| Objective: To explore the molecular mechanism of reducing the activity of IGF-1 in hepatoma cells,and elaborate the relationship between IGF-1 and B-lymphocytes in Ras oncogene-induced liver tumorigenesis.Methods:1、The liver tissues of 9-month-age non-transgenic male mice(Wt),peri-tumor tissues(P)and tumor tissues(T)of 9-month-age H-ras12 V transgenic male mice were collected.The expression levels of p-ERK,ERK,P-PI3 K,PI3K,p-AKT,AKT in the liver tissue and were detected by western blot.Fluorescence quantitative PCR was used to detect the IGFBP1、IGFBP3、GHR、IGF-1 m RNA levels in tissues.Detection of different months males’ IGF-1,Ig G1 and Ig G2 levels in the serum by ELISA.2、The ratio of subtypes and activation of T and B lymphocytes in peripheral white blood cells of 3,5,9 month-old normal wild type C57BL/6J(non-transgenic)and H-ras12 V transgenic males were detected by using flow cytometry(FCM)and the pathological changes of the liver tissues were also examined.Results:1、Western blot showed that,the protein levels of p-ERK in T tissues were 21.09±6.57,significantly increased than that of Wt(0.08±0.02),t=5.80,P=0.004,and P(8.42±3.21),t=4.95,P=0.005,the protein levels of p-ERK in P tissues were significantly increased than that of Wt,t=3.75,P=0.042;The protein levels of ERK in T tissues were 15.98±1.94,significantly increased than that of Wt(4.72±1.00),t=4.02,P=0.029,and P(4.55±1.12),t=4.92,P=0.005;The protein levels of P-PI3 K in T tissues were 0.47±0.02,significantly increased than that of Wt(0.06±0.00),t=9.02,P=0.008,and P(0.34±0.05),t=3.09,P=0.042,the protein levels of P-PI3 K in P tissues were significantly increased than that of Wt,t=3.32,P=0.045;The protein levels of PI3 K in T tissues were 0.78±0.11,significantly increased than that of Wt(0.03±0.00),t=11.05,P < 0.001,and P(0.29±0.08),t=3.96,P=0.029;the protein levels of PI3 K in P tissues were significantly increased than that of Wt,t=3.42,P=0.016;The protein levels of p-AKT in T tissues were 0.23±0.05,significantly increased than that of Wt(0.11±0.02),t=3.89,P=0.037;The protein levels of AKT in T tissues were 0.18±0.04,significantly increased than that of Wt(0.06±0.03),t=2.87,P=0.042,the protein levels of AKT in P tissues were 0.23±0.06,significantly increased than that of Wt,t=4.92,P=0.010;RT-q PCR results showed that,the levels of IGFBP1 and IGFBP3 m RNA were increased in T;the levels of GHR and IGF-1 m RNA were reduced in T;ELISA analysis showed that 9-month-old transgenic male serum IGF-1 levels(1.27 ± 0.35)compared with the same months in the control group(1.90 ± 0.34)was lower,the difference was statistically significant,t =-4.225,P = 0.020;9-month-old transgenic male serum Ig G1 levels(39.91 ± 4.86)compared with the same months in the control group(49.65 ± 7.14)was lower,the difference was statistically significant,t =-3.845,P = 0.011;9-month-old transgenic male serum the Ig G2 levels(28.80 ± 2.45)compared with the same months in the control group(34.86 ± 4.29)was lower,the difference was statistically significant,t =-3.024,P = 0.006.2、No obvious pathological changes was detected in the liver tissues of 3-month-old H-ras12 V transgenic males and 3,5,9-month-old non-transgenic males.Multiple hepatic nodules and hepatic tumors were found in the liver tissues of 5 and 9-month-old H-ras12 V transgenic males,respectively.No pathological change was detected in other organs apart from liver in transgenic and non-transgenic males.For B lymphocytes,the ratio of s Ig M+CD23+ and s Ig M+B220+ subtypes in peripheral white blood cells of 3,5,9-month-old transgenic males were significantly decreased compared with that of non-transgenic males(P<0.01).The activation ratio of CD19+CD69+ B lymphocytes were also significantly reduced in transgenic 5,9-month-old males(P<0.05).Additionally,the ratio of total and activated B lymphocytes decreased gradually along with aging and hepatic tumor development in H-ras12 V transgenic males.For T lymphocytes,no significant difference was detected in the ratio of T lymphocytes(CD3e+)and activated T lymphocytes(CD3e+CD69+、CD3e+CD25+)between 3 and 5 month-old transgenic and non-transgenic males(P>0.05).However,the total and activated T lymphocytes were significantly decreased in 9-month-old transgenic males compared to non-transgenic males(P<0.05).Conclusion: The vivo experiments confirmed the activation of ras signaling pathway caused IGFBP1 and IGFBP3 m RNA levels increased,GHR and IGF-1 m RNA levels decreased.Along with the malignancy of hepatic tumors,the ratio of subtypes and activation of T and B lymphocytes in peripheral white blood cells were significantly reduced. |
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