GITRL联合IL-21基因真核表达载体的构建及体外研究

Construction and In Vitro Study of Eukaryotic Expression Vector Carrying GITRL and IL-21 Gene

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)联合白介素-21(IL-21)基因真核表达载体,为探讨目的基因对慢性髓系白血病(CML)来源树突状细胞(DC)的影响。应用脂质体介导法将重组真核表达载体转染至纯化的DC,而此纯化的DC来源于经伊马替尼治疗有效或无效的慢性期CML患者或健康志愿者。应用ELISA法检测转染后DC细胞因子的浓度变化。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,应用乳酸脱氢酶释放法观察其对靶细胞的杀伤率。结果表明:所获目的基因经测序与GenBank比对序列一致,PCR及限制性核酸内切酶检测显示,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。成功转染相应载体后的DC分别表现出IL-2和IFN-γ分泌量增加;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:DC能够通过转染IL-21和GITRL基因的方式获得自我活化、上调自身细胞因子分泌的能力,为酪氨酸激酶抑制剂治疗无效的CML患者的免疫治疗奠定了理论依据和实验基础。

The aim of this study was 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） dervied dendritic cell （DC）,so as to provide an effective platform for exploring the function of target gene in CML.The recombinant eukaryotic expression vector was transfected to the purified DC by Liposome-mediated method,the interleukin-2 （IL-2） and Interferon-γ （IFN-γ） expression of transfected DC were analyzed by ELISA.Further,the transfected DC with purified NK were mixed and cultured to be DC-CIK,the lactate dehydrogenase release assay was performed to measure the killing activity of DC-CIK.The results indicated that the sequence of cloned target gene was same as that in GenBank.The size of endonuclease products by restriction enzyme were same as the predict one.The concentration of Interleukin-2 （IL-2） and interferon-γ （IFN-γ） in transfected DC all increased.The NK kill activity became stronger while induced by transfected DC.It is concluded that DC transfected by IL-21 and GITRL gene has the ability of self-activation,up-regulate cytokine secretion.Further,the results would be help to provide the theoretical evidence of advanced immunotherapy for treatment of CML patients who showed no reaction to tyrosine kinase inhibitor.