术后慢性疼痛大鼠脊髓背角组织的差异蛋白质组学研究

发布时间：2018-08-20 17:22

【摘要】：目的建立大鼠开胸术后慢性疼痛模型。利用蛋白质组学技术对经由手术侧脊髓背角组织提取的蛋白进行比较筛选,鉴定出表达存在差异的蛋白质,最后采用Western免疫印迹法进一步验证部分手术后慢性疼痛(chronic post-surgical pain, CPSP)相关蛋白的表达情况。方法 1.建立大鼠开胸术后慢性疼痛模型,采用更精确的行为学试验进行验证。 SPF级雄性SD大鼠,体重250g～350g,随机分为3组(n=12)：空白组、假手术组和模型组。麻醉后行气管内插管机械通气,于右胸第四五肋间做一小切口,用显微牵开器将两肋骨撑开8mm并持续1小时,建立开胸手术模型。假手术组除不进行牵引外,其余操作同模型组。于术前1天、术后每天共40天测量体重及大鼠对机械刺激和冷丙酮刺激的反应。 2.脊髓背角组织的差异蛋白质组学研究建立大鼠开胸术后慢性疼痛模型。建模成功后将动物分为2组,即手术后慢性疼痛组(CPSP组)以及无手术后慢性疼痛组(non-CPSP组)。分别提取两组大鼠脊髓背角组织中的总蛋白。利用蛋白质组学技术对提取的蛋白质进行比较筛选,鉴定与CPSP发生相关的蛋白质,并采用western免疫印迹法进一步验证部分CPSP相关脊髓背角蛋白的表达情况。结果 1.大鼠开胸术后慢性疼痛模型：与术前基础值比较,行开胸手术并肋骨牵开的12只大鼠中有5只在术后出现了明显的机械和冷刺激的异常性疼痛(P0.05)且疼痛持久；手术后慢性疼痛发生率为41.7%,与文献报告类似,提示建模成功。 2.差异蛋白质谱分析：经鉴定得到的58个差异蛋白质根据功能可分为5类：1.细胞骨架蛋白：微管蛋白(Tubulin alpha-1A chain, Tubulin beta-2C chain, Tubulin beta-3chain,Tubulin beta-2A chain, Microtubule-associated protein)、原肌球蛋白(Isoform1of Tropomyosin alpha-3chain)、β-肌动蛋白(Actb Actin, cytoplasmic1)、\肌动蛋白相关蛋白3(Actin-related protein3)、桥粒联结蛋白(Ahnak2)、突触小体相关蛋白(Snap91);2.能量产生／转运系统：甲基丙二酸半醛脱氢酶(Methylmalonate-semialdehyde dehydrogenase)、钠／钾转运ATP酶亚基alpha-3(Sodium/potassium-transporting ATPase subunit alpha-3)、磷酸甘油酸酯变位酶1(Pgaml Phosphoglycerate mutase1)、过氧化氢酶(Catalase)、NADP依赖性苹果酸酶(NADP-dependent malic enzym)、脂肪酸结合蛋白(Fatty acid-binding protein, epidermal)、丙酮酸激酶同工酶M1／M2(Pyruvate kinase isozymes M1/M2)、酰基辅酶A合成酶长链家族成员6(Long-chain-fatty-acid-CoA ligase6)、4-氨基丁酸氨基转移酶(4-aminobutyrate aminotransferase)、ATP合酶(ATP synthase gamma chain)、核苷二磷酸激酶B (Nucleoside diphosphate kinase B)和神经元特异性烯醇化酶(Gamma-enolase);3.信号传递分子：鸟嘌呤核苷酸结合蛋白α-2亚基(Isoform Alpha-2of Guanine nucleotide-binding protein G (o) subunit alpha)、ras相关蛋白Rap-1b (Ras-related protein Rap-lb)、多能蛋白聚糖核心蛋白亚型3(versican core protein isoform3)和转化蛋白(Transforming protein RhoA);4.运输蛋白：血清铁传递蛋白亚型1(血清铁传递蛋白亚型1)；5.其他：髓鞘碱性蛋白亚型1(Isoform1of Myelin basic protein S)、髓鞘相关糖蛋白(Myelin-associated glycoprotein)、发动蛋白-1(2型)(Isoform2of Dynamin-1)和WDR1蛋白(WD repeat-containing protein1)等。 3. Western验证结果：与蛋白质组学分析和鉴定基本一致,谷氨酰胺酶呈免疫阳性且CPSP组的谷氨酰胺酶含量高于non-CPSP组(P＜0.05)。结论： 1.采用肋骨牵引方法建立的大鼠开胸术后慢性疼痛模型具有良好的稳定性和重复性,可为研究开胸术后慢性疼痛的机制和治疗提供较好的动物模型。 2.本研究筛选和鉴定出的CPSP相关蛋白,可能是与CPSP发病有关的重要候选蛋白分子。
[Abstract]:objective
A rat model of chronic pain after thoracotomy was established. Proteins extracted from the dorsal horn of the spinal cord on the operative side were compared and screened by proteomics technique. The differentially expressed proteins were identified. Finally, the chronic post-surgical pain (CPSP) related eggs were further validated by Western blotting. The expression of white.
Method
1. to establish a model of chronic pain after thoracotomy in rats.
SPF male SD rats weighing 250g to 350g were randomly divided into three groups (n=12): blank group, sham operation group and model group. After anesthesia, endotracheal intubation and mechanical ventilation were performed, and a small incision was made between the fourth and fifth Costas of the right thorax. The two ribs were distracted by a microretractor for 8 mm for 1 hour to establish a thoracotomy model. The rest of the operations were the same as those in the model group. The body weight was measured one day before operation and 40 days after operation. The rats'responses to mechanical stimulation and cold acetone stimulation were also measured.
