【作者】 贾晓明；

【导师】 杨建平；

【作者基本信息】 苏州大学 ， 麻醉学， 2011， 硕士

【摘要】 第一部分:胫骨癌痛大鼠脊髓TrkB受体和KCC2的表达变化目的观察胫骨癌痛大鼠脊髓背角TrkB受体和KCC2表达变化。方法24只雌性SD大鼠,体重180～220g,随机分为2组:假手术组(J组,n=8)和模型组(M组,n=16)。假手术组和模型组大鼠分别于左胫骨近端骨髓腔注射生理盐水或Walker256乳腺癌细胞(1×10~7/ml)10μl。两组均在建模前、建模后第3、6、9、12天用机械触诱发痛测量仪测定大鼠左后肢机械缩足阈值,假手术组大鼠于建模后第12天处死,模型组于建模后第6、9、12天各处死4只大鼠,取脊髓L4～6组织,冰冻切片,用免疫组织化学方法检测脊髓背角TrkB受体和KCC2表达的变化。结果建模后6～12天,模型组大鼠左后肢机械缩足反射阈值显著下降,且随时间延长进行性发展;脊髓背角TrkB受体表达水平进行性升高,KCC2表达水平进行性降低;结果均有统计学差异(P<0.05)。结论脊髓背角TrkB受体和KCC2可能参与了大鼠胫骨癌痛的产生和维持。第二部分:鞘内注射TrkB中和抗体对胫骨癌痛大鼠痛行为学及脊髓KCC2表达的影响目的观察鞘内注射TrkB中和抗体后,胫骨癌痛大鼠痛行为学与脊髓背角KCC2表达的变化,并探讨其可能机制。方法40只雌性SD大鼠,体重180～220g,随机分为5组:Ⅰ组(n=8)为生理盐水对照组;Ⅱ组(n=8)为骨癌痛组;Ⅲ组(n=8)为对照组+TrkB中和抗体;Ⅳ组(n=8)为骨癌痛组+IgG;Ⅴ组(n=8)为骨癌痛组+TrkB中和抗体。Ⅰ组、Ⅲ组大鼠左胫骨近端骨髓腔注射生理盐水10μl,其他各组左胫骨近端骨髓腔均注射Walker256乳腺癌细胞(1×107/ml)10μl,建模当天所有大鼠行鞘内置管。Ⅲ组和Ⅴ组大鼠建模后第4、5天鞘内注射TrkB受体的中和抗体(2μg/10μl);Ⅳ组大鼠鞘内注射IgG(2μg/10μl),Ⅰ组、Ⅱ组大鼠不进行鞘内注射。各组在建模前、建模后第1～6天用机械触诱发痛测量仪测定大鼠左后肢机械缩足阈值(MWT),各组大鼠均于建模后第6天取L4～6脊髓组织,冰冻切片,用免疫组织化学方法检测脊髓KCC2的表达。结果建模后第6天,与Ⅰ组相比,Ⅱ组、Ⅳ组和Ⅴ组大鼠的MWT显著下降(P<0.01),Ⅲ组大鼠的MWT无变化(P> 0.05);与Ⅱ组、Ⅳ组相比,Ⅴ组大鼠的MWT显著增高(P<0.01)。与Ⅰ组相比,Ⅱ组、Ⅳ组大鼠脊髓背角中KCC2表达显著降低(P<0.01),Ⅲ组和Ⅴ组大鼠KCC2表达无显著差异(P>0.05);Ⅴ组大鼠脊髓背角中KCC2表达明显高于Ⅱ组、Ⅳ组(P < 0.01)。结论鞘内注射TrkB中和抗体可以部分缓解肿瘤诱发的机械性痛敏,这种效应可能与阻断TrkB途径从而阻止脊髓背角KCC2的表达下调有关;这提示脊髓背角KCC2可能参与了骨癌痛的形成。TrkB-KCC2途径可能是骨癌痛形成的重要机制之一。更多还原

