Gene therapy of pain by expressing endogenous opioid peptide

本研究以表达内源性阿片肽基因治疗疼痛为目标，在2002-2003年完成了三个主要项目。首先，为了阐明福尔马林致伤后大鼠脊髓中fos表达的意义，我们采用北方印迹和免疫组织化学技术，研究了福尔马林致伤后大鼠脊髓中c-fos和FOS表达的时程和定位变化。行为学测试显示出典型的两个阶段，由福尔马林实验诱导。福尔马林注射后0.5h，c-fos表达增加，6 h后恢复到对照组水平。FOS的表达在0.5h开始增加，并持续到6 h以后。FOS表达的上调在早期主要发生在背角浅层（I-II层），之后则发生在背角深层（V-VI层）。在这项研究中，我们证明，福尔马林注射引起的疼痛诱导c-fos mRNA翻译成FOS蛋白在脊髓神经元。FOS表达的部位变化 ...更多信息 在后一阶段，可能意味着福尔马林试验中枢致敏机制的改变。第二，我们研究了大鼠足底主人引起的脊髓后角Fos表达的时空模式以及局部麻醉药（0.5%布比卡因，2%利多卡因）预处理的影响。分别于术后1、3、6、24、48、72和120 h观察大鼠的疼痛行为和L_4-L_5脊髓Fos表达。与生理盐水组相比，在1 h至120 h，机械刺激的缩足阈值显著降低，并且局麻药预处理在1 h和6 h显著增加阈值。生理盐水组Fos主要表达于I ~ II和V ~ VI层，总表达在1h达高峰，随后逐渐下降。局部麻醉剂预处理抑制Fos表达显着在所有层，这种抑制持续了几天。本研究提供了足底控制器诱导Fos表达的时空变化的证据。此外，虽然局部麻醉剂预处理抑制Fos表达几天在术后期间，镇痛效果只观察到预期的持续时间的局部麻醉剂。第三，我们通过向大鼠注射携带GFP的腺病毒证实了在脊髓细胞中产生GFP（绿色荧光蛋白）。少

For aiming at gene therapy of pain by expressing endogenous opioid peptide, we achieved three main projects in 2002-2003. First, to clarify the significance of fos expression in the spinal cord of rats after the formalin test, we investigated the time course and location changes of c-fos and FOS expression, by using Northern blotting and immunohistochemical technique. The behavior test showed typical two phases induced by the formalin test. The amount of c-fos increased at 0.5 h after the formalin injection and returned to control level at 6 h. Also, the expression of FOS increased at 0.5 h, and the increase continued more than 6 h. At the early phase, the upregulation of FOS occurred in the superficial layers(laminae I-II ), and later it occurred in the deep layers (laminae V-VI) of the dorsal horn. In this study, we demonstrate that the pain caused by formalin injection induces c-fos mRNA translated to FOS protein in the spinal cord neurons. And the location changes of FOS expression … More in the later phase would imply the change of the mechanism of the central sensitization on the formalin test. Second, we investigated the spatial and temporal patterns of Fos expression in the dorsal horn of the spinal cord caused by plantar master in the rat and the effects of pretreatment with local anesthetics(0.5% Bupivacaine, 2% lidocaine). Pain behavior and Fos expression in the L_4-L_5 spinal cord were assessed at 1, 3, 6, 24, 48, 72, and 120 h after the master. The withdrawal threshold to mechanical stimulation decreased significantly at 1 h until 120 h, and pretreatment with local anesthetics increased the threshold significantly at 1 h and 6 h in comparison to that in the saline group. In the saline group, Fos expression was detected predominantly in laminaes I-II and V-VI, and the total Fos expression was maximal at 1 h and then decreased gradually. Pretreatment with local anesthetics suppressed Fos expression significantly in all layers and this suppression continued for several days. This study provides evidence of spatial and temporal changes in Fos expression induced by plantar master. Furthermore, although pretreatment with local anesthetics suppresses Fos expression for several days in the postoperative period, the analgesic effect is observed only for the expected duration of the local anesthetic used. Third, we confirmed that GFP(green fluorescent protein) was produced in spinal cord cells by injection of adenovirus with GFP to rats. Less