發(fā)布時間：2018-04-17 20:36

  本文選題：hIL-10 + 基因克隆　； 參考：《吉林大學》2007年碩士論文

【摘要】： 本研究以hIL-10為研究對象,從基因治療的角度入手,研究hIL-10的免疫抑制活性。為重組hIL-10與其他免疫抑制基因聯(lián)合治療免疫性疾病奠定實驗基礎。采用分子克隆技術(shù)建立hIL-10基因克隆并構(gòu)建真核表達載體pcDNA3.0-hIL-10;將其成功瞬時轉(zhuǎn)染人胚腎細胞HEK293;在此基礎上,采用MTT比色法檢測pcDNA3.0-hIL-10轉(zhuǎn)染HEK293細胞的上清對小鼠脾臟和胸腺細胞增殖的影響,實驗結(jié)果表明,pcDNA3.0-hIL-10轉(zhuǎn)染HEK293細胞的上清對小鼠脾臟和胸腺細胞增殖有一定的抑制作用;同時采用pcDNA3.0-hIL-10轉(zhuǎn)染HEK293細胞的上清刺激小鼠脾細胞,對上清中Th1型細胞因子進行了檢測,實驗結(jié)果表明,上清中Th1型細胞因子TNF-αIFN-γIL-2活性降低;應用RT-PCR的方法檢測了轉(zhuǎn)染后細胞中免疫抑制因子VEGFTGF-βFasL mRNA表達,實驗結(jié)果表明,重組基因轉(zhuǎn)染后HEK293細胞中有VEGF mRNA表達,而轉(zhuǎn)染前后TGF-βFasL mRNA表達無變化。 本研究在hIL-10基因成功轉(zhuǎn)染HEK293細胞后,表明hIL-10基因轉(zhuǎn)染對小鼠脾細胞和胸腺細胞增殖及Th1型細胞因子的活性均有抑制作用;hIL-10基因轉(zhuǎn)染可促進VEGF mRNA的表達,提示hIL-10基因可能參與了VEGF mRNA表達的調(diào)節(jié)。本研究為開發(fā)重組hIL-10轉(zhuǎn)基因治療的臨床應用及基因工程藥物制備奠定了實驗基礎。
[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- 偽 and IFN- 緯-緯 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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R392

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