人外周血内皮祖细胞转染自杀基因杀伤恶性胶质瘤细胞的体外研究  

An vitro study on killing maglinant glioma cell by human peripheral blood EPCs transfected with suicide gene

作　　者：李罡[1] 杨学军[1] 纪延伟[1] 张文高[1] 潘强[1] 高松[1] 

机构地区：[1]天津医科大学总医院神经外科

出　　处：《中国现代神经疾病杂志》2008年第5期465-470,共6页Chinese Journal of Contemporary Neurology and Neurosurgery

基　　金：国家自然科学基金资助项目(项目编号:30571904);国家自然科学基金资助项目(项目编号:30772228)

摘　　要：目的观察5-氟胞嘧啶诱导基因修饰型内皮祖细胞群发生自身死亡及其旁效应对共培养人脑胶质瘤细胞系U251的杀伤作用。方法采用密度梯度离心法分离经体外诱导培养的人外周血内皮祖细胞,经转染获得基因修饰型内皮祖细胞。RT-PCR法鉴定胞嘧啶脱氨酶(CD)基因在基因修饰型内皮祖细胞中的表达变化,观察野生型和基因修饰型内皮祖细胞在不同浓度5-氟胞嘧啶作用下的细胞存活率,以及基因修饰型内皮祖细胞所诱导的旁效应。结果真核细胞表达载体pCEA-CD-CAUP转染成功,CD基因在基因修饰型内皮祖细胞中表达阳性;在不同浓度5-氟胞嘧啶(0.01～1000.00μg/ml)作用下,基因修饰型内皮祖细胞存活率低于野生型内皮祖细胞,组间差异有统计学意义(均P<0.05)。不同比例的基因修饰型内皮祖细胞与人脑胶质瘤细胞系U251共培养72h后,其细胞生长抑制率均高于基因修饰型内皮祖细胞所占的比例,当基因修饰型内皮祖细胞比例占细胞总数的50%时,细胞生长抑制率即已>80%。结论人外周血内皮祖细胞转染CD基因后,在5-氟胞嘧啶的作用下可发生自身死亡并触发旁效应而杀伤共培养的人脑胶质瘤细胞系U251细胞,为抗血管生成药物与自杀基因联合应用治疗恶性胶质瘤的体内实验及后续研究提供了依据。Objective To investigate thekiliing action on the co-cultured glioma cell line U251 in the occurrence of autophagocytosis and the related bystander effect of gene modified type endothelial progenitor cells （EPCs） group induced by prodrug 5-fluorocytosine （5-FC）. Methods In vitro cultured human peripheral blood EPCs were separated by gradient centrifugation, and obtained cytosine deaminase （CD） gene modified type EPCs. The expression of CD gene in gene modified type EPCs was detected by RT-PCR. Observed the cell survival rate of gene modified and wild type EPCs in different 5-FC concentration and the bystander effect induced by gene modified type EPCs. Results Eucaryotic cell expressed carrier pCEA-CD-CAUP was successfully transfected into EPCs and CD gene expression was positive in gene modified type EPCs. Under different 5-FC concentrations （0.01-1000.00 μg/ml）, the cell survival rate in gene modified type EPCs was lower than that in wild type EPCs. The difference between 2 groups was significant under the same concentration of 5-FC exposure （P〈0.05, for all）. After 72 h-culture, the cell growth inhibitory rate in different ratio of co-cultured CD gene modified type EPCs and human cerebral glioma cell line U251 was higher than that of gene modified type EPCs. When the ratio achieved 50%, the cell growth inhibitory rate was already more than 80%. Conclusion After human peripheral blood EPCs transfected with CD gene, the administration of 5-FC can induce autophagocytosis and trigger bystander effect, and kill the co-cultured human cerebral glioma cell line U251. It would support the further work for therapeutic modalities of combined anti-angiogenesis agent With suicide gene therapy in vivo.

关 键 词：干细胞 内皮 基因 转染 氟胞嘧啶 神经胶质瘤 

分 类 号：R739.41[医药卫生—肿瘤]