【作者】 刘思兰；

【导师】 杨建平；

【作者基本信息】 苏州大学 ， 外科学， 2010， 博士

【摘要】 长久以来,以神经系统为靶点的镇痛药并不能完全有效控制疼痛尤其是神经病理性疼痛。科学家们已经证实了循环系统的免疫细胞对疼痛感觉神经起着类似于汽车调光器开关的作用。进一步研究大脑和脊髓的类免疫细胞,在分子水平上建立起“免疫—疼痛”关联性实验的基本原理,将为更彻底地治疗疼痛奠定理论基础。在连接中枢神经系统(central nervous system,CNS)和先天性免疫中TLRs (Tol1-1ike receptor,TLRs)起着关键性作用。TLR4主要表达CNS中小胶质细胞上,有研究证实神经病理性疼痛的形成过程中小胶质细胞上的TLR4是关键受体。本研究拟采用基因干扰技术,抑制大鼠脊髓TLR4基因的表达,研究TLR4信号转导通路在大鼠胫骨癌痛中的作用,为研发以TLR4信号通路为靶点镇痛新药提供理论基础。第一部分大鼠胫骨癌痛模型的建立及评估目的通过对大鼠痛行为学测试、胫骨X摄片和HE染色等方法,建立并评价大鼠胫骨癌痛模型。方法雌性S-D大鼠,体重150-180g,左侧胫骨内注射大鼠体内腹水传代Walker256乳腺癌细胞,接种肿瘤细胞后2d、4d、6d、9d、12d、14d、18d和21d观测大鼠的机械触诱发痛阈值(paw withdrawal threshold,PWT)和行走痛评分(ambulatory score),并在实验结束后取双侧胫骨,X摄片观察骨质的破坏情况;病理切片,HE染色,显微镜下观察肿瘤的生长情况。结果①大鼠种瘤6d后开始出现明显的机械痛敏和自发痛,并随着荷瘤时间的延长疼痛更加严重。②大鼠种瘤6d时X线摄片可见大鼠左侧胫骨上端骨小梁轻微缺损;12d时出现多出骨小梁缺损,骨皮质有破坏;18d时骨破坏进一步加重,胫骨上端骨皮质明显破坏,大片骨质缺损。③HE染色见种瘤后6d和12d大鼠胫骨骨小梁有不同程度的破坏;18d时可见肿瘤细胞充满骨髓腔,骨小梁广泛破坏,正常骨结构缺失,严重的肿瘤细胞穿破骨皮质,侵犯周围肌肉和软组织。结论成功建立了大鼠胫骨癌痛模型。第二部分胫骨癌痛模型大鼠脊髓TLR4及其下游细胞因子表达的变化目的观察胫骨癌痛模型大鼠脊髓TLR4及其下游细胞因子表达的变化,以及脊髓小胶质细胞的激活状况。方法正常组、假手术组以及接种肿瘤细胞后6d、12d和18d大鼠的脊髓,利用免疫组化、western-blot、real time -PCR检测TLR4表达的变化;real time-PCR检测小胶质细胞激活标志(CD11b和CD14)及细胞因子(IL-1β、IL-6、TNF-α和IFN-β)表达水平的变化。结果荷瘤后6d大鼠脊髓TLR4mRNA表达和蛋白质水平即显著上调(P<0.05),12d、18d更甚(P<0.01);荷瘤后6d大鼠脊髓CD11b、CD14mRNA表达即显著升高(P<0.01);IL-1β和TNF-αmRNA表达显著升高也在荷瘤后6d(P<0.01),而IL-6和IFN-βmRNA表达显著升高则在荷瘤后12d(P<0.01)。结论大鼠脊髓小胶质细胞在胫骨癌痛发展的早期即被激活,TLR4表达上调,其下游细胞因子表达水平均进行性升高,提示该通路可能参与了大鼠胫骨癌痛的发生和发展。第三部分SiRNA抑制大鼠小胶质细胞上TLR4的表达目的体外转录合成的small interference RNA(siRNA)转染大鼠小胶质细胞(HAPI细胞株),诱导TLR4基因表达沉默,从而筛选出有效抑制TLR4基因表达的siRNA。方法根据RNA干扰的原理及siRNA的设计原则,设计4个长度为21bp的TLR4siRNA(siRNA312、siRNA439、siRNA1495和siRNA2062),另选择相同长度的无义双链RNA作为阴性对照RNA,体外转录合成siRNA,利用LipofectamineTM2000将siRNA导入HAPI细胞中,于转染后24h提取细胞的RNA, 48h提取蛋白质, real time-PCR和western-blot检测HAPI细胞上TLR4的表达。结果2μg TLR4 siRNA可减少HAPI细胞上的TLR4mRNA分别为:83%(siRNA439)、61% (siRNA312)、55% (siRNA1495)和53% (siRNA2062)。Western-blot检测结果也显示对HAPI细胞上TLR4蛋白质表达抑制效果最好的是siRNA43(9P<0.001)。结论siRNA转染小胶质细胞可有效抑制TLR4基因的表达。siRNA439效果最好。第四部分鞘内注射TLR4siRNA可减轻大鼠胫骨癌痛目的观察不同时间点鞘内注射TLR4siRNA对大鼠胫骨癌痛的影响。方法大鼠鞘内置管。荷瘤后4d或9d时给予鞘内注射TLR4siRNA( 2μg/10μl)、阴性对照siRNA(2μg/10μl)或溶媒in vivo jetPEITM10μl,1次/d,连续3d,末次鞘内注射后24h观察大鼠PWT值和行走痛评分的变化。痛行为学测试后,取大鼠脊髓L4~6,western-blot、real time-PCR检测TLR4表达的变化;real time-PCR检测CD11b、CD14、IL-1β?IL-6?TNF-α和IFN-β表达水平的变化。结果荷瘤后4d鞘内注射TLR4siRNA可完全抑制大鼠的胫骨癌痛,荷瘤后9d鞘内注射TLR4siRNA能缓解大鼠胫骨癌痛,但不能完全阻止疼痛的发展。鞘内注射TLR4siRNA可显著降低模型大鼠脊髓的TLR4mRNA的表达和蛋白质水平,CD11b、CD14、IL-1β?IL-6、TNF-α和IFN-βmRNA表达也显著降低。结论鞘内注射TLR4siRNA可有效抑制大鼠脊髓TLR4及其下游细胞因子的表达,抑制小胶质细胞的激活,从而缓解大鼠胫骨癌痛。第五部分TLR4/NF-κB信号转导通路在大鼠胫骨癌痛中的作用目的研究鞘内注射NF-κB抑制剂二硫代氨基甲酸吡咯烷(pyrrolidine dithiocarbamate,PDTC)和TLR4siRNA对大鼠胫骨癌痛的作用。