| Research BackgroundOver the past decade,the level of cancer detection and treatment by leaps and bounds,so that the survival time of cancer patients greatly extended.However,cancer-induced pain has seriously affected the quality of life of cancer patients.Opioids are the most commonly used and most effective drugs for cancer pain treatment,but prolonged or high-dose use is likely to cause tolerance or even hyperalgesia.As there is no more effective than opioid analgesic drugs,opioid tolerance as cancer pain treatment to solve the most urgent problem.At present,almost all of the studies on opioid tolerance at home and abroad are based on simple morphine tolerant animal models,which are not based on the pathological state of cancer pain and are not consistent with clinical status.Cancer pain is a category of chronic pain,and inflammatory pain and neuropathic pain mechanism compared to similarities,but also has its unique and complex.Therefore,it is very important to establish a suitable animal model for in-depth study of the mechanism of cancer tolerance.Chemokines as secreted small molecular weight proteins(about 8 to 12 ku)are the ones that were first described as involved in immune cell recruitment,maturation and activation.Recent studies have shown that chemokine MCP-1 plays an important role in the production and maintenance of pain.It is known that microglia plays an important role in the formation of neuropathic pain.It was found that MCP-1 was induced by intrathecal MCP-1 in normal mice and could induce mechanical sensitization in a dose-dependent manner by CCR2,but the neutralizing antibody of MCP-1 could prevent the activation of microglia And the occurrence of mechanical pain.In addition,the expression of CCR2 was detected by immunofluorescence double labeling,and the intrahepatic administration of MCP-1 neutralizing antibody inhibited the activation of microglia in the spinal dorsal horn of the neuropathic pain model and effectively relieved neuropathology pain.Inflammatory cytokines play an important role in the regulation of chronic pain.IL-1 and IL-6 are one of the important mediators in the neuro-endocrine and immune function system.It is a sensitive indicator of acute inflammatory response.Nociceptive stimulation can cause IL-1 and IL-6 levels,The degree is closely related to pain.TNF-a can induce prostaglandin E through the cyclooxygenase-2 pathway and induce the release of bradykinin,substance P,catecholamine,leading to hyperalgesia;activation of glial cells and neurons on IL-1,IL-6 and TNF-a receptors can increase neuronal excitability,resulting in central sensitization,resulting in reduced pain threshold,resulting in increased sensory sensitivity.However,whether or not MCP-1 is involved in morphine tolerance on morphine tolerance rats with bone cancer pain,whether or not to activate inflammatory cytokines by activating microglia through CCR2,leading to morphine tolerance on morphine tolerance rats with bone cancer pain?This is the subject to be studied.Part one:The Effect of MCP-1 on Morphine Tolerance Rats with Bone Cancer PainObjective To investigate the expression of MCP-1 and CCR2 in spinal cord of bone cancer pain rats with morphine tolerance after intrathecal injection of MCP-1 antibody.To ivestigate pain behavioral indicators paw withdrawal threshold(PWT)and thermal withdrawal latency(TWL)of bone cancer pain rats with morphine tolerance after intrathecal injection of MCP-1 neutralizing antibody.Methods 24 adult female SD rats were randomly divided into four groups(n =6):shame group(group S),morphine tolerance group(group M),bone pain(group B),bone cancer pain morphine tolerance group(group BM).Group S tibial marrow cavity injection NS 5μl,intrathecal injection of NS 4μ1 from the 9th day,2 times/d,continuously for 9d;group M tibia bone marrow cavity injection NS5μl,intrathecal injection of 20μg/kg of morphine from the 9th day,2 times/d,continuously for 9d;group B tibial bone marrow cavity injection Walker 256 cells 5μl(2×107 cells/mL),intrathecal injection NS4μl from the 9th day,2 times/d,continuous for 9d;group BM tibia bone marrow cavity of rats were injected Walker 256 cell 5μl(2×107 cells/mL),intrathecal injection 20μg/kg morphine from the 9th day,2 times/d,continuously for 9d.Each group of rats were sacrificed on postoperative 18d,take L4-5 spinal cord tissue for MCP-1 and CCR2 expression by western-blot and RT-PCR method.48 adult female SD rats were randomly divided into six groups(n = 8):bone cancer pain group(B group),bone cancer pain and morphine tolerance group(BM