Effect Of Tacrolimus Associated With 5-Fu On The Proliferation And The Expression Of VEGF In Hepatocellular Carcinoma

Objective:Tumor recurrence rate determines the long prognosis of HCC patients after orthotopic liver transplantation.Our purpose is to investigate the effects of prograf and fluorouracil on remnant hepatocellular carcinoma cells in the liver receptors with HCC after OLT, and to investigate the effects of FK506 and 5-fu on the proliferation and the expression of VEGF in hepatocellular carcinoma cells which are cultured in vitro.So we can make clear that how FK506 make influence on the internal multiple signal conducting mechanism in hepatocellular carcinoma cells except the CD4 T lymphocyte.To hope that we can determine the biological activity of the remaining hepatocellular carcinoma after OLT with the effect of anti-rejection and 5-fu . Whether can order for hepatocellular carcinoma patients after liver transplantation, anti-rejection drugs and antitumor drugs, under the action of residual tumor cells in the biological activity of judgment, and preliminary speculation whether can inhibit tumor remnants cells of vascular endothelial growth factor expression, reduce the recurrence of hepatocellular carcinoma metastasis rate after transplantation, improve liver transplantation for patients with long-term prognosis.In the end ,we can instruct the use of anti-vegf to inhibit the expression of vascular endothelial growth factor in hepatocellular carcinoma cells.Methods: The SMMC-7721 cells used in the experiment were cultured in vitro and the MTT assay, RT-PCR and Western blot were used to examine the expression of VEGF in the hepatocellular carcinoma cell under the condition of FK506 detection, and 5-Fu lines for hepatocellular carcinoma. And cultivation of liver cancer in nude mice tumor mass, volume, and plasma VEGF levels in mice analysis, supplemented by the cultivation of tumor tissue biopsy VEGF expression and microvessel density analysis, research, and tacrolimus fluorouracil on hepatocellular carcinoma cell line SMMC - 7721 planting tumor proliferation and vascular endothelial growth factor expression.Results:1.the determination of hepatocellular carcinoma proliferation with MTT method: compared with no drugs, FK506 group,5-Fu group and its 5–Fu + Fk506 group that have been cultivated 72 hours, its group of hepatocellular carcinoma cells are different degree of proliferation, FK506 + 5-Fu inhibiting proliferation phenomenon is more obvious than other three groups. In control group with A450 value 0.728士0.07 , FK506 group with A450 value 0.570士0.38, 5 -Fu group with A450 value 0.386士0.21,and FK506 + 5-Fu group with A450 value 0.242士0.47. P < 0.05, That was statistically significant differences.2. ELISA method in detecting the level of VEGF with no drugs group, FK506 group, 5-Fu group and its 5–Fu + Fk506 group that have been cultivated 24 hours . In control group with VEGF density value 194.3pg/ml±5.53pg/ml , FK506 group with VEGF density value 165.7pg/ml±6.32pg/ml, 5 -Fu group with VEGF density value 144.3pg/ml±3.62pg/ml,and FK506 + 5-Fu group with VEGF density value 132.3pg/ml±4.56pg/ml. P < 0.05, That was statistically significant differences.3. the detection of liver cell mRNA VEGF expression:each test object OD value and beta actin OD value ratio: no drug gr oup with the ratio of1.60士0.187, FK506 group with the ratio of 1.00士0.20, 5 -Fu group with the ratio of 0.58士0.08 and FK506 + 5-Fu group with the ratio of 0.38士0.08. P < 0.05, That was statistically significant differences.4.the immune imprinting method to detect liver cell VEGF protein expression level of liver cells:each test object OD value and beta actin OD value ratio: no drug group with the ratio of 1.38士0.084, FK506 group with the ratio of 0.98士0.130, 5 -Fu group with the ratio of 0.60士0.087 and FK506 + 5-Fu group with the ratio of 0.36士0.089. P < 0.05, That was statistically significant differences.5.the liver cell VEGF protein expression in quantitative analysis of protein expressi on value: In control group with VEGF density value 840.7pg/ml±54.36pg/ml, FK506 group with VEGF density value 630.5pg/ml±36.84pg/ml, 5 -Fu group with VEGF density value 510.5pg/ml±29.10pg/ml,and FK506 + 5-Fu group with VEGF density value 341.5pg/ml±38.14pg/ml.P<0.05, That was statistically significant differences.6. FK506 and 5-Fu's effect on human hepatocellular carcinoma xenografts in nude mice : FK506 has no significant inhibition of tumor growth. 5-Fu has on significant tumor growth inhibition, FK506 and 5-Fu has no obvious synergies. FK506 on VEGF expression in tumor tissue had no significant inhibitory effect ,5-Fu has an obvious expression of VEGF inhibition, FK506 and 5-Fu has no obvious synergies. FK506 had no significant inhibition of microvascular on the formation of tumors, there is an obvious inhibition on tumor angiogenesis of 5-Fu, FK506 and 5-Fu has obvious synergies.With the product electrophoresis, compared with the control group, FK506,5-Fu and FK506 + 5 -Fu ,its group of liver cell and to cultivate the vascular endothelial growth factor,mRNA and protein expression is in different degree, FK506 + 5 -Fu inhibiting proliferation is clearly group. Conclusion: FK506(5ug/L) representaties can answer for hepatocellular carcinoma SMMC 7721 cell proliferation and in vascular endothelial growth factor expression were inhibited,fluorouracil(75mg/L)representaties can answer FK506(5ug/L) the pretreatment of liver cell proliferation and SMMC 7721 in vascular endothelial growth factor expression has inhibitory effect. With 5–Fu and FK 506 of the liver cell growth and its ascular endothelial growth factor inhibition, there may be synergistic effect. However, tacrolimus has no significant inhibition on cultivation of liver tumor growth in nude mice, VEGF expression and tumor microvessel formation,5-Fu has obvious role in this regard.