方法荷瘤后9d给予鞘内注射PDTC(20μg/10μl)或生理盐水10μl,2次/d,连续3d,末次鞘内注射后6h观察大鼠PWT值和行走痛评分的变化。痛行为学测试后,取大鼠脊髓L4~6,结合第四部分的实验标本,免疫组化、western-blot、real time-PCR检测NF-κB p65表达的变化;real time-PCR检测IL-6和TNF-α水平的变化。结果鞘内注射PDTC可缓解大鼠的机械痛敏和自发痛;且能显著降低模型大鼠脊髓NF-κB p65 mRNA的表达和蛋白质水平(P<0.01);同时,IL-6和TNF-αmRNA的表达平也显著降低(P<0.01)。鞘内注射TLR4siRNA也可以显著降低模型大鼠脊髓NF-κB p65、IL-6和TNF-α的表达。结论TLR4/NF-κB信号通路可能参与了大鼠胫骨癌痛的发生和发展。第六部分TLR4/p38MAPK信号转导通路在大鼠胫骨癌痛中的作用目的研究鞘内注射p38MAPK抑制剂4-(4-fluorophenyl )-2- (4-methylsulfonylphenyl)-5-(4-pyridyl)-1H-imidazole( SB203580)和TLR4siRNA对大鼠胫骨癌痛的作用。方法①荷瘤后4d给予鞘内注射SB203580(2μg/10μl)或5%二甲基亚砜10μl,2次/d,连续3d,末次鞘内注射后6h观察大鼠PWT值和行走痛评分的变化。痛行为学测试后,取大鼠脊髓L4~6,结合第四部分实验标本,免疫组化、western-blot、real time-PCR检测p38MAPK表达的变化;real time-PCR检测IL-1β和TNF-α水平的变化。结果鞘内注射SB203580可缓解大鼠的机械痛超敏和自发痛;并且能显著降低模型大鼠脊髓的p38MAPKmRNA的表达和蛋白质水平(P<0.01);同时,IL-1β和TNF-αmRNA的表达也显著降低(P<0.01)。鞘内注射TLR4siRNA也可以显著降低模型大鼠脊髓p38MAPK、IL-1β和TNF-α的表达。结论TLR4/p38MAPK信号通路可能参与了大鼠胫骨癌痛的发生和发展。更多还原

【Abstract】 Part one:Establish and evaluation a rat model of bone cancer painObjective To establish and evaluate the rat model of tibial bone cancer pain using behavioral tests, X-ray scan and tibia HE staining and other methods.Methods A rat model of bone cancer pain was developed by intr-tibial inoculations of Walker256 breast cancer cells in female Sprague-Dawley rats(weight 150-180g).The mechanical allodynia (paw withdrawal threshold, PWT) and spontaneous pain (ambulatory score) were observed on the day 2, 4, 6, 9, 12, 14, 18 and 21 after inoculation(AI).The development of the bone tumor and structural damage in the bone were assessed by X-ray scan and HE stainning on the 6dayAI, 12dayAI and 18dayAI.Results①Rats with tibia tumors after Walker 256 cells inoculation displayed both tactile allodynia and spontaneous pain on the 6dayAI. Subsequently, the bone cancer pain was progressive severe.②Radiography of the left proximal epiphysis of the tibiae in the tumor-bearing group showed that there were bone density reduction, and moth-eaten-like small defects on the 6dayAI,significant bone destruction on the 12dayAI,and bilateral cortical bone damage and large bone defects on the 18dayAI③Histological studies showed that the trabecular bone was destroyed in varying degrees on the 6dayAI and 12dayAI;the bone marrow cavity was full of tumor cells,and the cortical bone was destroyed on the 18dayAI.Conclusion The present study successfully established a female rat model of bone pain from metastatic bone cancer. Part two: The expression of TLR4 and its downstream cytokine in spinal cord of the rat model of