【作者】 陈晓玲；

【导师】 王永祥；

【作者基本信息】 上海交通大学 ， 药理学， 2012， 硕士

【摘要】 D-型氨基酸氧化酶(D-amino acid oxidase,DAAO)是一种以FAD为辅基的氧化酶,它在体内参与的生化反应为催化D-型氨基酸分解产生相应的α-酮酸、NH3和H2O2。先期研究通过基因敲除和化学抑制剂的方法先后证明DAAO在多种慢性疼痛中,可能发挥了作用。本研究为了进一步验证DAAO可以作为治疗慢性疼痛的靶点,选用特异性较强的RNA干扰的方法,进行如下探索:第一,利用计算机软件设计针对DAAO的siRNA及非特异序列siRNA(阴性对照)。我们构建并包装腺病毒作为基因转运载体。以shRNA作为siRNA的前体,利用其转染进入细胞体内,在Dicer酶的作用下生成siRNA。由于shRNA在结构上需存在一个反向的互补碱基序列,故在设计shRNA时,我们利用siRNA的碱基序列设计了一段它的互补结构,并用一个loop结构连接这两段RNA。根据我们利用分子克隆技术,在体外合成编码DAAO基因的shRNA核苷酸序列单链,退火形成shRNA双链之后,利用限制性内切酶技术将其克隆到真核表达载体pDC316-EGFP-U6中,委托第二军医大学的钱其军教授课题组包装为腺病毒。第二,用腺病毒转染NRK-52E(大鼠肾小管上皮细胞)细胞,分别在24h和48h提取总RNA和蛋白质。RNA做RT反应获得cDNA,cDNA以GAPDH作为参照,进行Realtime PCR ,结果表明,实验组NRK-52E中的DAAO mRNA表达相对空白组和阴性对照组分别被抑制了51.7%和49.1%;DAAO蛋白以β-actin为参照,进行Western blot,DAAO蛋白表达相对空白组和阴性对照组分别被抑制了50.2% or 51.2%。确定了设计的该条有效的siRNA序列后,我们分别用腺病毒,PEI(Polyetherimide聚醚酰亚胺)为载体进行大鼠鞘内基因给药。连续给药7天后,我们通过皮下注射福尔马林溶液来建立慢性疼痛动物模型,测定大鼠福尔马林疼痛,并检测大鼠脊髓DAAO的沉默效果。结果显示:两种载体给药效果相当,大鼠福尔马林急性疼痛不受影响,而慢性疼痛抑制了约60%,用Real-time PCR和Western blot检测脊髓DAAO沉默效率约50%。我们又选用了骨癌痛模型,用PEI载体鞘内给药,测试了沉默大鼠脊髓DAAO后的足底痛阈变化。鞘内基因给药对骨癌痛抑制了约40%。另外,我们监测了骨癌痛给药的时效曲线,发现给药5天后开始出现镇痛作用,在第7天达到最大镇痛药效,继续给药不能增强镇痛效果。给药10天后镇痛作用逐渐降低,停止给药后第8天镇痛作用消失。该结果将对siRNA的基因治疗提供临床依据。沉默DAAO在骨癌痛实验中达到最大镇痛作用,即给药第10天时单次给药CBIO,非特异序列组(对照组)也出现40%的镇痛效果,而siRNA/ DAAO组(实验组)镇痛作用没有继续增强。结果说明CBIO对DAAO的抑制作用是特异性的,抑制脊髓DAAO对骨癌痛有40%的镇痛作用。第四,由于前期研究发现星形胶质细胞在疼痛状态中处于激活状态,DAAO大量存在于星形胶质细胞。我们测试DAAO沉默后星形胶质细胞的变化,发现随着DAAO沉默后表达降低,星型胶质细胞的生物标记物GFAP(glial fibrillary acidic portein胶质纤维酸性蛋白)的表达也降低。说明星形胶质细胞的激活受到抑制。DAAO参与疼痛过程可能与星形胶质细胞的状态有关。综上,我们用siRNA基因沉默的方法进一步证明了DAAO是慢性疼痛的潜在靶点,并推测DAAO参与疼痛过程可能与星形胶质细胞的状态有关。更多还原

