摘要
脊髓损伤后中枢神经病理性疼痛(central neuropathic pain, CNP)是发生于脊髓损伤平面以下、痛觉已经消失区域的疼痛,是脊髓损伤后的顽固并发症,可自发地产生或者由皮肤刺激所引发,且疼痛持续存在并难以忍受,给患者带来极大痛苦,,使患者不能完成自我照顾活动、不能履行治疗性康复锻炼计划,严重影响患者的生活质量。CNP的临床治疗非常棘手,传统的药物和手术治疗往往无明显效果,这是因为其发生机制目前仍不明确。脊髓损伤后CP的病理变化是非常复杂的过程,而有关脊髓损伤后中枢神经免疫机制在CNP的发病中的作用则是目前疼痛学领域研究的热点。Janus kinase (JAK)/signal transducer and activators of transcription (STAT)介导的信号途径是一条重要的信号通道,可以发挥信号转导和基因转录活化子蛋白的双重作用,介导多种细胞因子和生长因子的细胞内信号转导过程并活化相应靶基因,从而产生生物学效应。其中STAT3参与了许多细胞因子及生长因子介导的细胞增殖及分化过程；STAT3主要表达在CNS中小胶质细胞上,有研究证实外周神经损伤所致神经病理性疼痛的早期形成过程中脊髓背角的小胶质细胞上的STAT3是关键信号转导分子。本研究拟采用基因干扰技术,抑制大鼠脊髓STAT3基因的表达,研究JAK/STAT3信号转导通路在大鼠脊髓损伤后中枢神经病理性疼痛中的作用,为研发以JAK/STAT3信号转导通路为靶点镇痛新药提供理论基础。
第一部分脊髓损伤后中枢神经痛模型大鼠脊髓背角STAT3表达变化及意义
目的观察T10脊髓钝性损伤后中枢神经痛模型大鼠后肢疼痛行为学改变,腰4-6节段脊髓背角STAT3信号通路的表达分布变化,以及脊髓背角胶质细胞的激活状况。
方法雌性SD大鼠42只,随机分为CNP组、假手术组和正常对照组,CNP组建立大鼠T10节段脊髓钝性损伤后中枢神经痛(CNP,below-level pain)模型,假手术组仅暴露T10节段脊髓不钝性打击,模型制作前一天及术后28天内每隔7天观察各组大鼠后肢疼痛行为学变化进行机械痛阈、热缩足持续时间及后肢运动功能检测,并于术后第3、7、14、21、28天处死取脊髓腰4-6节段,利用免疫组化、荧光双标染色、western-blot及qRT-PCR方法STAT3与胶质细胞的激活和共表达情况,同时检测脊髓背角STAT3mRNA和蛋白表达的变化,探讨STAT3的表达上调与时间相关的大鼠痛阈程度改变之间是否相关。
结果术前各组动物基础痛阈值之间无统计学差异(P>0.05)；CNP组与normal组和sham组相比,术后第3天痛阈值即明显下降,并于术后第14天降至最低点,持续至第28天仍下降明显,差异有统计学意义(P<0.05),同时CNP组大鼠后肢运动BBB评分脊髓损伤后第一天为0,损伤后3天开始恢复,28天时恢复至损伤前80%;CNP组大鼠腰4-6节段脊髓背角处免疫组化显示STAT3阳性细胞以灰质表达为主,并可见于灰质全层均有较多表达,尤其在脊髓背角高表达,与sham组相比,差异有统计学意义(P<0.01)；荧光双标染色提示CNP组脊髓背角浅层的STAT3阳性细胞主要为小胶质细胞,共表达从术后第3天开始明显增多,第14天表达最多,并持续至第28天与sham组相比,差异有统计学意义(P<0.01)；Westemblot分析结果与免疫组织化学结果基本相一致,即在术后第7d、21d、28d, CNP组大鼠腰4-6节段脊髓背角中STAT3蛋白的表达水平与术前相比明显增加,差异有统计学意义(P<0.01)；STAT3的mRNA表达于术后14天明显增高达最大值,并持续至第28天,于第28天仍明显高于sham组,差异有统计学意义(P<0.01)。相关性分析结果表明脊髓背角STAT3表达水平与脊髓损伤中枢神经痛大鼠疼痛程度之间具有明显的正相关。
结论脊髓T10钝性损伤后中枢神经痛大鼠腰段脊髓背角小胶质细胞激活,且小胶质细胞内STAT3蛋白表达明显上调,并与大鼠的疼痛学行为变化相平行。
第二部分靶向STAT3小分子干扰RNA慢病毒载体的构建和鉴定
目的以小分子干扰RNA技术构建大鼠STAT3siRNA慢病毒载体,并在大鼠小胶质细胞(HAPI细胞株)中行体外干扰效率鉴定。
方法分别设计STAT3-shRNA干扰序列及阴性对照序列,BLAST验证基因同源性。设计的序列经体外退火形成双链寡核苷酸,将其与以HpaI/Xhol酶切后的载体pGCL连接,转化DH5a感受态细胞,抽提质粒后测序鉴定。以HpaI单酶切鉴定并克隆质粒,即为重组慢病毒载体。将慢病毒包装系统三质粒和重组慢病毒载体混合,导入293T细胞,包装成病毒后,采用系列梯度稀释法,测定病毒滴度。同法构建阴性干扰慢病毒载体。将包装好的病毒转染HAPI细胞,分为干扰组,阴性干扰组和空白对照组3组,干扰组和阴性干扰组分别加入构建的干扰载体和阴性干扰载体,空白对照组加入等量不含病毒的培养基。转染96h后以westemBlot检验STAT3蛋白表达验证慢病毒载体干扰效率。并分别取3实验组HAPI细胞滴片,以STAT3多克隆抗体为一抗,FITC标一记的山羊抗兔IgG为二抗,间接免疫荧光法观察STAT3表达情况。
结果测序结果显示,STAT3siRNA干扰序列成功插入pGCL载体,该重组质粒与pHelper1.0载体重组、转化、氨节青霉素抗性筛选阳性克隆,酶切验证质粒重组成功。在293T细胞中进行病毒包装,系列梯度稀释法检测病毒悬液的滴度,测定慢病毒滴度为2×108Tu/ml。Western Blot检验STAT3蛋白表达,干扰组明显低于阴性干扰组(P<0.05)和空白对照组(p<0.05),抑制效率为75%。免疫荧光显示阴性干扰组和空白对照组的荧光则呈较强表达,而干扰组的绿色荧光表达则明显减弱。
结论成功构建小鼠STAT3siRNA慢病毒载体LV-GFP-STAT3,可以有效体外抑制HAPI细胞内STAT3蛋白的表达。
第三部分鞘内注射STAT3siRNA可减轻大鼠脊髓损伤后中枢神经病理性疼痛
目的观察携带STAT3siRNA片段重组慢病毒对脊髓损伤后中枢神经痛大鼠镇痛作用。
方法我们选取36只成年雌性SD大鼠制作T10脊髓钝性损伤中枢神经痛(CNP, below-level pain)模型,术后第1天经蛛网膜下腔内置管后将模型大鼠随机分为3组：生理盐水组(鞘内注入10μl生理盐水)、STAT3siRNA组(鞘内注入10μl重组慢病毒载体)和阴性对照siRNA组(鞘内注入10μ慢病毒空载体),连续给药3天。观察各组脊髓损伤术后7d、14d、28d的疼痛行为学和后肢运动功能的改变。不同时间点取腰段脊髓进行荧光显微镜检测GFP(绿色荧光蛋白)的表达,qRT-PCR检测STAT3基因的mRNA含量、WestemBlot测定STAT3蛋白含量以观察RNA干扰效应,qRT-PCR-PCR检测OX-42、AFT-3和MCP-1表达水平的变化,及给药后4周通过组织学观察脊髓损伤神经修复情况。
