【作者】 千年松；

【导师】 窦科峰； 陶开山； 王亚云；

【作者基本信息】 第四军医大学 ， 外科学， 2011， 博士

【摘要】 慢性胰腺炎是临床常见病,80-90%的慢性胰腺炎病人都有持续性腹部疼痛,其严重的、反复发作的腹部疼痛是病人的主要症状之一。然而,慢性胰腺炎导致疼痛的机制尚不明确,解释其引起疼痛的机制也有很多,包括胰腺本身和胰腺外部原因。关于神经病理性疼痛的研究表明中枢神经系统的疼痛传导通路发生了异常。有研究发现在某些胰腺病变,包括慢性胰腺炎、胰腺癌和胰性脑病,可以导致“神经重塑“和胰腺神经支配发生变化。以上结果提示中枢神经系统可能参与了慢性胰腺炎导致的慢性疼痛。脊髓背角神经-免疫系统和神经元-神经胶质细胞的相互作用在神经可塑性变化和神经病理性痛中起了十分重要的作用。有研究表明神经-免疫系统相互作用在慢性胰腺炎疼痛中非常重要。以往表明脊髓背角星形胶质细胞在大鼠慢性胰腺炎模型发生了活化,同时抑制星形胶质细胞的活性可以减轻慢性胰腺炎引起的疼痛。我们推测星形胶质细胞在此过程中可能被某些受体活化,然后导致一系列分子表达变化和信号通路的活化,参与了慢性胰腺炎引起的疼痛。但是具体那种分子参与了慢性胰腺炎引起的星形胶质细胞的活化未见报道。Toll样受体(TLRs)在慢性痛病理条件下脊髓背角神经-免疫相互作用和神经元-神经胶质细胞相互作用中起到了十分重要的作用。有学者表明TLRs在中枢神经系统中,特别是神经胶质细胞,有广泛表达。在哺乳动物神经胶质细胞中TLRs的表达已经被确认。在这些受体中,TLR2-4被认为主要介导了神经病理性疼痛。通常来说,在内源性或是外源性信号通路的刺激下,活化的TLRs能够导致神经胶质细胞的活化,调节疼痛相关的炎症因子。有趣的是,TLRs同样有可能在胰腺炎的发生过程中发挥了作用。最近有报道表明腹腔内注射TLR3可以成功的引起慢性胰腺炎样的病理变化。在本实验中,我们假设TLRs,尤其是TLR2-4,参与了慢性胰腺炎病理条件下星形胶质细胞的活化和疼痛的发生。我们的实验结果表明脊髓TLR3参与了大鼠慢性胰腺炎模型的疼痛。慢性胰腺炎病理条件下脊髓的“TLR3-星形胶质细胞- IL-1β/MCP-1”的正反馈通路可能为TLR参与慢性胰腺炎疼痛的机制,或许可以成为临床慢性胰腺炎病人疼痛的缓解的治疗靶点。目的:慢性胰腺炎导致的疼痛的机制目前为止尚不明确。以前的很多结果表明星形胶质细胞在疼痛过程中得到了活化。尽管如此,星形胶质细胞在慢性胰腺炎病理过程中是否发生了活化以及其发生活化的分子机制尚不明确。本实验的目的为探讨星形胶质细胞在慢性胰腺炎病理过程中是否发生了活化以及TIRs是否参与星形胶质细胞的活化和慢性胰腺炎引起的疼痛发作。方法:1.动物分为慢性胰腺炎实验组,假手术组和正常组。雄性Sprague–Dawley大鼠,体重250-300g,由第四军医大学动物实验中心提供。术前大鼠禁食水12h。麻醉实验动物,常规消毒,沿腹白线做正中切口剖腹,寻找十二指肠并找到胰腺,用微型动脉夹阻断胰胆管肝门部。用4号静脉头皮针经肠壁穿刺入胰胆管,并用丝线固定静脉针,通过恒流静脉输液泵注射含2%TNBS的PBS(PH 7.4),共0.4ml,持续60min,注毕后关闭恒流泵,维持压力50min。对照组注射等体积的生理盐水。除去动脉夹及丝线,逐层关闭腹腔。整个手术操作过程严格执行无菌操作。检测各组血清和胰腺组织细胞因子(TNF-?、IL-1。COX-1和α-SMA)的表达变化。分别于0点,1周和5周对各组大鼠胰腺HE染色后进行病理评分。同时从0点到第5周实时监测各组大鼠的RFs值。利用免疫荧光和RE-PCR检测星形胶质细胞标志物在不同时间点的表达,west blotting检测TLR2-4在不同时间点的表达。免疫荧光双标研究TLR3与星形胶质细胞标志物的共存关系。2.慢性胰腺炎组给予LAA拮抗星形胶质细胞的活性,利用RT-PCR验证LAA拮抗效果;同时检测RFs值的和星形胶质细胞标志物的变化情况。3.慢性胰腺炎组给予TLR3反义探针拮抗TLR3的表达,利用west blotting验证TLR3反义探针拮抗效果;同时检测RFs值的和星形胶质细胞标志物的变化情况。4. RT-PCR检测各组大鼠脊髓细胞因子,如TNF-?, IL-1, IL-6,COX2和MCP-1的变化情况。结果:1.在假手术组和正常组,胰腺组织HE染色表现正常。在TNBS注射5周后,实验组大鼠胰腺HE染色表现出明显的胰腺腺泡萎缩,炎细胞浸润,组织纤维化和间质增生。血清淀粉酶、TNF-?和IL-1在实验组显著升高。胰腺组织的TNF-?、IL-1、COX-1和α-SMA显著升高。在此同时,TNBS注射可导致RFs值的从开始到第5周的持续性升高。而假手术组和正常组RFs值比较无统计学意义。TNBS注射组5周时RFs值与刺激正相关。与临床慢性胰腺炎病人症状非常相似。因此我们确认TNBS可导致大鼠慢性胰腺炎以及腹部疼痛敏感度的增高。2.我们在不同时间点处理各组大鼠观察星形胶质细胞活化标志物GFAP表达的变化情况。免疫荧光和RT-PCR结果显示在假手术组和正常组,GFAP在胸椎背角的表达很低。在实验组,有趣的是,脊髓GFAP的表达从TNBS注射后第1周到第5周显著升高,第5周达到高峰,其表达的变化与疼痛的变化成正相关。因此我们假设GFAP可能介导慢性胰腺炎导致的疼痛。3.我们在不同时间点处理各组大鼠观察TLR2-4蛋白表达的变化情况。Western blotting结果显示在假手术组和正常组,TLR2-4在胸椎背角的表达很低。在实验组,有趣的是,脊髓TLR3的表达从TNBS注射后第一周到第五周显著升高,第5周达到高峰,其表达的变化与疼痛的变化成正相关,这与以往我们的研究表明脊髓星形胶质细胞的表达变化非常一致。因此我们假设TLR3可能在星形胶质细胞上表达,同时介导慢性胰腺炎导致的星形胶质细胞的活化。免疫双标显示星形胶质细胞特异性标志物GFAP与TLR3的共表达,初步证实了我们的假设。4.接下来我们鞘内注射GFAP的活性抑制剂LAA。RT-PCR证实了其拮抗效果。鞘内注射LAA在一定程度上可以缓解慢性胰腺炎引起的疼痛,这种效应为计量依赖性。在假手术组或正常组,鞘内注射LAA对RFs无影响。这些结果提示GFAP有可能参与到慢性胰腺炎导致的疼痛。接下来我们鞘内注射TLR3的反义探针拮抗TLR3的功能,western blotting证实了其拮抗效果。鞘内注射TLR3的反义探针在一定程度上可以缓解慢性胰腺炎引起的疼痛,这种效应为计量依赖性。在假手术组或正常组,鞘内注射TLR3反义探针对RFs无影响。这些结果暗示了TLR3有可能参与到慢性胰腺炎导致的疼痛。考虑到TLR3在脊髓星形胶质细胞的高表达,接下来我们探讨了TLR3反义探针对于慢性胰腺炎介导的星形胶质细胞活化的作用。我们实验结果发现TLR3反义探针显著抑制了脊髓星形胶质细胞的活化。然后我们又检测了脊髓炎症因子的表达情况,脊髓IL-1?,TNF-?,IL-6和MCP-1在慢性胰腺炎组表达明显升高,而只有TNF-?和IL-6的mRNA表达不受TLR3反义探针的影响。有趣的是,脊髓COX2在正常组和胰腺炎组表达无显著差异,TLR3反义探针也不影响其在脊髓的表达。结论:以上结果表明脊髓星形胶质细胞和其表达的TLR3参与了大鼠慢性胰腺炎模型的疼痛。慢性胰腺炎病理条件下脊髓的“TLR3-星形胶质细胞- IL-1β/MCP-1”的正反馈通路可能为TLR参与慢性胰腺炎疼痛的机制,或许可以成为临床慢性胰腺炎病人疼痛的缓解的治疗靶点。更多还原

