Exploration Of Action Concentration And Anti-FBL-3 Mechanism Of Crude Extracting(antimicrobial Peptide) From Musca Domestica Larvae

Objective:1.In this experiment, through constructing the model of C57BL/6 mouse erythroleukemia, get closer to the in vivo state primary cells. But many tumor cells are used in experiment of treated cell lines. The construction of erythroleukemia animal model is an important prerequisite for the subsequent experimental primary FBL-3 cells were cultured successfully.2.Adjust the antibacterial peptides extracts from Musca domestica larvae to different concentrations,then use them to effect on the primary FBL-3 cells, in order to explore the best concentration of killing the primary FBL-3 cells.3.Under the microscope, we can observe the amount of cellular debris, and see the cytoplasmic streaming outside. Which way does Musca domestica larva antibacterial peptide effect on tumor cells through? We connect Fluorescein isothiocyanate(FITC) to the antibacterial peptide and effect on FBL-3 cells and another tumor cell MCF-7. By Laser confocal fluorescence microscopy, explore the mode of action about antimicrobial peptides inhibiting tumor cell.Methods:Select healthy mice for building the animal model, then construct of red leukemia animal model. C57BL/6 mice are suitable for constructing leukemia animal model which is good repeatability, short cycle. We choose the method of allograft transplantation to fabricate animal model. Firstly, recover and cultivate FBL-3 cells used for transplantation.Secondly, when the cells are in logarithmic phase, digest cells, count the cell number and adjust them to 2.25 × 106/m L. Thirdly, via the tail vein inject cells into the target mice,observe at any time mental state of mice, and touch the lymph nodes and thymus of mice.Execute model mouse, strip the tumor, shearing and grinding mass to obtain tumor cells. Transfer cell suspension obtained into cell culture flask,culture it in 37 ℃、5 %CO2cell culture medium and observe cells growth conditions.Dip into the Escherichia coli, induce three instar larvae of Musca domestica by acupuncture, culture 24 h. Through grinding, centrifugal, solid phase extraction and vacuum freezing and drying, we can get the crude extract of Musca domestica antibacterial peptide.The antibacterial peptide concentrations are adjusted of 150, 250, 350, 450 μg/m L,then effect in primary FBL-3 cells. The cell morphology was observed under the microscope after 24 h. By using trypan blue staining, cell survival rate was calculated. Use CCK-8 kit to detect cytotoxic of primary FBL-3 cells at different concentrations of antibacterial peptide. Finally, carry on statistics analysis.Label antibacterial peptide by fluorescein isothiocyanate(FITC), effect on FBL-3cells and MCF-7 cells, and incubate for 4 h. Make temporary cell specimen, and observe tumor cells using laser confocal fluorescence microscopy, in order to know mechanism of antibacterial peptides to the non solid tumor cell FBL-3.Results:By using the method of allograft transplantation, we can find that there are lumps in armpit of C57BL/6 mouse about a week or so, which spread throughout the thymus. At the same time, the state of mouse is dispirited. After stripping of mass, we cultivate FBL-3cells. The biological nature of these cells is rapidly proliferating and repeated passages.Under the effect of antibacterial peptide, we can find the emergence of a large number of cell debris in the field of vision, and see signs of cytoplasmic streaming with the inverted microscope observation. After trypan blue staining, and count, calculate the cell survival rate. With the increase of concentration of antibiotic peptide, the trend of survival rate of tumor cells is first increased and then decreased. Use CCK-8 kit to detect cytotoxic of primary FBL-3 cells at different concentrations of antibacterial peptide. The statistical results show that there is significant difference between each experimental group and the normal group(P<0.05). 250, 300, 350 μg/m L,pairwise comparison,the difference was not statistically significant(P<0.05). With the increase of antibacterial peptide mixture concentration, cell toxicity increased first and then decreased. When the concentration ofantibiotic peptide is 300 μg/m L, cell toxicity is the maximum.Which mechanism does antibacterial peptide inhibit and kill FBL-3 cell by?Connecting the antibacterial peptide with fluorescein isothiocyanate(FITC), and using laser confocal fluorescence microscope, we can see that tumor cells fluoresce green. At the same time, we verify the antibacterial peptides have effect on the MCF-7 cells.Conclusion:Through the screening of healthy C57BL/6mice, using the method of allograft,injecting of FBL-3 cells via the tail vein, the model of red leukemia is constructed successfully in C57BL/6 mice. The most important is that we get the FBL-3 primary cells.By using the animal model of acquired FBL-3 primary cells, we interfer with tumor cell through the different concentrations of antimicrobial peptides, and find that the antimicrobial peptides for primary FBL-3 cells does not increase with the concentration of the enhancement of antibacterial peptide. According to the different concentrations, the experiment group is divided into 150, 200, 250, 300, 350, 400 μg/m L group. 300 μg/m L group is detected the cytotoxicity of maximum. The statistical results suggest that cytotoxic firstly increases with increasing concentration of antibiotic peptide and then decreases, and reachs the peak value at 300 μg/m L. Thus, the best concentration of antibacterial peptide which effects on primary FBL-3 cells can be got, and this result provides help for animal experiment.It has been confirmed once again that Musca domestica antibacterial peptide can inhibit and kill tumor cells. At the same time, explore the best concentration of inhibition of FBL-3 cells. Musca Domestica antibacterial peptides have inhibitory or killing effects on FBL-3 and solid tumor cell MCF-7. The way is to enter the cell through the dissolved cell membrane, as a result of cell apoptosis.Antibacterial peptide with marking FITC effects of FBL-3 cells, and under the fluorescence microscope cells show green fluorescence. Under inverted microscope,through effect of antibacterial peptide, we can see a large number of cellular debris, and cell membrane appear fracture. By inference, antibacterial peptide can enter tumor cells through the cell membrane, which is the mechanism of antibacterial peptides to inhibit or kill non solid tumor cell FBL-3.