白细胞介素-10基因转染对大鼠肝细胞表达细胞因子的影响

Effect of interleukin-10 gene transfection on the cytokine expression of rat hepatocyte in vitro

目的观察转染白细胞介素-10(IL-10)基因后肝细胞表达细胞因子的变化,探讨IL-10基因修饰的大鼠肝细胞抑制肝星状细胞(hepatic stallete cells,HSC)活化的可能机制。方法体外培养大鼠肝细胞系BRL,分3组:对照组(C组)、转染pc DNA3.0空质粒组(P组)、转染pc DNA 3.0-r IL-10重组质粒组(I组)。转染48 h后取培养上清液用抗体芯片筛选各组间细胞因子的差异性表达。用ELISA试剂盒分别检测上清中血管表皮生长因子(VEGF)和转化生长因子-β(TGF-β)的变化。抽提3组BRL细胞的总蛋白,用Western blotting方法检测细胞内VEGF蛋白表达的变化。结果抗体芯片筛选显示,I组较P组IL-10表达上调大于50倍,而VEGF下调大于2倍。进一步检测显示,I组培养上清液中VEGF的表达(136.824±34.150)pg/ml较P组(252.026±33.300)pg/ml显著下降(P<0.05),I组培养上清液中TGF-β的表达(220.296±42.115)pg/ml较P组(363.366±17.434)pg/ml显著下降(P<0.01),I组细胞中VEGF的表达较P组也显著下降(P<0.01)。结论转染IL-10基因的大鼠肝细胞除了通过高分泌IL-10直接抑制HSC的增殖与活化,也可能通过下调VEGF和TGF-β等促肝纤维化细胞因子的表达而间接作用。

Objective To observe the effect of interleukin-10 （IL-10） gene transfection on the cytokine expression of rat hepatoeye in vitro. Methods BRL cells, a normal rat hepatocyte line cultured in vitro, were divided into three groups： control group （group C）, transfected with plasmid pcDNA3.0 group （group P） and transfected with plasmid peDNA3.0-rIL-10 group （group I）. After 48 hours, the supernatant of each group was collected, difference of eytokines expression was analyzed by cytokines antibody chip. The expressions of vascular endothelial growth factor （VEGF） and transforming growth factor-β （TGF-β） in supernatant of each group were detected by ELISA. The expression of VEGF in BRL cells of each group was detected by Western blotting. Results Compared with group P, the expression of IL-10 in supernatant of group I was upregulated more than 50 times, the expression of VEGF was downregulated more than 2 times. Compared with group P, the level of VEGF in supernatant of group I was decreased [ （252. 026 ±33. 300） pg/ml vs （ 136. 824± 34. 150） pg/ml （P 〈 0.05） ], and the level of TGF-13 was decreased [ （363. 366 ± 17. 434） pg/ml vs （220. 296 ±42. 115） pg/ml （P 〈0.01 ）]. Compared with group P, the expression of VEGF in BRL cells of group I was decreased （P 〈 0.01）. Conclusion Rat hepatoeytes transfeeted with IL-10 gene suppress the proliferation and ac- tivation of hepatic stallete cells （HSC） not only by high expression of IL-10, but also by inhibiting the expressions of VEGF and TGF-β.