电离辐射促羟基磷灰石纳米载体介导的hGM-CSF基因在人肝癌HepG2细胞中的转移及表达

目的观察电离辐射对羟基磷灰石(HA)纳米载体介导的质粒pOR F-hGM-CSF基因转染的影响,并探索能提高HA转染效率的方法。方法转染细胞前,依据不同辐射剂量下的HepG2细胞剂量-存活曲线选出2、4和6 Gy3种不同剂量进行分组照射。48 h后R T-PCR检测HepG2细胞内hGM-CSF的mR NA表达水平以比较转染效率。结果电离辐射可以促进HA纳米载体介导的基因在HepG2细胞中的转移和表达,在辐射剂量为6和4 Gy时,hGM-CSF基因mRNA相对表达量可达(0.9941±0.0703)和(0.9501±0.5619),与剂量为0 Gy的表达量差异有显著性(P<0.05),但与Lipofectamin 2000组比较差异无显著性(P>0.05),故当辐射剂量为6和4 Gy时,HA纳米载体的转染效率可接近Lipofectamin 2000的转染效率。辐射剂量为6、4、2和0 Gy时,培养基中hGM-CSF的分泌量分别为(499.20±5.76)、(497.06±4.54)、(472.53±3.91)和(428.65±7.16)ng/106cell。结论电离辐射可以提高HA纳米载体的转染效率,并且HA纳米载体转hGM-CSF基因的自体肿瘤疫苗联合放疗治疗人类肝细胞癌有望成为可能。

Ionizing irradiation enhanced delivery and expression of nano-HA mediated hGM-CSF gene in HepG2 cells

【Objective】 To observe the effects of irradiation on the delivery and expression of HA-pORF-hGM-CSF complexes in HepG2 cells and find out a way to improve gene transfection.【Methods】 Prior to hydroxyapatite(HA) transfection,HepG2 cells were irradiated at dose of 2,4,6 Gy according to the cell survival curve of HepG2 cells after being exposure to different dosage of radiation.After 48 hours of nano-HA transfection,the expressions of mRNA in HepG2 cells were tested by RT-PCR.【Results】 Ionizing radiation could enhance delivery and expression of nano-HA mediated gene in HepG2 cells.The relative expression of hGM-CSF mRNA were(0.9941±0.0703),(0.9501±0.5619) at the radiation dose of 6,4 Gy,respectively,which were significantly different from the radiation dose of 0 Gy,but were not significantly different from Lipofectamin 2000 vector.Combined radiation and nano-HA gene delivery achieved the approximate transfection efficiency of Lipofectamin 2 000 vector,when radiation doses were 6,4 Gy.The level of GM-CSF secreted by stably transfected HCC cells were(499.20±5.76),(497.06±4.54),(472.53±3.91),(428.65±7.16) ng/106 cell,when the doses of irradiation were 6,4,2 Gy.【Conclusion】 Ionizing radiation could be a useful way of improving the transfection efficiency of HA-mediated gene delivery.And autologous tumor vaccine of the transfected hGM-CSF gene mediated by nano-HA vector and radiation combination may have the potential to treat hepatocellular carcinoma.