The Effect And Mechanism Of Glutamate And Its Receptors In Photodynamic Therapy On C6Glioma Cells

Objective:To evaluate the lethal effect of glutamate and its receptors in hepatoprophyrin derivative(HpD) mediated photodynamic therapy (PDT) on C6glioma cells so as to provide the furtherevidence for the clinical application.Methods:The first part: we established the model of SD rat. The suspension of C6glioma cellswas stereotaxically injected into the right caudate of SD rat.10days after the model wasestablished,the rat were divided into7groups: control group, PS group, PDT group,PS+MK801group, PDT+MK801group, PS+CNQX group, PDT+CNQX group. The gliomawere irradiated at628nm,20mW/cm2for5min after being incubated with HpD(10mg/L)respectively. The drug of MK801(0.2mM) and CNQX (0.2mM) were sent into glioma30min before PDT,by Microdialysis. The behavior, survival time of the rats and volume oftumor detected by MRI were observed. We detected the concentration of glutamate at0,0.5,1,2,4,8,12and18hours in tumor and normal brain after PDT by CMA600Microdialysis.The second part: C6glioma cells were irradiated at628nm,20mW/cm2for5min afrer beingincubated with HpD(10mg/L)respectively. We Observed the growth under microscopy anddetected the concentration of glutamate at0,0.5,1,2,4,8,16and24hours after PDT;1.3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolium bromide(MTT)assay was used todetermine the cell survival rate of C6glioma cells4hrs after PDT;Apoptosis measured byflow cytometry;the expressed of NMDAR1,NMDAR2,AMPAR的GluR1and GluR2byPCR and Western blot.The third part:4hours after photodynamic therapy (PDT) on C6glioma cells, the [Ca2+]i measured by flow cytometry and ImmunocytochemistryResults:1. The tumor volume of MRI10days after PDT had significance difference between thethree groups (control group, PS group, PS+CNQX group) and the PS+MK801group(P<0.05).The control group had significance difference with the PDT groups(PDT group, PDT+MK801group, PDT+CNQX group)(P<0.01).There also had significance differencewithin the PDT groups(PDT+MK801group<PDT group <PDT+CNQX group)(P<0.05).2. The averge survival time was the same with the tumor volume of MRI. There also hadsignificance difference within the PDT groups(PDT+MK801group>PDT group> PDT+CNQX group)(P<0.05).3. Glioma released glutamate and the level of glutamate intracellular increased after PDTin vivo, it get the peak at2h after PDT. There was significance difference between within thePDT groups(PDT+MK801group<PDT group <PDT+CNQX group) and those withoutirradiation groups(control group, PS group, PS+CNQX group and the PS+MK801group)(P<0.01).4. Morphological changes of C6glioma cells after PDT were found within44hour and16h after PDT cells became spherical or oval protruded membrane and fragmentation andfloating in the medium. But CNQX＋PDT groups deleted the time, it was began at8h and24h. There no significance difference between the control group, PS group, PS+CNQX groupand the PS+MK801group.5. Glioma released glutamate and got the peak at1h after PDT in vitro. There was nosignificance difference in those without irradiation groups(control group, PS group,PS+CNQX group and the PS+MK801group) and the PDT groups(PDT+MK801group<PDT group <PDT+CNQX group), but there was significance difference between thetwo groups.6. MTTassay was used to determine the cell survival rate of C6glioma It wassignificance difference between within the PDT groups(PDT+MK801group<PDT group <PDT+CNQX group) and those without irradiation groups(control group, PS group,PS+CNQX group and the PS+MK801group)(P<0.01).7. Apoptosis measured by flow cytometry get the same results compared with themortality of MTT. PDT induced C6glioma cells apoptosis, while AMPAR antagonistprolonged the survival time8. PDT increased [Ca2+]i in C6glioma cells, which was partly blocked by AMPARantagonist.(P<0.01).9. The Ca2+influx out of cells, and K+in normal, but it was reversed after PDT, and thiereversed can be block by CNQX.(P<0.01). 10. NMDAR1did not expressed on the C6glioma cells; the GluR1and GluR2increased after PDT. FITC was used to visualize GluR1. Cy3wasused to visualize GluR2.Nuclei were stained for30min with the nuclear dye DAPI during Immunocytochemistry. Wefind the GluR1and GluR2increased after PDT and expressed on membrane.Conclusion:1. HpD-PDT had lethal effective to C6glioma cells in vivo or in vitro.2. The C6glioma cells release glutamate in vivo or in vitro, especially during PDT. Ithad no relationship with AMPAR and NMDAR3. MK801postpone the averge survival time of rats in vivo, but CNQX is the on thecontrary4.NMDAR1did not expressed on the C6glioma cells; the AMPAR increased theapoptosis of C6glioma cells during PDT.5. The C6glioma cells increased the release of glutamate after PDT, witch activated theAMPAR, increased the [Ca2+]iis the important pathway to induced the apoptosis of C6gliomacells.