【Abstract】 Chronic pancreatitis (CP) is a severe inflammatory and painful disease of the exocrine pancreas. Constant, recurrent and serious abdominal pain is one of the most common symptoms in CP, present in 80–90% of the patients. However,the pain mechanisms in CP are incompletely understood and probably are multifactorial,including pancreatic and extrapancreatic causes. Experimental human pain studies show that pain processing in the central nervous system (CNS) is abnormal in neuropathic pain disorders. A recent study shows that in the patients of CP and pancreatic cancer, pancreatic neuropathy could bring "neural remodeling" and alter pancreatic innervation. These results highly suggest that neuroplastic changes in the CNS are probably important contributors to the CP-induced chronic pain. Neuron-immune interactions and neuron-glial crosstalk in the spinal dorsal horn play a pivotal role in neuroplastic changes and neuropathic pain. It has been pointed out that neuroimmune interactions are significant in pain generation in CP. Our recent study observed that astrocytes are activated in the thoracic spinal cord in a rat model of CP induced by intrapancreatic infusion of trinitrobenzene sulfonic acid (TNBS). While inhibiting astrocytic activation could attenuate pain of CP. We can estimate that, like in that of nerve injury-induced neuropathic pain states, astrocytes might be activated through some receptors on it, and then produce signaling molecules that can feed back to enhance neuronal activity, contributing to pain facilitation. However,it was still unclear,in CP conditions:which receptor(s) mediated astrocytic activation. Toll-like receptors (TLRs) play a key role in neuron-immune interactions and neuron-glial crosstalk in the spinal dorsal horn in chronic pain conditions . It has been demonstrated that TLRs are widely expressed in the CNS, particularly by glial cells. In mammals, 12 members of the TLR family have been identified so far. Among them, TLR2-4 have been clarified to be major mediators in neuropathic pain. In general,in response to stimulation by endogenous and exogenous signals,activation of TLRs could induce glial activation in which multiple TLRs could trigger and tailor innate immune responses of glia by altering production of pain-associated pro-inflammatory cytokines/chemokines. However, there is still no report on the contribution of TLRs in CP related pain. Interestingly, TLRs have been implicated in the process of pancreatitis. A recent study even showed that intraperitoneal injection of TLR 3 activator could successfully induce CP-like pathological changes. In the present study, we hypothesized that TLRs,especially TLR2-4,contributed to astrocytic activation and pain behavior in the process of CP-induced pain. To test our hypothesis, we first observed the expression changes of TLR2-4 following TNBS-induced CP. We found that TLR3, but not TLR2 or 4 was increased in the thoracic spinal dorsal horn in the process of CP. Then we detected the cellular localization of TLR3 with double immunostaining and observed these TLR3 were highly expressed on spinal astrocytes. We further used a kind of TLR3 antisense oligodeoxynucleotide (ASO) to decrease