水流动力学注射介导的IL-12基因在小鼠体内的高效表达

Efficient expression of interleukin-12 gene transfered by hydrodynamic injection in mice

目的:评估水流动力学注射法转移表达小鼠白细胞介素12(mIL-12)质粒pCMV-mIL-12的有效性,以及质粒DNA重复注射后转基因的表达情况。方法:通过小鼠尾静脉大体积快速注射pCMV-mIL-12质粒后取血及主要器官,应用ELISA、PCR、RT-PCR及免疫组织化学染色等方法,检测质粒在组织器官中的分布与表达。为了研究重复注射质粒后mIL-12的表达,分别在首次注射后第7天、第14天和第30天二次注射相同剂量的质粒。每次注射前及注射后均取血检测mIL-12的表达及其诱导产生的IFN-γ及NO。结果:注射后10小时血清中的mIL-12蛋白水平最高,第7天恢复到基线值,其表达呈DNA剂量依赖性。IFN-γ及NO的表达高峰均出现在注射后第3天。首次注射后第14天及第30天重复注射可获得mIL-12的再次高效表达。结论:水流动力学注射方法是一种简便、有效的体内IL-12基因转移方式。重复应用水流动力学注射方法体内转移表达mIL-12的裸DNA后,可以获得转基因的再次高效表达,两次注射间隔时间至少为14天。

Objective:To evaluate the efficiency of transferring plasmid DNA encoding mouse interleukin-12 (mIL-12) by hydrodynamic injection and investigate the transgene expression after repeated injection of plasmid DNA.Methods:Varying doses of pCMV-mIL-12 plasmid were injected into mice by a hydrodynamic injection. Thereafter, the blood and organs were collected at specific time interval. The distribution and expression of pCMV-mIL-12 were assayed by ELISA, PCR, RT-PCR and immunohistochemical staining. To investigate the expression of mIL-12 after plasmid re-administration, the same amount of pCMV-mIL-12 was repeatedly injected into the mice 7 days, 14 days and 30 days respectively after the initial injection. Following each injection, blood was collected for determination of mIL-12, and induction of IFN-γ and NO levels. Results:mIL-12 was detected 4 h after injecting pCMV-mIL-12 by a hydrodynamic injection. Then it reached a peak level 10 h and abruptly declined to the minimal level on day 3. It was undetectable on day 7. The peak expression level of IFN-γ and NO appeared on day 3 after plasmid injection. A similar high expression of mIL-12 could be obtained after repeated pCMV-mIL-12 injection with an interval of 14 days or 30 days after the initial injection. In contrast,repeated injection 7 days after the first injection did not result in as high level of mIL-12 production as the first injection.Conclusion:The hydrodynamic injection is a convenient and efficient gene transfer method for mIL-12 gene in vivo.Similar high level of mIL-12 is attained after repeated administration of plasmid DNA encoding mIL-12 with at least a 14 day interval following initial injection.