Efficient Retrovirus‐mediated Cytokine‐gene Transduction of Primary‐cultured Human Glioma Cells for Tumor Vaccination Therapy
Efficient Retrovirus‐mediated Cytokine‐gene Transduction of Primary‐cultured Human Glioma Cells for Tumor Vaccination Therapy
复制标题
原代培养的人胶质瘤细胞的高效逆转录病毒介导的细胞因子基因转导用于肿瘤疫苗治疗
DOI：
--
复制
发表时间：
1997
期刊：
影响因子：
--
通讯作者：
H. Hamada
中科院分区：
文献类型：
--
作者：
H. Wakimoto;Y. Yoshida;M. Aoyagi;K. Hirakawa;H. Hamada
为了利用基因修饰表达某些细胞因子的肿瘤细胞实现恶性神经胶质瘤的新型疫苗接种治疗，必须实现对原代培养细胞的有效基因转导。我们研究了通过逆转录病毒介导的基因转导制备神经胶质瘤疫苗的可行性。神经胶质瘤细胞主要从六名患者手术切除的肿瘤组织中培养。在所有 6 个案例中，我们在 8 周内获得了超过 1000 倍的培养物增殖。体外感染携带大肠杆菌 β-半乳糖苷酶标记基因的重组逆转录病毒 GKlacZ 导致超过 65% 的基因转移至原代培养的神经胶质瘤细胞。通过重复感染或使用带有neo标记基因的载体，可以进一步富集转导细胞（~90%）。通过用两个单表达 GK 载体顺序转导，将两种细胞因子基因（粒细胞-巨噬细胞集落刺激因子和白细胞介素-4）引入神经胶质瘤细胞中。转导的神经胶质瘤细胞产生高水平的两种细胞因子。我们还评估了使用包含内部核糖体进入位点（IRES）或内部启动子（PGK）的双表达 GK 载体同时引入两个基因。虽然双表达载体转导的细胞分泌两种细胞因子，但IRES或PGK后的基因产物水平比上游基因产物水平低10倍。 100 Gy 照射后，转导细胞在体外保留细胞因子分泌 14 天。我们的数据表明逆转录病毒介导的从临床神经胶质瘤材料制备基因修饰肿瘤疫苗的可行性，这可能有助于增强神经胶质瘤患者的抗肿瘤免疫力。
In order to realize a novel vaccination treatment for malignant gliomas using tumor cells genetically modified to express certain cytokines, it is essential to achieve an efficient gene transduction into primary cultured cells. We investigated the feasibility of preparing a glioma vaccine through retro‐virus‐mediated gene transduction. Glioma cells were cultured primarily from surgically resected tumor tissues of six patients. We obtained more than 1000‐fold proliferation of cultures within eight weeks in all six cases. In vitro infection with a recombinant retrovirns GKlacZ carrying an Escherichia coli β‐galsictosidase marker gene resulted in over 65% gene transfer to the primary cultured glioma cells. Further enrichment (∼90%) of transduced cells was possible by employing repeated infections or using vectors with neo marker gene. Two cytokine genes, granulocyte‐macrophage colony‐stimulating factor and interleukin‐4, were introduced into glioma cells by sequential transduction with two single‐expression GK vectors. The transduced glioma cells produced high levels of both cytokines. We also evaluated simultaneous introduction of two genes with double‐expression GK vectors containing internal ribosomal entry site (IRES) or internal promoter (PGK). Although the cells transduced with double‐expression vectors secreted both cytokines, the level of the gene product following IRES or PGK was 10 times lower than that of the upstream gene product. The transduced cells retained cytokine secretion in vitro for 14 days after 100 Gy irradiation. Our data indicate the feasibility of retrovirus‐mediated preparation of gene‐modified tumor vaccines from clinical glioma materials, which could be useful for potentiating antitumor immunity in glioma patients.
影响因子：
4.1
作者：
MAHALEY, MS;BIGNER, DD;BYNUM, JM
通讯作者：
BYNUM, JM