group),bone cancer pain and morphine tolerance intrathecal injection neutralizing antibody group(BM+Ab group),bone cancer pain and morphine tolerance intrathecal injection IgG group(BM+IgG group),bone cancer pain intrathecal injection neutralizing antibody group(B+Ab group),bone cancer pain intrathecal injection IgG group(B+IgG group).Group B tibial bone marrow cavity injection Walker 256 cells 5μl(2×107 cells/mL),intrathecal injection NS4μl from the 9th day,2 times/d,continuously for 9d;group BM tibia bone marrow cavity of rats were injected Walker 256 cell 5μl(2 ×107 cells/mL),intrathecal injection 20μg/kg morphine from the 9th day,2 times/d,continuously for 9d;group BM+Ab tibia bone marrow cavity of rats injected Ab Walker 256 cells 5μ1(2×107 cells/mL),intrathecal 20μg/kg morphin from the 9th day,2 times/d,continuously for 9d,the injection of MCP-1 antibody 10μg daily for 3d from 15th day;group BM+IgG tibia bone marrow cavity of rats injected IgG Wa lker 256 cells 5μl(2×107 cells/mL),intrathecal 20μg/kg morphine from 9th day,2 times/d,continuously for 9d,injection IgG 10μg day for 3d from 15th day;group B+Ab tibia bone marrow cavity of rats injected Ab Walker 256 cells 5μl(2×107 cells/mL),intrathecal NS4μl from the 9th day,2 times/d,continuously for 9d,the injection of MCP-1 antibody 10μg daily for 3d from 15th day;group B+IgG tibia bone marrow cavity of rats injected IgG Wa lker 256 cells 5μl(2×107 cells/ml),intrathecal NS4μl from 9th day,2 times/d,continuously for 9d,injection IgG10μg day for 3d from 15th day.Preoperative and postoperative 3d,6d,9d,12d,15d,18d each group rats’ paw withdrawal threshold(PWT)and thermal withdrawal latency were detected.Results The expression of MCP-1 and CCR2 in spinal cord of BM group rats was significantly increased(P<0.05).The paw withdrawal threshold(PWT)and thermal withdrawal latency(TWL)of BM+Ab rats after intrathecal injection of MCP-1 neutralizing antibody were significantly increased(P<0.05).Conclusion MCP-1 may play an important role in morphine tolerance with bone cancer painPart two:Effects of intrathecal MCP-1 neutralizing antibody on expression of spinal cord OX-42 and the inflammatory cytokines TNFa,IL-1,IL-6 in bone cancer pain rats with morphine toleranceObjective To investigate the expression of spinal cord OX-42 and the inflammatory cytokines TNFa,IL-1,IL-6 in bone cancer pain rats with morphine tolerance.Methods 72 adult female SD rats were randomly divided into four groups(n =12):bone cancer pain group(B group),bone cancer pain and morphine tolerance group(BM group),bone cancer pain and morphine tolerance intrathecal injection neutralizing antibody group(BM+Ab group),bone cancer pain and morphine tolerance intrathecal injection IgG group(BM+IgG group),bone cancer pain intrathecal injection neutralizing antibody group(B+Ab group),bone cancer pain intrathecal injection IgG group(B+IgG group).Group B tibial bone marrow cavity injection Walker 256 cells 5μl(2×107 cells/mL),intrathecal injection NS4μl from the 9th day,2 times/d,continuously for 9d;group BM tibia bone marrow cavity of rats were injected Walker 256 cell 5μl(2 ×107 cells/mL),intrathecal injection 20μg/kg morphine from the 9th day,2 times/d,continuously for 9d;group BM+Ab tibia bone marrow cavity of rats injected Ab Walker 256 cells 5μl(2×107 cells/mL),intrathecal 20μg/kg morphin from the 9th day,2 times/d,continuously for 9d,the injection of MCP-1 antibody 10μg daily for 3d from 15th day;group BM+IgG tibia bone marrow cavity of rats injected Walker 256 cells 5μl(2×107 cells/mL),intrathecal 20μg/kg morphine from 9th day,2 times/d,continuously for 9d,the injection of IgG 10μg daily for 3d from 15th day;group B+Ab tibia bone marrow cavity of rats injected Walker 256 cells 5μl(2×107 cells/mL),intrathecal NS4μl from the 9th day,2 times/d,continuously for 9d,the injection of MCP-1 antibody 10μg daily for 3d from 15th day;group B+IgG tibia bone marrow cavity of rats injected Walker 256 cells 5μl(2×107 cells/ml),intrathecal NS4μl from 9th day,2 times/d,continuously for 9d,the injection of IgG 10μg daily for 3d from 15th day.Each group rats were killed after 18ds,OX-42 expression of L4-5 spinal cord tissue was detected by immunohistochemistry,RT-PCR method was used to detect the expression of TNFa,IL-1,IL-6.Results The expression of BM+Ab group rat spinal OX-42 and inflammatory cytokines TNFa,IL-1,IL-6 was significantly reduced(P<0.05).Conclusion MCP-1 may cause inflammation of the microglia by activating microglia,leading to morphine tolerance with bone cancer pain. |
PDF Full Text Request