Comparative Study On Inhibiting Proliferation Of Hepatocellular Carcinoma In Vitro And In Vivo By Matrine And Oxmatrine

The chemical components from the extract of traditional Chinese herb medicine Sophora flavescens root mainly include flavones and alkaloid. Researches in recent years showed that the alkaloid has a anti-tumor effect in vitro,and the matrine and oxmatrine are the main active components. Today the injections whose main components are matrine and oxmatrine are on sale. But they are mainly used as assistant drug of other anti-tumor drugs and anti-HBV drug. The reports about on their anti-tumor effects remain few both at home and abroad. This project take matrine and oxmtrine as inducer, human hepatoma cell HepG2 and mice with tumors as experimental model to observe the differences of the effects and the possible mechanisms in inhibiting proliferation of tumor by Matrine and Oxmatrine in vitro and in vivo. Mainly include the following 2 aspects:1. Comparative study on the inhibiting effect of matrine and oxmatrine on the proliferation of HepG2 in vitro.(1) The survival rate and changes in number of cells were detected with MTT assay. The cell's necrosis was observed with Trypan blue staining method. With above experiments; The study determined the differences between marine's and oxmatrine's effects that inhibited the proliferation of HepG2. (2) Study on the apoptosis of HepG2 cell induced by matrine. The apoptosis peaks were detected with flow cytometric assay. DNA's break was detected with TUNEL.The expression of Bcl-2 and Bax gene were investigated with immunohistochemical analysis.(3) Study on signal transduction of apoptosis. The changes of ATPase were detected with the assay of phosphor. The changes of cAMP and cGMP were found with immunoradiometric assay. The changes of Ca2+ in cells were detected with the laser scanning confocal microscope.2. Comparative study on the anti-tumor functions in vivo of marine and oxmatrine:(1) We divided the nudemice into several groups, give them hypodermic with matrine and oxmatrine of deferent concentration, meanwhile, design the negative control of water and positive control of cyclophosphoramide.Survey the anti-tumor rate of each group.(2) Taking the KunMing mice with H22 tumor as experimental model,give them celiac injections with matrine and oxmatrine of deferent concentration. The negative and positive controls are equal to that of nudemice. The anti-tumor rate of each group was deteted.The changes of ATPase of mice's red blood cells were detected with assay of phosphor. The functions of mice's livers of each group were detected with automatic biochemical analyzer. The differences of expression of Bcl-2 and Bax in the tissue of tumor were found with IHC.The changes of immunity function of mice's after injected matrine and oxmatrine were discovered by WBC count , determination of spleen coefficient,experiment of the lymphocyte proliferations in vitro, experiment of hemolysin and the experiment of eliminating of powder of carbon.The main results of the research can be represented as follows:1. Experimental study on the inhibiting of matrine and oxmatrine on the proliferation of HepG2.According to MTT and cell court,it could be shown that after being treated with 0.8g/L Martine for 3 days,the proliferation of HepG2 could be inhibited apparently with its cell's survival rate remaining 51% While the proliferation of HepG2 could not be inhibited after being treated with 1.5g/L oxmatrine for 3 days ,with its cell's survival rate reaching 84.2%.2. Study on the apoptosis of tumor cells induced by matrine and oxmatrine based on FCM,the apoptosis of tumor cells could be induced by matrine when the concentration was 1.5g/L,while lots of cells were blocked in the S phage. The apoptosis peak appeared when the concentration of oxmatrine reached 8.g/L. The TUNEL experiment showed matrine could apparently induce the apoptosis of tumor cells when the concentration was 1.5g/L while oxmatrine couldn't at the same concentration. It was found that after being treated with 1.5g/L matrine, the expression of Bax protein increased and the expression of Bcl-2 protein w...