【Abstract】 D-amino acid oxidase (DAAO) is a peroxisomal enzyme with FAD as cofactor, which catalyzes the oxidization of D-amino acids to the corresponding imino acids, producing ammonia and hydrogen peroxide. Early studies have shown that DAAO may play a role in chronic pain by inhibitors of DAAO and gene mutation mice. To further investigate the potential of DAAO as a new drug target for chronic pain, I applied RNA interference as a more special tool to this study as below:In this article, we describe a method for construction of recombinant advenovirus shRNA expression vector targeting rat DAAO gene.the expression vector can transcribe to shRNA, and shRNA can degrade to siRNA with the help of Dicer as the Dicer can recognize the structure of“loop”we designed specially according some principles. Then the oligonucleotides were synthesized cloned into a plasmid pDC316-EGFP-U6 which was one type of recombinant adenovirus expression vectors.We employed NRK-52E rat kidney tubule epithelial cells to test the efficacy of Ad-shDAAO to knock down DAAO expression. Expressions of mRNA and protein of DAAO were tested by Real-time PCR and Western blotting at 24 and 48 hrs after transfection, respectively. Transfection with Ad-shDAAO significantly inhibited DAAO mRNA by 51.7% or 49.1%, respectively, compared to the saline or nonspecific Ad-shControl group (P<0.05 by ANOVA followed by post-hoc Student-Newman-Keuls test). Furthermore, Ad-shDAAO significantly inhibited DAAO protein expression by 50.2% or 51.2%, respectively, compared to the saline or nonspecific Ad-shControl group (P<0.05) Multiple-daily intrathecal injections of both siRNA/DAAO and Ad-shDAAO for 7 days significantly inhibited spinal DAAO expression by 50-80% as measured by real-time quantitative PCR and Western blot, and inhibited spinal DAAO enzymatic activity by approximately 60%. Meanwhile, both siRNA/DAAO and Ad-shDAAO prevented formalin-induced tonic phase pain by approximately 60% but not acute phase nociception.In addition, multi-daily intrathecal injections of the DAAO gene silencer siRNA/DAAO in the regimen to effectively block spinal DAAO expression also yielded anti-allodynic effects by approximately 40% and the analgesia remained for at least 6 days after stopping administrations of drugs.In order to test whether the anti-allodynic effect of CBIO is specifically via inhibition of the spinal DAAO, we further examined the effect of CBIO on bone cancer pain in the above two groups of rats on the 10th day postdose of oligonucleotides. Both groups of rats received intrathecal single bolus injection of CBIO (10μg) and their mechanical withdrawal thresholds were measured for 4 hours. As shown in Fig. 4B, CBIO blocked mechanical allodynia in a time-dependent manner in ipsilateral paws by approximately 40% maximum possible effect (peak) compared to the pre-injection control in nonspecific oligonucleotide-treated rats However, CBIO was not effective in reducing mechanical withdrawal thresholds in rats multi-daily pretreated with siRNA/DAAO. The results indicated that CBIO produced bone cancer allodynia specifically via inhibition of spinal DAAO.In the spinal cord, DAAO is almost restrictively found in astrocytes. This study tested whether intrathecal administration of siRNA/DAAO or Ad-shDAAO blocked activation of astrocytes marked by GFAP (glial fibrillary acidic protein) in the spinal cord. GFAP gene expression in the homogenates of the spinal cord was determined by Real-time quantitative PCR. Multiple-daily intrathecal injections of siRNA/DAAO (5μg/kg/day) but not nonspecific oligonucleotide for 7 days significantly reduced GFAP gene expression by approximately 40.6%, compared to the PEI-treated rats (P<0.05 by ANOVA followed by post-hoc Student-Newman-Keuls test). Spinal GFAP protein expression was also detected by Western blot and its ratio scanned withβ-actin was calculated. siRNA/DAAO but not nonspecific oligonucleotide significantly reduced GFAP protein expression by approximately 32.0%, compared to the PEI-treated rats (P<0.05). The same inhibitory effect on GFAP expression of astrocytes was confirmed by application of Ad-shDAAO.These results further suggest that down-regulation of spinal DAAO expression and enzymatic activity leads to analgesia with its mechanism potentially related to activation of astrocytes in the spinal cord.更多还原