Research On The Inhibitory Effects Of Oligochitosan Combined With Earthworm Fibrinolysin On Hepatocellular Carcinoma Cells In Vivo And In Vitro

ObjectiveTo investigate the inhibitory function in vivo and in vitro of oligochitosan combined with earthworm fibrinolysin on hepatocellular carcinoma cells.MethodsVarious methods were used to detect the growth condition of hepatocelluiar carcinoma cell in vivo and in vitro when they were affected by different concentration and time of oligochitosan and earthworm fibrinolysin alone and by the combinative use of the drugs above. The procedures were the following:1. Estimating the inhibition of proliferation by MTT-test.2. Observing the morphologic changes by light microscope.3. Detecting apoptosis cells by flow cytometry.4. Detecting the expression of Bcl-2 mRNA by RT-PCR.5. Observing the effect of the drugs on the growth of H22 cell subcutaneous transplanting tumor in mice in experiment in vivo.6. Measuring the expression of apoptosis-associated gene proteins Bcl-2 in the tumor tissues by immunohistochemical technique.Results1. Oligochitosan could inhibit the proliferation of SMMC-7721 cell lines, and the inhibition depended on the exposure time and dose, but the inhibition was weak. Earthworm fibrinolysin could inhibit the proliferation of SMMC-7721 cell lines, and this effect increased with the increasing dosage and time. There were additive effects when oligochitosan of lmg/ml was combined with earthworm fibrinolysin of 2.5uku/ml and 5uku/ml. There were synergistic effects when oligochitosan of 2mg/ml was combined with earthworm fibrinolysin of 2.5uku/ml and 5uku/ml.2. Observed by light microscope, the cells shrank and turned round, and the contacts among cells became loose when oligochitosan was combined with earthworm fibrinolysin.3. Both oligochitosan and earthworm fibrinolysin could induce the apoptosis of SMMC-7721 cell lines. The apoptosis rate markedly increased and reached to 23.60% when oligochitosan was combined with earthworm fibrinolysin.4. The expression of Bcl-2 mRNA in SMMC-7721 cells was suppressed by both oligochitosan and earthworm fibrinolysin and obvious strengthening effects were observed when oligochitosan was combined with earthworm fibrinolysin.5. In experiment in vivo, both oligochitosan and earthworm fibrinolysin could inhibit the growth of H22 tumor cells, the tumor inhibiting rates were 17.13% and 18.65%, respectively. The tumor inhibiting rate was 49.07% and obvious strengthening effects were observed when oligochitosan was combined with earthworm fibrinolysin. Compared with those of the control group, the inhibitory effect was significant (P<0.05).6. The expression of Bcl-2 was down-regulated in the tumor tissues by both oligochitosan and earthworm fibrinolysin, while obvious strengthening effects were observed when oligochitosan was combined with earthworm fibrinolysin.ConclusionBoth oligochitosan and earthworm fibrinolysin can inhibit the proliferation and induce the apoptosis of hepatocellular carcinoma cells both in vivo and in vitro. When oligochitosan is combined with earthworm fibrinolysin, it strengthens the inhibition of proliferation and inducement of apoptosis. The mechanism is related to down-regulation of the expression of Bcl-2. This research provides the experimental foundation for the combination of TCM with western medicine to cure hepatocarcinoma.