| As chronic pain,cancer pain is seriously affecting the quality of life of cancer patients.Cancer pain has its own complex and unique mechanism.Tissue acidification is an important factor causing pain and hyperalgesia caused by tumor.The pain signal is produced by the nerve endings of the nociceptive receptor,and there are various ion channels and receptors on the cell membrane of these subgroups of sensory neurons,which can affect the stimulation of extracellular damage and make it change into action potential and mediate the production of pain.Acid-sensing ion channels(ASICs)is a class of proton channel proteins widely distributed on the membrane of sensory neurons.The purpose of this study is to establish a mouse metastatic skin cancer pain model by using RT-PCR and Western blotting techniques to determine the changes in the expression of ASIC3 at different time points in the tumor growth period in mice,and to explore the correlation between ASIC3 and cancer pain.At the same time,the effects of pararecoxib sodium on the expression and activity of ASIC3 and the therapeutic effect in cancer pain was studied to provide a new multi-mode analgesic scheme for the treatment of clinical cancer pain and to provide a new evidence for the study of its mechanism.Chapter one:The establishment of a model of metastatic cutaneous cancer pain in miceObjective:To establish a model of metastatic cutaneous cancer pain in mice.Methods:24 Balb/c mice(clean grade,female,8 weeks old,body weight 21?25g)were randomly divided into 3 groups:blank control group,sham group and CIP(Cancer induce pain)group,with 8 rats in each group.Each mouse subcutaneously injected 20?l PBS suspension solution contained about 2 x 105 4Tl breast cancer cells into skin of left paw in the CIP group.Each mouse subcutaneously injected 20?l PBS in sham group and don't make any change in blank control group.The general situation before and first to eleventh days after inoculation,the appearance of the inoculated toe,the limb activity of the inoculated side and the sie of mice were observed.Changes in walking gait(walking pain score)and changes of paw thermal withdrawal latency(PTWL).Results:There were no significant differences in behavioural walking pain score and PTWL between blank control and sham groups(P>0.05).Compared with the blank control group and the sham group,the tumor body of the CIP group increased from the fourth day of the tumor inoculation,and the score of the walking pain was increased gradually,the difference was statistically significant(P<0.05).After seventh days of inoculation,the expression of PTWL was shortened gradually and the difference was statistically significant(P<0.05).The pain score and the PTWL of the mice in the CIP group reached the peak on the eleventh day after the inoculation(P<0.01).The research had to stop due to the ulceration or bleeding of soft tissue in some CIP group mice because of the fast enlargement of solid tumor after the 14th day.Conclusion:The mice model of metastatic cutaneous cancer pain can be successfully established.Chapter two:The change of expression of ASIC3 in a mouse model of metastatic cutaneous cancer painObjective:To observe the change of expression of ASIC3 in a mouse model of metastatic foot plantar cutaneous cancer pain.Methods:The establishment of cutaneous cancer pain model in mice is the same as in the first part of the experiment.After modeling,the mice in the blank control group,the sham group and the 5th,8th,11th day group after the tumor inoculation were executed and the DRG tissues were removed.The relative changes of the ASIC3 mRNA and protein expression on the DRG of the spinal cord of the mice were measured by RT-PCR and Western blotting at different time after the tumor inoculation.Results:There were no significant differences in expression of ASIC3 mRNA and Protein between blank control and sham groups(P>0.05).Compare to blank control and sham group,the expression of ASIC3 mRNA and Protein gradually increased from the 5th day after tumor inoculated(P<0.05),and the expression began to obviously enhance from the 8th day to the 11th day(P<0.01).Conclusion:After inoculation with 4Tl tumor cells in the CIP group,the expression of ASIC3 mRNA and protein on spinal cord DRG neurons increased gradually.Chapter three:Analgesic effect of intrathecal injection of parecoxib sodium on metastatic cutaneous cancer pain miceObjective:To research the analgesic effect of parecoxib sodium intrathecal injection on metastatic cutaneous cancer in mice.Methods:The establishment method of metastatic cutaneous cancer pain model in mice is the same as the first chapter of experiment.32 Balb/c mice(clean,female,8 weeks old and 22 to 25g)were randomly divided into 4 groups after modeling:The control group(intrathecal injection of NS 5?l)(group A),20?g/?L parecoxib sodium group(group B),40?g/?L parecoxib sodium group(group C)and 80?g/?L parecoxib sodium group(group D),8 mice in each group.At the 11th days after modeling,group B,group C and group D were intrathecally injected with corresponding dose of parecoxib sodium,and 5?l of saline was intrathecally injected into the group A.Record the changes of PTWL of hot plate test of each group at 15,30,60,90,120,150,180 min after intrathecal injection,and to detect the changes of ASIC3 mRNA and protein expression in spinal cord DRG of each group by RT-PCR and Western blotting.Results:Compared with the control group(group A),the PTWL in group B,C and D was significantly prolonged at 60 min after intrathecal injection,the difference was statistically significant(P<0.05);The difference of PTWL reached the peak value at 120 min after intrathecal injection(P<0.01).There was a significant difference between the three groups in group B,group C and group D(P<0.05).The PWTL in mice was positively correlated with the concentration of pareoxib sodium in the intrathecal injection.With the increase of intrathecal injection of parecoxib sodium in group B,group C and group D,the expression of ASIC3 mRNA and protein on DRG decreased gradually.Conclusion:Intrathecal injection of parecoxib sodium has analgesic effect on metastatic skin cancer pain mice.The inhibitory effect of parecoxib sodium intrathecal injection on the expression of ASIC3 mRNA and protein on DRG in mice with metastatic skin cancer pain model was positively correlated with the concentration of parecoxib sodium,suggesting that ASIC3 plays an important role in the analgesic mechanism of metastatic cutaneous cancer pain in mice. |
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