the expression of TLR3 and observed the behavioral and biochemical changes in the spinal cord.OBJECTIVE:Mechanisms underlying pain in chronic pancreatitis (CP) are incompletely understood. Our previous data showed that astrocytes were actively involved. However, the molecular basis for astrocytic activation was unclear. The objective of this study is to investigate whether toll-like receptors (TLRs) contributed to astrocytic activation and pain behavior in CP-induced pain.METHODS:1. Induction of pancreatitisMale Sprague–Dawley rats were used in the study. Animals were given free access to drinking water and standard food pellets until 12 h prior to induction of pancreatitis, at which point food was withdrawn. Briefly, the common bile duct was closed temporarily near the liver with a small vascular clamp. A blunt 28 gauge needle with PE 10 tubing attached was inserted into the duodenum and was guided through the papilla into the duct and was secured with suture. TNBS solution (0.5 ml, 2%) in 10% ethanol in phosphate buffered saline (PBS,pH 7.4) was infused into the pancreatic duct over a period of 2–5 min at a pressure of 50 mmHg. After 30 min exposure to TNBS, needle and tubing were removed. The hole in the duodenum was sutured and the vascular clamp was removed restoring the bile flow. All the procedures in the sham group were same to that of the TNBS group, except that the same volume of saline instead of TNBS was infused into the duct.2. Administration of drugsFor single intrathecal injection, we performed spinal cord puncture under sevo uorane anesthesia with a 27 gauge needle between the T9 and T12 level to deliver the reagents (20 l) to the cerebrospinal ?uid. Immediately after the needle entry into subarachnoid space, a brisk tail ?ick could be observed. Intrathecal injection of LAA (Sigma, St. Louis, MO, USA; 50, 100, and 150 nmol in 10 l saline) or saline (10 l) was performed on the 34th d (1 d before 5 weeks post-operation) after pancreatitis induction. TLR3 ASO (5’- AACAATTGCTTCAAGTCC TCAAGTCC-3’) and TLR3 mismatch ODN (5’-ACTTCAACAGTAGACTACTAGACTAC-3’) were synthesized by Sangon Biotechnology Co.. For intrathecal infusion, laminectomy was performed at the level of the thoracic vertebrae, under pentobarbital anesthesia (45 mg kg-1 ,i.p.). A polyethylene (PE10) catheter (I.D. 0.28 mm and O.D. 0.61 mm) was passed caudally from the T9 to the T12 level of the spinal cord,and 2 cm of the free ending was left exposed in the upper thoracic region. The catheter was connected to an osmotic pump was filled with AS-ODN (0.01, 0.1or 1 nmol/μL) or MM-ODN (1 nmol/lL) in normal saline.All the rats were divided into three groups: TNBS group (n = 54); sham group (n =42) and na?ve controls (n = 6). In order to study the time course of TLRs change,we sacrificed every 6 rats in TNBS group at 1,2,3,4 and 5 w after TNBS infusion. At 4 w after surgery,other rats in TNBS and sham groupwere each further divided for drug injection:TNBS-ASO (or sham-ASO) group: TLR3 ASO was intrathecally infused on pancreatitic (0.01,0.1 or 1 nmol/μL) or sham operated rats (1 nmol/μL); TNBS-MO (or sham-MO) group: TLR3 MO was injected on pancreatitic or sham operated