结果1.鞘内注射LV-GFP-STAT3重组慢病毒载体后,CNP大鼠机械痛敏和热痛敏明显得到改善,但不能完全阻止疼痛的发展；2.鞘内注射LV-GFP-STAT3重组慢病毒载体可显著降低模型大鼠腰段脊髓的STAT3mRNA的表达和蛋白质水平,OX-42. AFT-3和MCP-1mRNA表达也显著降低。(P<0.05)；3.大鼠脊髓损伤后运动功能BBB评分,所有实验大鼠在损伤后第1天都表现为丧失所有后肢运动,BBB评分均为0分,但损伤14天后STAT3siRNA组恢复情况明显好于空慢病毒组及生理盐水组,虽然至损伤后第28天各实验组大鼠的神经功能均有一定程度的神经恢复,但STAT3siRNA组和生理盐水组及空慢病毒组之间神经功能评分结果差异显著(P<0.01),生理盐水组和空慢病毒组之间神经功能评分结果无明显差异(P>0.05)。
结论鞘内注射STAT3siRNA慢病毒重组体,重组体能够在体内T10钝性脊髓损伤后CNP模型中实现RNA干扰,较长时间下调脊髓背角小胶质细胞内源性STAT3及其下游细胞因子mRNA表达,从而有效缓解大鼠脊髓损伤后中枢神经痛,并在一定程度上促进脊髓神经轴突再生和大鼠后肢运动功能的恢复。
Central neuropathic pain after spinal cord injury (central neuropathic pain, CNP) is a kind of pain occurs below the level of spinal cord injury, where pain have has disappeared. CNP is the intractable complication after spinal cord injury. It generates spontaneously or is induced by skin irritation,. Besides, these kind of pain is persistent and unbearable, bringing great suffering to patients and makes patients lose the ability of self-care and unable to perform therapeutic rehabilitation exercise program, which affects the quality of life of patients seriously. For the mechanism is not clear at present, clinical treatment of CNP is very difficult, traditional drugs and surgical treatments are often not significant effect. The pathogenesis of CNP after spinal cord injury is a very complex process, during which the immune mechanism is currently a hot research field of pain. Janus kinase (JAK)/signal transducer and activators of transcription (STAT)-mediated signaling pathway is an important signal channel, thatplays a dual role on signal transduction and gene activation. It mediates the process of intracellular signal transduction ofmultiple cytokines and growth factors, then activatesof the corresponding target genes, resulting in biological effects. STAT3is involved in cell proliferation and differentiation mediated by many cytokine and growth factor. STAT3is expressed mainly in microglia in the CNS. Research has shown that STAT3in microglia in spinal dorsal horn cells is the key transduction signal during the early formation of molecules neuropathic pain. In this study, we used gene interference to inhibit the expression of STAT3gene in rat spinal cord, Studying the role of JAK/STAT3signal transduction pathway on central neuropathic pain after spinal cord injury in rats, which provided a theoretical basis for use the JAK/STAT3signal transduction pathway as an analgesic drug target.
Part Ⅰ The significance of STAT3expression in dorsal horn in the model of central neuropathic pain after spinal cord injury
Objective:To observe the changes of pain behavior of the animals after T10spinal cord blunt injury,the changes of STAT3expression in lumbar4-6spinal cord dorsal horn, the expression of the downstream cytokines of STAT3and also the activation of glia in spinal cord dorsal horn.