【Abstract】 Part one: The changes of TrkB receptor and KCC2 in the spinal cord of tibial bone cancer pain ratsObjective To investigate expression change of tyrosine receptor kinase B(TrkB) and potassium-chloride cotransporter2(KCC2) in the spinal cord of tibial bone cancer pain rats.Methods 24 female SD rats weighing 180～220g were randomly divided into 2 groups: Sham-operated group(J, n=8)、Model group(M, n=16).J and M groups were respectively injected 10μl normal saline(NS) or Walker 256 breast cancer cells(1×107/ml) into the left proximal tibial medullary cavity.At the day before and 3、6、9、12d after inoculation, response of the left hind paw to mechanical stimulation with dynamic plantar aesthesiometer were measured. Rats of J group were executed at the twelfth day after inoculation, Rats were executed at the sixth,Four rats of M group were respectively executed at sixth,ninth day,eight rats were executed at twelfth day after inoculation,the L4-6 spinal cord was removed.The expression of the spinal TrkB receptor and KCC2 was detected by immunohistochemistry assay.Results From the 6 to 12th day post inoculation, MWT in M group was significantly decreased(P<0.05),appeared visible mechanical allodynia and became more and more severe. At the same time, spinal TrkB expression levels were progressive increased(P<0.05)and spinal KCC2 protein levels were progressive reduced(P<0.05).Conclusion TrkB receptor and KCC2 in dorsal horn of Spinal cord may be involved in the generation and maintenance of rat tibial bone cancer pain. Part two: The effect of intrathecal injection of TrkB Ab on pain behavior and spinal KCC2 expression in a rat model of tibial bone cancer painObjective To observe the effection of intrathecal injection of TrkB Ab on pain behavior and spinal KCC2 expression in a rat model of tibial bone cancer pain, then explore its possible mechanisms.Methods 40 female SD rats weighing 180～220g were randomly divided into 5 groups(n=8), groupⅠ: control group; groupⅡ: model group; groupⅢ: control group+ TrkB Ab; groupⅣ: model group+IgG; groupⅤ: model group+ TrkB Ab. GroupⅠand groupⅢwere injected 10μl normal saline(NS) into the left proximal tibial medullary cavity, groupⅡ, groupⅣand groupⅤw ere injected 10μl Walker 256 breast cancer cells(1×107/ml) into the left proximal tibial medullary cavity. Set pipes into subarachnoid space of all rats at establishing model day. TrkB Ab or IgG was injected intrathecal (it) at the fourth and fifth day after inoculation. At the day before and 1 to 6 d after inoculation, response of the left hind paw to mechanical stimulation with dynamic plantar aesthesiometer were measured.Rats were killed at the sixth day after establishing model and L4～6 lumbar segment of the spinal cord were removed for determination of KCC2 expression in the spinal cord dorsal horn by immunohistochemical methods.Results At the sixth day after inoculation, mechanical withdrawal threshold (MWT) was significantly decreased in rats of groupⅡ,ⅣandⅤvs groupⅠ; MWT was significantly increased in rats of groupⅤcompared with those in groupⅡandⅣg roup. KCC2 protein levels in the dorsal horn of spinal cord from groupⅡandⅣwere significantly decreased compared with those in groupⅠ(P<0.05);spinal KCC2 protein levels in groupⅤhigher relative to groupⅡandⅣ; spinal KCC2 expresssion was no found remarkly difference between groupⅢand groupⅤ.Conclusion Intrathecal TrkB Ab can partial remission mechanical hyperalgesia induced by tumor, this effect may be related to prevent the downregulation of spinal KCC2 expression by blocked BDNF-TrkB pathway. These results also indicate that KCC2 in dorsal horn of spinal cord may be involved in the development of bone cancer pain. TrkB-KCC2 pathway may be an important mechanism of bone cancer pain.更多还原