【作者】 刘磊；

【导师】 傅志俭；

【作者基本信息】 山东大学 ， 临床医学（麻醉学）（专业学位）， 2017， 博士

【摘要】 研究背景随着医学科学技术水平的发展,癌症的检出率越来越高,治疗方法和疗效也得到了提高,生存期逐渐延长。然而,癌性疼痛却严降低了恶性肿瘤病人的生存质量。迄今为止,阿片类药物仍然是癌性疼痛治疗最有效和最常用的药物,但大剂量长时间的应用阿片类药物极易造成耐受甚至痛觉过敏。临床上还没有比阿片类药物效果更好的治疗癌性疼痛的药物,因此阿片类药物耐受是癌性疼痛治疗中急需解决的棘手问题。目前单纯吗啡耐受动物模型几乎是国内外关于阿片耐受的研究的全部基础,但此动物模型并不能与癌性疼痛临床情况完全相符合。癌痛是最复杂的慢性疼痛,既有炎症痛和神经病理性疼痛的参与,又比二者而复杂的多。所以要想深入的研究癌痛吗啡耐受机制,必需建立能模拟临床实际情况的动物模型。趋化因子是参与免疫细胞募集、成熟和激活的重要介质,其化学成分是分泌型的小分了量蛋白质(约8～12 ku),。有研究发现趋化因子 MCP-1在疼痛的产生和维持过程中发挥重要的作用。也有研究证实小胶质细胞在神经病理性疼痛的形成中有重要的作用。既往研究还发现正常小鼠鞘内给注射MCP-1能激活CCR2活化脊髓小胶质细胞,并能诱发机械痛敏且成剂量依赖性,但预先鞘内注射MCP-I的中和抗体就能阻止小胶质细胞的激活并且预防机械性痛敏的发生。此外,免疫荧光双标发现脊髓小胶质细胞OX-42和CCR2共表达,鞘内注射MCP-1中和抗体能够抑制神经病理性疼痛模型中脊髓背角小胶质细胞的激活和有效缓、解神经病理性疼痛。炎性细胞因子在慢性痛的调控中具有重要作用。IL-1、IL-6不但在神经-内分泌免疫功能系统中发挥重要作用,也是急性炎性反应的敏感指标,伤害性刺激可引起IL-1、IL-6水平升高,其升高的程度与疼痛关系密切。TNF-a可通过激活环氧化酶-2诱导前列腺素E的合成导致痛觉过敏,也可通过诱发缓激肽、P物质、儿茶酚胺的释放导致痛觉过敏。通过激活神经元和胶质细胞上的TNF-a、IL-1和IL-6受体可致神经兴奋性提高,从而导致中枢敏化和痛觉敏感性增加。然而,骨癌痛吗啡耐受大鼠中MCP-1是否参与吗啡耐受的产生,是否通过CCR2激活小胶质细胞释放炎症细胞因子,从而导致吗啡耐受呢?这正是本课题要研究的问题。第一部分:MCP-1在骨癌痛吗啡耐受大鼠中的作用目的观察骨癌痛吗啡耐受大鼠脊髓MCP-1、CCR2的表达情况。研究鞘内注射MCP-1中和抗体后骨癌痛吗啡耐受大鼠疼痛行为学指标机械缩足阈值(PWT paw withdrawal threshold)和热缩足潜伏期(PWLpaw withdrawal latency)的变化。方法成年雌性SD大鼠,体重150～170g,24只大鼠,随机分为4组(n=6):对照组(S组)、吗啡耐受组(M组)、骨癌痛组(B组)、骨癌痛吗啡耐受组(BM组)。S组大鼠胫骨髓腔注射NS5μ1,第9天鞘内注射NS4W,2次/d,连续9d;M组大鼠胫骨髓腔注射NS5μl,第9天鞘内注射20μg/kg吗啡,2次/d,连续9d;B组胫骨髓腔注入Walker 256细胞5μl(2×1 07个细胞/mL),第9天鞘内注射NS4μl,2次/d,连续9d;BM组大鼠胫骨骨髓腔注入Walker 256细胞5μl(2×107个细胞/mL),第9天开始鞘内给予20μg/kg吗啡,2次/d,连续9d。于术后18d处死各组大鼠,取L4-5脊髓组织,采用Western-blot法检测大鼠脊髓MCP-]表达情况,采用RT-PCR法测定大鼠脊髓MCP-1、CCR2表达情况。成年雌性SD大鼠48只,随机分为6组(n=8):骨癌痛组(B组)、骨癌痛吗啡耐受组(BM组)、骨癌痛吗啡耐受注射中和抗体组(BM+Ab组)、骨癌痛吗啡耐受注射IgG组(BM+IgG组)、骨癌痛注射中和抗体组(B+Ab组)、骨癌痛注射IgG组(B+IgG组)。