Methods:Forty two female SD rats were selected and randomly divided into3groups:CNP group, sham operation group and control group. Rats in the CNP group were induced central neuropathic pain (below-level pain) by T10spinal cord blunt injury. Rats in tthe sham group were only exposed the spinal cord segment T10without blunt injury. we observed the changes of pain behavior index, such as he mechanical withdrawal threshold、thermal withdrawal duration and hindlimb function, on1day pre-operation and every interval7days after injury until the28th day.Besides, the animals were killed on day3th,7th,14th,21st,28th and then thelumbar4-6segments of spinal cord were taken. Glia activation and the changes of STAT3mRNA and protein expression in lumbar4～6spinal cord dorsal horn were detected by immunohistochemistry, double-labeling immunofluorescene.western-blotand qRT-PCR Relationship between the up-regulation of STAT3and time-related changes in pain threshold levels in the rats was also studied.
Results:There was no significant difference of basic pain thresholdamong all groups before operation (P>0.05). Compared CNP group with the sham group, the pain threshold is observably decreased form the3rd day to the14th day, and until to the28th day, the decreases remained significantly (P<0.05. The immunohistochemistry result displayed that the expression of STAT3-positive cells at lumbar4-6spinal cord were mainly in the gray matter in CNP group, and we found that it has more expression in all gray matter layer, particularly high expression in the spinal cord dorsal horn. Compared with the sham group, the difference was statistically significant (P<0.05). The fluorescent double staining suggest that the STAT3positive cells in spinal cord dorsal horn were mainly microglia. Co-expression of STAT3and OX42starting from the1st dayafter injury and then increased markedly on the3rd day, thenup to the most on the14th day, and last to the28th day.Compared with the sham group, the difference was statistically significant (P<0.05); Results of western blot analysis were in coincidence with those of the immunohistochemistry showed that STAT3protein expression was significantly increased fromthe7th day tothe21st day andthe28th daySTAT3mRNA significantly increased after surgery and it up to the maximum on the14th day and until to the28th day, it was still significantly higher than the sham group (P<0.01). Corelation analysis indicated that the STAT3expression in dorsal angle in rats had a significant positive correlation relationship with the central neuropathic pain after spinal cord injury (P<0.05).
Conclusion:The STAT3protein expression in microglia at lumbar spinal dorsal horn in the rats with central neuropathic pain were significantly increased after T10spinal cord injury, which paralleled with the changes of pain behavior in these rats.
