rhIL-24质粒的构建及rhDCN联合rhIL-24对人外周血单个细胞影响的体外研究

发布时间：2018-05-22 15:41

  本文选题：DCN + IL-24　； 参考：《山西医科大学》2010年硕士论文

【摘要】： 目的：探讨核心蛋白聚糖(DCN)基因联合白介素-24(IL-24)基因在体外对PBMC功能的影响。 方法：1.构建重组质粒pcDNA3.1(+)-DCN和pcDNA3.1(+)-IL-24。经双酶切、测序鉴定基因序列,将重组质粒转入大肠杆菌DH-5α中,大量提取pcDNA3.1(+)-DCN和pcDNA3.1(+)-IL-24重组质粒；2.利用脂质体2000将重组质粒pcDNA3.1(+)-DCN和pcDNA3.1(+)-IL-24转入PBMC,鉴定转染效率,并通过RT-PCR检测细胞内DCN和IL-24的mRNA表达；3.测定重组质粒对PBMC的影响：MTT法检测转染后PBMC的增殖、ELISA测定转染细胞IFN-r分泌水平、流式细胞技术(FCM)检测PBMC表面PD-1的表达。 结果：1.构建的质粒经EcoRⅠ.NotⅠ双酶切及PCR鉴定后,所得的基因片段与预期的基因片段大小相符；经DNA测序证实：序列与GenBank中IL-24的序列一致；2.通过脂质体2000将重组质转入PBMC后,荧光显微镜下可见细胞内绿色荧光的表达；RT-PCR检测到细胞内有DCN和IL-24的mRNA表达；3.MTT法检测细胞增殖：转染后96h、120h、144h,DCN与IL-24联合组的细胞增殖率明显高于DCN组,IL-24组,空质粒组,空白对照组,脂质体组；ELISA显示：DCN与IL-24联合组分泌IFN-γ的量显著高于其他组,有统计学差异(P0.05)；流式细胞技术(FCM)显示：PD-1受体的表达水平,联合组高于其它组。 结论：成功构建了pcDNA3.1(+)-IL-24重组质粒；利用脂质体转染法将DCN和IL-24重组质粒转入PBMC,二者能显著促进PBMC的增殖,并增加IFN-γ的分泌及其表面PD-1受体的表达。提示DCN和IL-24联合对PBMC活性能显示更强的促进作用。
[Abstract]:Objective : To investigate the effect of core protein ( DCN ) gene and interleukin - 24 ( IL - 24 ) gene on the function of PBMC in vitro .



Methods : 1 . The recombinant plasmid pcDNA3.1 ( + ) - DCN and pcDNA3.1 ( + ) - IL - 24 were constructed .
2 . The recombinant plasmid pcDNA3.1 ( + ) - DCN and pcDNA3.1 ( + ) - IL - 24 were transferred into PBMC by liposome 2000 to identify the transfection efficiency , and the mRNA expression of DCN and IL - 24 in the cells was detected by RT - PCR ;
3 . The effect of recombinant plasmid on PBMC was determined : MTT assay was used to detect the proliferation of PBMC , and the level of IFN - r secretion was determined by ELISA , and the expression of PD - 1 was detected by flow cytometry ( FCM ) .



Results : 1 . The constructed plasmid was digested with EcoR鈪，

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