Differential proteomics study of spinal dorsal horn tissue in 2.
Rats were divided into two groups: the CPSP group and the non-CPSP group. Total proteins were extracted from the spinal dorsal horn tissues of the two groups. Proteomics was used to screen the proteins and identify CPSP. The expression of CPSP-related spinal dorsal keratin was further confirmed by Western blot.
Result
1. Rat model of chronic pain after thoracotomy: Comparing with the preoperative baseline, 5 of 12 rats undergoing thoracotomy with rib retraction had abnormal pain (P 0.05) and persistent pain, and the incidence of chronic pain after thoracotomy was 41.7%, which was similar to that reported in literature, suggesting that the model was successful.
2. Differential protein spectrum analysis: 58 differentially identified proteins can be divided into five categories according to their functions: 1. Cytoskeleton: Tubulin alpha-1A chain, Tubulin beta-2C chain, Tubulin beta-3chain, Tubulin beta-2A chain, Microtubule-associated protein, Isoform 1 of Tropomyosin alpha-3. Chain, beta-actin (Actin, cytoplasmic 1), actin-related protein 3 (Actin-related protein 3), desmosome-associated protein 2 (Ahnak2), synaptosome-related protein 91 (Snap91); 2. Energy production/transport system: methylmalonate-semialdehyde dehydrogenase, sodium/potassium ATPase subunit Alpha-3 (Sodium/potassium-transporting ATPase subunit alpha-3), phosphoglyceride mutase 1 (Pgaml Phosphoglycerate mutase 1), catalase, NADP-dependent malic enzyme, fatty acid-binding protein (epidermal), pyruvate kinase isozymes M1/M2 (Pyruvate k) Inase isozymes M1/M2, long-chain-fatty-acid-CoA ligase 6, 4-aminobutyrate aminotransferase, ATP synthase gamma chain, nucleoside diphosphate kinase B and neuron-specific enolase Signal transduction molecule: Isoform Alpha-2 of Guanine nucleotide-binding protein G (o) subunit alpha, Ras-related protein Rap-1b (Ras-related protein Rap-lb), polypotent glycan core protein isoform 3 (versican core protein isoform 3) and transforming protein RhoA; 4. Transporter proteins: serum transferrin subtype 1 (serum transferrin subtype 1); 5. Other: Isoform 1 of Myelin basic protein S, Myelin-associated glycoprotein, activator protein 1 (type 2) (Isoform 2 of Dynamin-1) and WDR 1 (WD repeat-containing protein 1) Wait.
3. Western test results: The results were consistent with proteomics analysis and identification. Glutaminase was immunopositive and the content of glutaminase in CPSP group was higher than that in non-CPSP group (P < 0.05).
Conclusion:
1. The rat model of chronic pain after thoracotomy established by rib traction has good stability and reproducibility, which can provide a better animal model for studying the mechanism and treatment of chronic pain after thoracotomy.
2. The CPSP-related proteins screened and identified in this study may be important candidate proteins related to the pathogenesis of CPSP.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R614;R-332

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