| Background and Objective Thrombin is a natural component of organism thromboxane system and multifunctional prolease developing at the injured vascular endothelial cells. Besides activating the other blood coagulation factors and participating hemostasis and thrombopoiesis, thrombin is an important mitogen and can induce many cell functions. Through the in vitro and in vivo experiments, we aim (1)to investigate the effects of thrombin on the proliferation inhibition and apoptosis induction of Hepatocellular carcinoma cell strain HepG2; (2)to observe the effects of thrombin on the growth suppression and apoptosis induction of the subcutaneously transplanted VX2 tumor in rabbits.Methods (1)HepG2 cells were cultured in vitro and planted at the 96-well plate with 1×105cells/well. The HepG2 cells were divided into 6 groups, among which 5 groups were treated with thrombin at the does of 25 U/ml, 50 U/ml, 100 U/ml, 150 U/ml and 200 U/ml, respectively, and the other one, without any treatment, was taken as control. Proliferation inhibition in each group was observed at 24,48 and 72h with MTT assay. The simultaneous procedure as above was performed at the 24-well plate and the apoptosis induction was detected with TUNEL assay at the same time points. (2) The model of subcutaneously transplanted VX2 tumor in rabbits was estabtish The tumor-bearing rabbits were divided into 6 groups randomly, which were treated with thrombin of 50U/ml, 100U/ml, 150U/ml, 200U/ml and 250U/ml, and 0.9% NS (control) , respectively. By ultrasound-guided puncture, thrombin was injected into the VX2 tumors in rabbit. The PT,APTT and FIB in blood were detected respectively on the 1std pretherapy,the 3rdd and 14thd post-treatment, which showed the change of blood clotting function of rabbit, the tumor volume in each group was measured by three-dimensional ultrasonography; the tumor vascularization was observed by contrast-enhanced ultrasound and three-dimensional power doppler ultrasonography; the tumor pathologic changes were observed by microscope; and the apoptosis induction was detected with TUNEL assay.Results (1) The proliferation inhibition increased with the dose and the treating time of thrombin when the dose was at 25U/ml-100U/ml. However, when the does increased to >100U/ml and the treating time was >48h, the inhibitory ratio of HepG2 cells was stopped rising and maitained at the level of (39.08±2.32)%, despite the continuous increase of dose and treating time.(2) HepG2 cells apoptosis was induced by thrombin, in a dose and time-dependent manner when the dose was between 25 to 100 U/ml. However, there was no significant difference between the different groups when the dose was higher than 100U/ml and the time exceeded 48 h. (3) There was no significant difference in the blood clotting function presented by PT and APTT between pre and post treatment (P<0.05), FIB was raised at the 3rdd post-treatment and dropped back to normal level at the 14thd(P>0.05).(4) Compared with the control group, the tumor growth was inhibited obviously in all the treating groups (P<0.05) while there was no significant difference between 200U/ml group and 250U/ml group (P>0.05). (5) Apoptosis was detected by TUNEL assay in all the treating groups, but there was no significant difference in apoptotic index between 200U/ml group and 250U/ml group (P>0.05).Conclusions (1) thrombin of high doses can inhibit the proliferation of the HepG2 cells and lead to apoptosis, in which there are a dose-dependent trend (<100U/ml) and a time-dependent trend (<48h). (2) The treatment with thrombin do not cause marked change of the blood clotting function including PT,APTT, FIB rises up temporarily and comes back to normal level fleetly. (3) Thrombin can inhibit the growth of the subcutaneously transplanted VX2 tumor in a dose-dependent manner when the dose is between 50 to 200U/ml. (4) The mechanism of thrombin inhibit tumor cell proliferation is that it can induce the tumor cell apoptosis. |
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