K562细胞总RNA转染对人树突状细胞抗原提呈、成熟及功能影响的初步研究

探讨人的树突状细胞(dendriticcell,DC)体外经K562细胞株总RNA转染后,能否诱导出p210蛋白表达,并诱导特异性CTL,为DC疫苗的临床应用提供理论基础。自健康人浓缩白细胞制剂分离有黏附特性的单核细胞,经GM-CSF、IL-4培养5d后,获得未成熟的DC(imDC);体外以脂质体DOTAP或直接电穿孔转染K562细胞总RNA。抽提后即时以及转染24h后的DC内RNA进行RT-PCR检测,Westernblot分析p210蛋白表达,流式细胞仪检测,以及诱导CTL杀伤K562细胞功能检测等。结果显示,RT-PCR检测表明:DC经K562细胞总RNA转染后,细胞内出现Bcr-Abl的cDNA条带,24h后消失。Westernblot实验表明:转染24h后,开始表达p210特征性蛋白。与对照组相比,转染K562细胞总RNA的DC,在24h后CD80、CD83、CD86、HLA-DR等表面标志均不同程度表达增高,并可促进T细胞对K562的杀伤活性。该研究从多方面角度说明应用肿瘤总RNA负载DC来制备DC疫苗的可行性,提示改良的DC疫苗抗肿瘤可能的应用前景。

Effects of transfection with total RNA of K562 cells upon the antigen presentation, maturation and function of human dendritic cells

To investigate whether the tranfection of human dendritic cells(DC) with total RNA of K562 cells could induce the expression of p210Bcr-Abl protein and was able to trigger the antigen-specific CTL responses in vitro, DC obtained from human peripheral blood monocytes were incubated in the presence of GM-CSF and IL-4 for 5 days, and then transfected with total RNA of K562 cells by using electroporation or DOTAP lipofection. To verify the success of transfection of DC with K562-RNA, the expression of the Bcr-Abl fusion gene with the specific characteristics of chrinic myelogenic leukemia(CML) in DC was detected by RT-PCR and Western blotting. The phenotype of DC was determined by flow cytometry, and the cytotoxicity of CTL was assayed by propidium iodide (PI) staining and flow cytometry. The results of RT-PCR assay indicated that the cDNA of the Bcr-Abl fusion gene could be found in DC after transfection of these cells with total K562-RNA , but 24 hours later, this Bcr-Abl mRNA from K562-RNA transfected DC disappeared. As demonstrated by Western blotting, the expression of p210Bcr-Abl was detected 24 hours after transfection. Meanwhile, 24 hours later after transfection, the expressions of CD80, CD83, CD86 and HLA-DR increased under different degrees. In addition, the transfected DCs could enhance T lymphocytes to kill target K562 cells significantly. It is concluded that human dendritic cells transfected with total RNA of K562 cells in vitro can induce effective p210Bcr-Abl protein-specific immune responses, and it is possible to use the tumor cell RNA loaded DC for the preparation of DC vaccine.