Experimental Study Of The Effect Of Pseudomonas Aeruginosa Injectionon Rat C6 Glioma Cells In Vitro

Objective:Take the C6 Glioma Cells of Rat as a test object,to observe the growth inhibition action of Pseudomonas aeruginosa(PA-MSHA) preparation for C6 glioma cells in vitro,explore the mechanism of tumor cell growth inhibition action caused by the PA-MSHA in vitro.To explore new Method of effectively postoperative adjuvant therapy for glioma.Methods:Through C6 glioma cells of rat were cultured in vitro,Determination of cell viability,cell attachment rate.And three concentrations C6 cells were inoculated with 1×10~3 / holes,3×10~3 / holes,5×10~3 / holes within 96-well plates.MTT colorimetric assay 6h,12h,24h,48h,72h,96h of the OD value,draw the growth curve,calculate cell doubling time,determine the optimum inoculation concentration of cells and the range of drug intervention.MTT colorimetric assay at different times, different concentrations of PA-MSHA on proliferation inhibition rate of C6 glioma cells to calculate the cell survival rate and the half inhibitory concentration (IC50).Use inverted microscope to observe changes in cell morphology.Hochest 33258 staining,detecte apoptosis by fluorescence microscopic,detecte the influence of PA-MSHA on the cell cycle by flow cytometry.Results:C6 glioma cells used in this experiment,were adherent growth under conventional culture conditions,growth in good condition and be determinated of dynamic before each passage,the survival rate were always over 95%.Detected the rates of attachment,respectively,are 20%,68%,82%,85%,92%and 98%,at different time points including 2h,4h,6h,8h,10h and 12h,in accordance with the requirements of this experiment,Determinate of the growth curve by MTT prompted the cells were inoculated in accordance with 5×10~3 / hole concentration in 96-well plates,100μL culture medium(containing 10%fetal calf serum in RPMI1640) can provide enough space and nutrient composition for cell growth,so that cells can adapt to the new environment and enter the intermediate propagation phase gradually 12 hours after being inoculated.In this experiment we select the cells from 24h to 72h after which were inoculated in 96-well plates,the range has always been to ensure that cells are in a logarithmic phase stability.In this experiment,we used MTT assay the influence on the C6 glioma cells of different concentrations PA-MSHA,the results suggest that cell growth inhibition_rate have time and dose-dependent manner, and calculate the IC50 values for 24h and 48h are respectively(5.80±1.79)×10~8cfu/ml and(3.90±2.14)×10~8cfu/ml.We have seen obvious morphological changes of apoptosis by Hochest 33258 Staining.Flow cytometry shows the typical hypodiploid apoptotic peak(AP),and S phase ratio increased obviously,which suggest that Pseudomonas aeruginosa(PA-MSHA) preparation can induce C6 cells to apoptosis and inhibit cell proliferation by making cell cycle arrest in S phase, Conclusion:1.Pseudomonas aeruginosa(PA-MSHA) preparation can inhibit the Proliferation of C6 cells by making cell cycle arrest in S phase,and the inhibition action of PA-MSHA for C6 glioma cells with time,dose-dependent manner.2.Pseudomonas aeruginosa(PA-MSHA) preparation can induce C6 cell to apoptosis 3.Pseudomonas aeruginosa(PA-MSHA) preparation may be promising anti-glioma drug,particularly in the local treatment of gliomas may be more widely used,it is necessary to further study.