hIL-10基因瞬时转染HEK293细胞介导的免疫学效应研究

发布时间：2018-04-17 20:36

本文选题：hIL-10 + 基因克隆　；参考：《吉林大学》2007年硕士论文

【摘要】： 本研究以hIL-10为研究对象,从基因治疗的角度入手,研究hIL-10的免疫抑制活性。为重组hIL-10与其他免疫抑制基因联合治疗免疫性疾病奠定实验基础。采用分子克隆技术建立hIL-10基因克隆并构建真核表达载体pcDNA3.0-hIL-10;将其成功瞬时转染人胚肾细胞HEK293;在此基础上,采用MTT比色法检测pcDNA3.0-hIL-10转染HEK293细胞的上清对小鼠脾脏和胸腺细胞增殖的影响,实验结果表明,pcDNA3.0-hIL-10转染HEK293细胞的上清对小鼠脾脏和胸腺细胞增殖有一定的抑制作用;同时采用pcDNA3.0-hIL-10转染HEK293细胞的上清刺激小鼠脾细胞,对上清中Th1型细胞因子进行了检测,实验结果表明,上清中Th1型细胞因子TNF-αIFN-γIL-2活性降低;应用RT-PCR的方法检测了转染后细胞中免疫抑制因子VEGFTGF-βFasL mRNA表达,实验结果表明,重组基因转染后HEK293细胞中有VEGF mRNA表达,而转染前后TGF-βFasL mRNA表达无变化。本研究在hIL-10基因成功转染HEK293细胞后,表明hIL-10基因转染对小鼠脾细胞和胸腺细胞增殖及Th1型细胞因子的活性均有抑制作用;hIL-10基因转染可促进VEGF mRNA的表达,提示hIL-10基因可能参与了VEGF mRNA表达的调节。本研究为开发重组hIL-10转基因治疗的临床应用及基因工程药物制备奠定了实验基础。
[Abstract]:In this study, hIL-10 was used to study the immunosuppressive activity of hIL-10 from the perspective of gene therapy.To establish the experimental foundation for the combination of recombinant hIL-10 and other immunosuppressive genes in the treatment of immune diseases.HIL-10 gene clone was established by molecular cloning technique and eukaryotic expression vector pcDNA3.0-hIL-10 was constructed.The effect of supernatant of HEK293 cells transfected with pcDNA3.0-hIL-10 on the proliferation of mouse spleen and thymocytes was detected by MTT colorimetry. The results showed that the supernatant transfected with pcDNA3.0-hIL-10 could inhibit the proliferation of mouse spleen and thymocytes.At the same time, the supernatant of HEK293 cells transfected with pcDNA3.0-hIL-10 was used to stimulate the spleen cells of mice, and the Th1 type cytokines in the supernatants were detected. The results showed that the activity of TNF- 伪 and IFN- 纬-纬 in the supernatants was decreased.The expression of immunosuppressive factor VEGF- 尾 -FasL mRNA was detected by RT-PCR. The results showed that the expression of VEGF mRNA was detected in HEK293 cells after transfection of recombinant gene, but the expression of TGF- 尾 -FasL mRNA did not change before and after transfection.In this study, the successful transfection of hIL-10 gene into HEK293 cells showed that hIL-10 gene transfection could inhibit the proliferation of mouse spleen cells and thymocytes and the activity of Th1 cytokines. HIL-10 gene transfection could promote the expression of VEGF mRNA.The results suggest that hIL-10 gene may be involved in the regulation of VEGF mRNA expression.This study laid an experimental foundation for the clinical application of recombinant hIL-10 transgenic therapy and the preparation of genetically engineered drugs.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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