| Objective: At present, the attack rate of malignant tumor increase year by year, malignant tumor has become a major cause which threatens people's health severely, the prevention and cure of malignant tumor become the important topic for medical workers. More and more researches indicate that tumor is a kind of disease with cell apoptosis disturbance.,The cell apoptosis gene get involve in the happen of malignant tumor, and regulate it's development. In this investigation, different methods were employed to study the effects of apoptosis-inducing and growth-inhibition on the human osteosarcoma cells line MG63 and ovarian cancer cells line A21-2 by matrine, at the same time we detect and analyse the apoptosis activator caspase-3. The purpose of this study is to clarify the anti-tumor mechanism of matrine, it may provide experimental foundation for matrine on anti-tumor medicine.Methods: A21-2cells and MG63 cells were cultured in the environment of 37℃, 5 % CO2 with Dulbecco Eagle's minimum essential medium (DMEM) and RPMI1640. The logarithmically growing A21-2 cells and MG63 cells were used.1 Using light microscope observe the change of morphology: A21-2 cells and MG63 cells were treated with different concentrations of matrine, the change of morphology was observed by inverted phase contrast microscope in different time and photographed.2 Morphology analysis by transmission electron microscopy: A21-2 cells and MG63 cells treated with 0.6mg/ml matrine for 72 hour were harvested , and pre-fixed with 2.5% glutaraldehyde at 4℃, The cells were postfixed for 1 hour with 1% osmium tetroxide, then dehydrated through agraded ethanol series, and embedded in Epon 812. The ultrastructure of cells was analyzed in ultrathin sections in a transmission electron microscope after the sections were stained with uranylacetate and lead citrate.3 The suppressive effects of matrine on the proliferaition of A21-2 cells and MG63 cells were evaluated in vitro by MTT colrimetric assay.4 Flow cytometric analysis: A21-2 cells and MG63 cells were treated with matrine for 72 hour. Experimental group and control group cells were harvested and washed twice with PBS. Cells were fixed with ice cold 70% ethanol at 4℃. Precipitates were digested with 0.5% pepsin after centrifugation. And then cells were resuspended in 0.5ml propideium iodide (PI)/RNAase A solution. Cells were incubated in the dark at room temperature for 15 min. The fluorescence emission of stained cells was measured with a flow cytometer. Data were analyzed with Multipcycle software. 5 Analysis of caspase-3 protein by flow cytometry: A21-2 cells and MG63 cells were treated with matrine for 72 hour.. At the end of the treatment, adherent and floating cells were combined and centrifuged, cells were stained by indirect immunofluorescence labing method. A histogram plot of FITC-fluorescence intensity (in logarithmic fluorescence intensity) (χ-axis) versus counts (у-axis) has been shown by flow cytometry. Fluorescence index was used to analysis the expression of caspase-3 proteins.6 Reverse transcription polymerase chain reaction (RT-PCR) were used to observed caspase-3mRNA expression. After A21-2 cells and MG63 cells were treated with matrine for 72 hour, total cellular RNA was extracted with TRIZOL reagent according to the manufacturer's instructions. Reverse transcription reaction and amplification were carried out. PCR products were electropheresed on 8% polyacrylamide gels(1×TBE running buffer ) and visualized by gels imaging system (BIO-PROFIL, VL company, France) stained by ethidium bromide. The fluorescence intensity ofβ-actin fragments served as the criterion for the fragments.Results: 1 A21-2 cells and MG63 cells were treated with 0.2mg/ml,0.4mg/ml,0.6mg/ml matrine for 72 hour. Inverted phase contrast microscope observed, comparing with control group cells, the growth of drug-treated group cells were inhibited, cell granulations were increased and thicken, some cells became round and suspension in the medium. Under transmission electron microscope, the nuclear chromatin of drug-treated group cells became condensed, marginated and segregated and the cytopolasm was vacuolated. The membrane of the cell, nucleus and cell organ were all complete.2 A21-2 cells and MG63 cells were treated with matrine at various concentrations for 48h and 72h respectively, the growth of cells was inhibited significantly in a time- and dose- dependent fashion. The IC50 of A21-2 cells of 48h and 72h were 1.017mg/ml,0.751mg/ml, The IC50 of MG63 cells of 48h and 72h were 1.019mg/ml,0.701mg/ml, respectively. The biggest inhibitory rate of matrine for A21-2 cells and MG63 cells were 71.89% and 71.16% at the concentration of 1.0 mg/ml for 72h(p<0.01)3 The analysis of cellular DNA content by FCM showed that there was a sub-G0/G1 peak in the graph of drug-treated groups. That was a typical apoptotic peak, which was not shown in the graph of control groups. After A21-2 cells were exposed to matrine of 0.2 mg/ml,0.4mg/ml,0.6mg/ml for 72h, the apoptosis rates were 13.54%±0.39%,17.93%±0.19%,26.35%±0.11%,respectively. while the apoptosis rates of control cell is 5.34%±0.41%. the quantity of treated cells in G0/G1 phase were 59.35 %±1.31% , 67.52 %±0.42% , 77.53 %±0.19%,respectively, while the quantity of control cells in G0/G1 phase were 54.12%±1.23%. After MG63 cells were exposed to matrine (0.2,0.4mg/ml,0.6mg/ml) for 72h, the apoptosis rates were 11.37%±0.53%,14.28%±0.16%,24.13% ±0.23% respectively, which were significantly higher than control (3.27%±0.11)(p<0.01).The quantity of treated cells in G0/G1 phase increased but that in S,G2/M phase decreased. Most MG63 cells were blocked at G0/G1stage (from 63.21%±1.52% to 69.14%±1.12%, 75.31%±0.33%,82.64%±0.16%, respectively). Expressions of caspase-3 protein was increased (Fluorescence index of A21-2 were 1.60±0.03,1.78±0.02,2.08±0.05, respectively. Fluorescence index of MG63 were 1.64±0.01,1.84±0.03,2.08±0.02, respectively.).4 The result of RT-PCR showed that expression of caspase-3mRNA was up-regulated .Conclusions: Mtrine can inhibit proliferation and induce apoptosis of A21-2 cells and MG63 cells.Its possible molecular mechanisms might be related to modulation the expression of caspase-3, Other mechanisms should be investigated further. |
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