B组大鼠胫骨髓腔注入Walker2 5 6细胞5μl(2×107个细胞/mL),第9天鞘内注射NS4μl,2次/d,连续9d;BM组大鼠胫骨骨髓腔注入Wa l k e r 2 5 6细胞5μl(2×107个细胞/mL),第9天开始鞘内给予20μg/kg吗啡,2次/d,连续9d;BM+Ab组大鼠胫骨骨髓腔注入Walk e r 2 5 6细胞5μl(2×107个细胞/mL),第9天开始鞘内给予20μg/kg吗啡,2次/d,连续9d,第15天开始每天注射MCP-1中和抗体10μg,连续3d;BM+IgG组大鼠胫骨骨髓腔注入Walk e r 256细胞5μl(2×1 07个细胞/mL),第9天开始鞘内给予20μg/k g吗啡,2次/d,连续9d,第15天开始每天注射IgG10μg,连续3d;B+Ab组大鼠胫骨髓腔注入Wa l k e r 2 5 6细胞5μl(2×1 07个细胞/mL),第9天鞘内注射NS4μl,2次/d,连续9d,第15天开始每天注射MCP-1中和抗体10μg,连续3d;B+IgG组大鼠胫骨骨髓腔注入Wa l k e r 256细胞5μl(2×107个细胞/mL),第9天鞘内注射NS4μl,2次/d,连续9d,第15天开始每天注射IgG10μg,连续3d。术前、术后3d、6d、9d、12d、15d、18d检测各组大鼠机械缩足阈值(PWT paw withdrawal threshold test)和热缩足阈值(TWL thermal withdrawal latency)。结果M组大鼠较S组、M组和B组大鼠脊髓MCP-1、CCR2表达明显增多(P<0.05)。BM+Ab组大鼠鞘内注射MCP-1中和抗体后机械缩足阈值(PWT paw withdrawal threshold test)和热缩足阈值(TWL thermal withdrawal latency)明显升高(P<0.05)。结论MCP-1可能在骨癌痛吗啡耐受中起重要作用。第二部分:鞘内注射MCP-1中和抗体对骨癌痛吗啡耐受大鼠脊髓OX-42和炎症因子TNFα、IL-1、IL-6表达的影响。目的研究鞘内注射MCP-1中和抗体后骨癌痛吗啡耐受大鼠脊髓OX-42和炎症因子TNFα、IL-1、lL-6表达的变化。方法成年雌性SD大鼠72只,随机分为6组(n=12):骨癌痛组(B组)、骨癌痛吗啡耐受组(BM组)、骨癌痛吗啡耐受注射中和抗体组(BM+Ab组)、骨癌痛吗啡耐受注射IgG组(BM+IgG组)、骨癌痛注射中和抗体组(B+Ab组)、骨癌痛注射IgG组(B+IgG组)。B组大鼠胫骨髓腔注入Wa l k e r 2 5 6细胞5μl(2× 107个细胞/mL),第9天鞘内注射NS4μl,2次/d,连续9d;BM组大鼠胫骨骨髓腔注入Walk e r 2 5 6细胞5μl(2× 1 07个细胞/mL),第9天开始鞘内给予20μg/kg吗啡,2次/d,连续9d;BM+Ab组大鼠胫骨骨髓腔注入Walker2 5 6细胞5μl(2×107个细胞/mL),第9天开始鞘内给予20μg/kg吗啡,2次/d,连续9d,第15天开始每天注射MCP-1中和抗体10μg,连续3d;BM+IgG组大鼠胫骨骨髓腔注入Wa 1 k e r256细胞5μl(2×1 07个细胞/mL),第9天开始鞘内给予20μg/kg吗啡,2次/d,连续9d,第15天开始每天注射IgG10μg,连续3d;B+Ab组大鼠胫骨髓腔注入Walker2 5 6细胞5μl(2×107个细胞/mL),第9天鞘内注射NS4μl,2次/d,连续9d,第15天开始每天注射MCP-1中和抗体10μg,连续3d;B+IgG组大鼠胫骨骨髓腔注入Walker256细胞5μl(2×107个细胞/mL),第9天鞘内注射NS4μl,2次/d,连续9d,第15天开始每天注射IgG10μg,连续3d。术后18d处死全部大鼠,取L4-5脊髓组织,采用免疫组化法测定OX-42的表达情况,采用RT-PCR法检测炎症因了 TNFα、IL-1、IL-6的表达情况。结果BM+Ab组大鼠鞘内注射MCP-1中和抗体后脊髓OX-42和炎症因子TNFα、IL-1、IL-6表达明显减少(P<0.05)。结论MCP-1可能通过激活小胶质细胞引发炎症反应从而导致骨癌通吗啡耐受。更多还原

【Abstract】 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.更多还原