rats. At 5 w after surgery,all the rats including na?ve rats and sham rats without intrathecal injection were sacrificed for further experiments.3. Behavioral testsMechanical allodynia was measured with von Frey filaments. Briefly, the belly was shaved prior to test and areas designated for stimulation were marked. Rats were placed in a plastic cage with a mesh floor and were given 30 min for adaptation before testing. The von Frey filaments were applied from underneath through the mesh floor,in ascending order to the abdominal area at different points on the surface. A single trial consisted of 10 applications each for 1–2 s with a 15 s interval between applications allow the animal to cease any response and return to a relatively inactive position. A positive response consisted of the rat raising its belly (withdrawal response). The data were expressed as a percentage of the number of positive responses with each filament for each rat. In the time-course study, behavioral tests were performed before and once weekly for up to 5 w after induction of pancreatitis with a single force of von Frey filament (40.7 mN). In the strength-response study, rats were tested 5 w after pancreatitis induction with a series of von Frey filaments (2.29, 2.75, 6.76, 16.6, 40.7, 69.2 and 120 mN).4. Pancreatic histologyRats were deeply anesthetized with sodium pentobarbital (60 mg/kg,i.p.) and the pancreas was obtained and then fixed in 4% paraformaldehyde in phosphate buffered (PB,pH 7.4) at 4℃overnight. Pancreatic tissue was then transferred to xylene washes twice and was placed in cassettes and embedded in paraffin. Paraffin blocks were cut in 5-μm sections and stained with hematoxylin and eosin. Histological sections were analyzed by a pathologist in a double blinded manner. The severity of CP was morphologically assessed by semiquantitative scores according to previous reports : graded glandular atrophy (0–3),intralobular,interlobular and periductal fibrosis (0–3) and inflammatory cells infiltration (0–3).5. reverse transcription polymerase chain reaction for Pancreatic tissuesRT-PCR was performed to analyze the mRNA expression levels of TNF-α, IL-1β, COX-2 and IL-10 in the pancreatic tissue. RNA was extracted from pancreatic tissue using Trizol reagent. One microgram of RNA was reverse transcribed to cDNA. Amplification was performed with the following cycles: 95°C for 30 s,followed by 40 cycles of denaturing at 95°C for 5 s and annealing at 60°C for 20 s. All of the reactions were performed in triplicate.6. Biochemical AssaysSerum TNF-α, IL-1βand IL-10 were determined by ELISA. The serum TNF-α, IL-1βand IL-10 levels were evaluated using ELISA kits.7. Western blottingAll animals were rapidly sacrificed and the thoracic (T) 10 spinal dorsal horn was rapidly harvested. Spinal cord was then homogenized with a hand-held pestle in SDS sample buffer (10 ml/mg tissue), which contained a cocktail of proteinase and phosphatase inhibitors. The electrophoresis samples were heated at 100°C for 5 min and loaded onto 10% SDS-polyacrylamide gels with standard Laemmli solutions. The proteins were electroblotted onto a polyvinylidene difluoride membrane. The membran