人GITRL和IL-21基因对CML来源树突状细胞功能的影响

Effect of GITRL and IL-21 gene on chronic myeloid leukemia derived dendritic cell

目的:本研究构建人糖皮质激素诱导的肿瘤坏死因子受体配体(GITRL)联合白细胞介素21(IL-21)基因真核表达载体,为探讨目的基因对慢性粒细胞白血病(CML)来源树突状细胞(DC)的影响。方法:脂质体介导法将重组真核表达载体转染经纯化的伊马替尼治疗有效、无效的慢性期CML患者及健康志愿者来源的DC,流式细胞仪检测转染后DC的CD1а、CD83、CD86表达率变化。将转染后的DC与纯化的自体NK混合培养使之成为DC-CIK。以DC-CIK细胞为效应细胞,乳酸脱氢酶释放法观察其对靶细胞的杀伤率。结果:所获目的基因经测序与GenBank比对序列一致,真核表达载体质粒经限制性酶切鉴定片段大小正确,并均可转染至伊马替尼治疗有效、无效的慢性期CML患者及健康志愿者来源的DC细胞。经转染DC的CD1а、CD83、CD86表达率增加,细胞毒活性试验表明转染后的DC可增加自体NK细胞对K562细胞的杀伤活性。结论:各组DC能够通过转染IL-21和GITRL基因的方式获得自我活化、上调自身细胞表面分子的表达,增强其对肿瘤细胞的细胞毒活性。

Objective： To construct the eukaryotic expression vector carrying glucocorticoid-induced tumor necrosis factor receptor ligand （GITRL） and interleukin-21 （ IL-21 ） gene for transfection into chronic myeloid leukemia （CML） derived dendritic cell （ DC）, which provides an effective platform for exploring the function of target gene in CML Methods ： The recombinant eukaryotic expression vector was transfected the purified DC by Liposome-mediated method. The surface molecular expression of transfected DC was analyzed by Flow Cytometry. Further, we mixed culture the transfected DC with purified NK to be DC-CIK, lactate dehydrogenase release assay was performed to measure the killing activity of DC-CIK. Results ： The sequence of cloned target gene was same as that in GenBank. The size of endonuclease products by restriction enzyme was same as the predict one. The surface molecular expression and cytokine secretion were all increased in transfected DC. The killing activity of NK became stronger while induced by transfected DC. Conclusion： DC transfected by IL-21 and GITRL gene has the ability to self-active, up-regulate cytokine secretion. Moreover, the results would be help to seek advanced immunotherapy theoretical evidence for CML.