肝癌细胞下调IGF-1活性因子对Ras癌基因诱导小鼠肝肿瘤影响分析

Effect of down-regulation of IGF-1 active factor on mouse liver cancer induced by Ras oncogene

目的分析肝癌细胞下调胰岛素生长因子(IGF-1)活性因子对Ras癌基因诱导小鼠肝肿瘤的影响。方法选取3、5、9月龄Ras癌基因诱导雄性小鼠各10只,同时选取非转基因3、5、9月龄雄性小鼠各10只,提取9月龄Ras癌基因诱导雄性小鼠肿瘤组织、肿瘤周围组织样品为实验组,同时提取9月龄非转基因小鼠肝组织样品作为对照组,提取两组样品总RNA和蛋白质,选用蛋白质印迹法对其细胞外信号调节激酶(ERK)、磷脂酰肌醇3-激酶(PI3K)、蛋白激酶B(AKT)蛋白表达进行检测;选用荧光定量PCR法对IGF-1、GHR、IGFBP3的mRNA表达水平进行检测;选用酶联免疫法对小鼠血清IGF-1水平进行检测;对3、5、9月龄Ras癌基因诱导雄性小鼠及非转基因小鼠外周血中B淋巴细胞、活性B淋巴细胞比例进行检测。结果 Ras癌基因小鼠诱导肿瘤组织ERK、PI3K、AKT平均蛋白表达水平与肿瘤周围组织比较,差异有统计学意义(P<0.05);Ras癌基因诱导小鼠肿瘤组织、肿瘤周围组织ERK、PI3K、AKT蛋白平均表达水平与对照组比较,差异有统计学意义(P<0.05);Ras癌基因诱导小鼠肿瘤组织IGF-1、GHR、IGFBP3的mRNA平均表达水平(0.27±0.05)、(0.23±0.02)、(19.48±2.54)与肿瘤周围组织(1.25±0.07)、(0.89±0.17)、(3.25±0.56)比较,差异有统计学意义(P<0.05);肿瘤周围组织IGF-1、GHR、IGFBP3的mRNA平均表达水平与对照组比较,差异无统计学意义(P>0.05);3月龄Ras癌基因诱导小鼠血清IGF-1水平(61.72±9.24)μg/mL与非转基因小鼠(58.89±8.16)μg/mL比较差异无统计学意义(P>0.05);月龄5、9个月Ras癌基因诱导小鼠血清IGF-1水平(51.28±5.27)、(40.28±6.28)μg/mL均低于非转基因小鼠(55.32±7.91)、(49.52±6.94)μg/mL,差异有统计学意义(P<0.05);3、5、9月龄Ras癌基因诱导小鼠B淋巴细胞比例均低于非转基因小鼠,差异有统计学意义(P<0.05);3、5月龄Ras癌基因诱导小鼠活性B淋巴细胞比例与非转基因小鼠比较差异无统计学意义(P>0.05);9月龄Ras癌基因诱导小鼠活性B淋巴细胞(CD19+CD69+、CD19+CD25+)比例(0.77±0.13)、(1.42±0.17)低于非转基因小鼠(0.87±0.14)、(2.39±0.22),差异有统计学意义(P<0.05)。结论 IGF-1的表达的抑制,降低了PI3K、AKT、GHR的表达,提高了ERK、IGFBP3的表达,进而导致Ras癌基因通路的活化。

Objective To investigate the effect of the down-regulation of IGF-1 active factor on Ras oncogene induced mouse liver tumor.Methods Each 10 Ras oncogene induced male mice with 3-,5-,9-month old were selected,meanwhile each 10 male and female mice with 3-,5-,9-month old were selected.The tumor tissues and peritumoral tissues in the Ras oncogene induced male mice with 9-month old served as the experimental group,meanwhile the liver tissue samples extracted from non-transgenic mice with 9-month old served as the control group.The total RNA and protein were extracted from the two groups.The ERK,PI3K and AKP proteins were detected by using Western blot.The expression levels of IGF-1,GHR and IGFBP3 mRNA were detected by using the fluorescence quantitative PCR.The level of serum IGF-1 was detected by the enzyme-linked immunosorbent assay(ELISA).The proportions of peripheral blood B lymphocytes and active B lymphocytes in Ras oncogene induced male mice and non-transgenic mice with 3-,5-,9-month old were detected.Results The average expression levels of ERK,PI3K and AKT proteins had statistically significant difference between the tumor tissues and peritumoral tissues in Ras oncogene mice(P<0.05),and the expression levels of ERK,PI3K and AKT had statistically significant difference between the tumor tissues and peritumoral tissues in Ras oncogene mice with the control group(P<0.05).The mean expression levels of IGF-1,GHR and IGFBP3 mRNA in tumor tissues of Ras oncogene induced mice were(0.27±0.05,0.23±0.02),(19.48±2.54),which in peritumoral tissues were(1.25±0.07),(0.89±0.17)and(3.25±0.56)respectively,the difference was statistically significant(P>0.05).The IGF-1,GHR and IGFBP3 mRNA level had no statistical difference between peripheral tissues and control group(P>0.05);the serum IGF level in Ras oncogene mice with 3-month old was(61.72±9.24)μg/mL,which in the non-transgenic mice was(58.89±8.16)μg/mL,the difference was not statistically significant(P>0.05);the serum IGF-1 levels in the Ras oncogene induced mice with 5-,9-month old were(51.28±5.27)μg/mL and(40.28±6.28)μg/mL,which were lower than(55.32±7.91)μg/mL and(49.52±6.94)μg/mL,the difference was statistically significant(P<0.05).The proportions of B lymphocytes in the Ras oncogene mice with 3-,5-,9-month old were lower than those in the non-transgenic mice,the difference was statistically significant(P<0.05);the proportions of B lymphocytes had no statistical difference between the Ras oncogene mice with 3-5-month old and non-transgenic mice Ras carcinomas was(P>0.05).The proportions of active B lymphocytes(CD19+CD69+,CD19+CD25+)in Ras oncogene mice with 9-month old was 0.77±0.13 and 1.42±0.17,which were lower than 0.87±0.14 and 2.39±0.22 in the non-transgenic mice,the difference was statistically significant(P<0.05).Conclusion Inhibition of IGF-1 expression reduces the expressions of PI3K,AKT and GHR,enhances the expressions of ERK and IGFBP3,and then leads to the activation of the Ras oncogene pathway.