Part Ⅱ Construction and identification of lentiviral vector expressing siRNA of STAT3
Objective:STAT3-shRNA lentivirus vectors targeting rat were built by small RNA interference technology and then identified their interference effects in vitro in rat microglia (HAPI cell strains).
Methods:The target sequences interference with STAT3-shRNA and the negative control sequences were designed individually.BLAST was used to verified the gene homology. After annealing, these double strand DNAs were connected with pGPU6vector which was digested by BamHI/Xhol, then converted into DH5a competent cells, extracted plasmid and then sequencing DNA. The recombinant lentivirus vector was made by plasmid that digested by a single restriction endonuclease BamHI and cloned. The three-plasmid lentivirus packaging systems and hybrid recombinant lentivirus vector were mixed, then the virus was produced in293T cell. The series of gradient dilution method was used to detect the virus titer. Use the same method to constructed the negative interference lentivirus vector. The packaged virus infected with HAPI cells were divided into3groups interference groups, negative interference group and control group. The interference group and the negative interference group were added to construct the interference vectors and negative interference vector, while the control group was added the same amount culture medium without virus in. Ninety six hours after transfection, westem-blot was used to test the expression of STAT3protein to identified the interference effects of lentivirus vectors. HAPI cells were collected form the3experimental groups and drops, the polyclonal antibodies STAT3was used as a the first antibody, a FITC labeled goat anti-rabbit IgG as the secondary antibody,and the indirect immunofluorescence was used to observe the expression of STAT3.
Results:DNA Sequencing demonstrated that, the STAT3-shRNA interference sequences were successfully inserted into pGPU6vector.The recombinant plasmid vector with pRsv-REV reorganization, transformation, resistance to ampicillin to screen positive clones. Digestion verified that the recombinant plasmid was success.Virus was packaged in293T cells. Series of gradient dilution method Showed that virus titer was measured4x108Tu/ml. Western Blot showed that the expression of STAT3protein was lower in the interference group than that in the negative interference group (P<0.05) and control group (p <0.05). The efficiency of inhibition is75%. The negative interference group and the control group showed a strong fluorescence of STAT3, while the STAT3fluorescence in the interference group was significantly decreased
Conclusion:Construction of STAT3-shRNA lentivirus vector targeting rat LV-GFP-STAT3could effectively inhibit the expression of STAT3protein in HAPI cells.
Part Ⅲ The intrathecal injection of STAT3siRNA can reduce central neuropathic pain after spinal cord injury
Objective:To observed the analgesic effect of lentivirus fragment carrying STAT3siRNA on central neuropathic pain after spinal cord injury.
Methods:Thirty six adult female SD rats were selected to make the model of central neuropathic pain. One day after T10spinal cord blunt injury (CNP, below-level), the subarachnoid catheter was done, then the rats were randomly divided into3groups:saline group (intrathecal injection of10μl saline), transfection reagent group (intrathecal injection of recombinant lentiviral vector10μl) and negative control siRNA group (intrathecal injection of lentiviral empty10μ, vector). The changes of pain behavioral of each group on post-operation7d,14d,21d,28d were observed. Fluorescence microscopy was used to detect the expression of GFP (green fluorescent protein) in lumbar spinal cord. Real time-PCR was carried out to detect the mRNA levels of STAT3gene. Meanwhile, western blot analysis was used to detect the content of STAT3protein in order to observe the effects of RNA interference. Also real time-PCR was used to detect the expression level of OX-42, AFT-3and MCP-1. Histology was used to observe the injury and recovery of the spinal cord4weeks after. intrathecal injection.
Results:It was showed that after the intrathecal injection of LV-GFP-STAT3lentivirus vector, the mechanical hyperalgesia and thermal hyperalgesia of CNP rats was significantly improved, but the injection can not completely prevent the progress of the development of pain. The intrathecal injection of LV-GFP-STAT3lentivirus vector could significantly reduce the expression of STAT3mRNA and protein in lumbar spinal cord after spinal cord injury. The expression of OX-42, AFT-3and MCP-1mRNA was also significantly reduced.(P<0.05). BBB score showed that the intrathecal injection of LV-GFP-STAT3lentivirus vector could greatly improve the recovery of the motor function.
Conclution:The Intrathecal injection of STAT3siRNA can inhibit microglia activation and the expression of STAT3and its downstream cytokine in lumbar spinal in the rats with central neuropathic pain after T10spinal cord blunt injury. Thus the central nervous pain was alleviated, and also axon regeneration of spinal cord neurons were improved.
引文
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