bone cancer painObjective To observe the expression of TLR4 and its downstream cytokine and the activation of microglia in spinal cord of the rat model of bone cancer pain.Methods The expression of TLR4 in spinal cord of nomal,sham and bone cancer pain group was detected with immunohistochemistry,western-blot, real-time-PCR on the 6dayAI, 12dayAI and 18dayAI. The expression of the microglial activation markers(CD11b and CD14) as well as cytokines (IL-1β, IL-6, TNF-αand IFN-β) was detected with real time-PCR in the same part and at the same time.Results The expression of TLR4mRNA and protein in spinal cord of the rat model of bone cancer pain significantly increased (P<0.05) on the 6dayAI, and further increase on the 12dayAI and 18dayAI (P<0.01); The expression of CD11b and CD14mRNA was significant increase on the 6dayAI in the bone cancer pain group compared with that in normal and sham group(P<0.01); The expression of IL-1βand TNF-αmRNA was also significant increase in the bone cancer pain group compared with that in normal and sham group on the 6dayAI (P<0.01), while the expression IL-6 and IFN-βmRNA was significantl increase on the 12dayAI (P<0.01).Conclusion In spinal cord of the rat model of bone cancer pain, the microglia were activated at the early stage of bone cancer pain,the expression of TLR4 was upregulation ,and the expression of its downstream cytokine was progressive increase, which suggested that the TLR4 signal transduction pathway minght be involved in the development of bone cancer pain.Part three: SiRNA inhibited the expression of TLR4 on the microgliaObjective To select the most effective siRNA which could inhibit TLR4 gene expression, siRNAs chemically synthesized in vitro were transfected the microglia (HAPI cell line) to induce TLR4 gene silencing.Methods Four different siRNA duplexes(siRNA312, siRNA439, siRNA1495 and siRNA2062) targeting the S-D rat TLR4 were selected using standard siRNA design rules. Additionally, a scrambled sequence was designed as a mismatch control. All siRNA duplexes were chemically synthesized in vitro. SiRNAs transfected the HAPI cells with LipofectamineTM2000. Forty-eight hours after transfection, the cells were harvested for western blot analysis of the level of knockdown proteins by the siRNAs. For targeting and detection of the mRNA levels, the cells were harvested after 24hr of transfection.Results 2μg TLR4 siRNA could reduce TLR4 mRNA by 83% (siRNA439), 61% (siRNA312), 55% (siRNA1495) and 53% (siRNA2062). Results from several western-blot test also showed