- T cells transfected with PTD-Foxp3. Conclusion: PTD-Foxp3 can regulate CD4~ CD25~- T cells, and may transform CD4~ CD25~- T cells into Treg-like cells. It is hoped that CD4~ CD25~- T cells can be used in the treatment of autoimmune diseases and anti-organ transplantation rejection.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R341

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