that The most effective siRNA for knocking down TLR4 expression was siRNA439 (P<0.001).Conclusion Transfection the microglia by siRNA could effectively inhibit the TLR4 gene expression. siRNA439 was the best one.Part four: Intrathecal injection of TLR4siRNA could attenuate bone cancer pain in the rat modelObjective To observe the effects of intrathecal TLR4siRNA on bone cancer pain in the rat model at different time points.Methods Rats were intrathecally catheterizated. TLR4siRNA (2μg/10μl), negative control siRNA (2μg/10μl) or vehicle(in vivo jetPEITM)10μl were injected intrathecally to rats on the 4dayAI or 9dayAI. Injections were given daily for three consecutive days. Nociceptive testing and tissue harvesting were carried out 24 hr after the last injection. The expression of TLR4 was detected with western-blot and real time-PCR, and the expression of CD11b,CD14,IL-1β, IL-6, TNF-αand IFN–βwas detected with real time-PCR. Results Intrathecal injection of TLR4 siRNA on the 4dayAI could prevent the initial development of bone cancer pain.However,intrathecal injection of TLR4 siRNA on the 9dayAI could alleviate, but not reverse, well-established bone cancer pain. Intrathecal injection of TLR4siRNA could significantly reduce the expression of TLR4mRNA and protein(P<0.01) in the spinal cord of the rat model.The expression of CD11b, CD14, IL-1β, IL-6, TNF-αand IFN-βmRNA also significant decreased(P<0.01). Conclusion Intrathecal injection of TLR4siRNA could effectively inhibit the expression of TLR4 and its downstream cytokines, inhibit the activation of microglia and attenuate bone cancer pain in the rat model.Part five: The role of TLR4/NF-κB signaling pathway on the bone cancer pain of the rat modelObjective To observe the effects of intrathecal NF-κB inhibitor pyrrolidine dithiocarbamate(PDTC) and TLR4siRNA on bone cancer pain in the rat model .Methods PDTC (20μg/10μl ) or saline 10μl was injected intrathecally to rats on the 9dayAI. Injections were given twice a day for three consecutive days. Nociceptive testing and tissue harvesting were carried out 6hr after the last injection(ALI). Combined the experiment samples in the quartus section,the expression of NF-κBp65 was detected with immunohistochemistry,western-blot and real time-PCR, and the expression of IL-6 and TNF-αwas detected with real time-PCR.Results Intrathecal PDTC could alleviate rats’mechanical allodynia and spontaneous pain, and significantly reduce the expression of NF-κB p65 mRNA and protein in spinal cord of the rat model (P<0.01).Aat the same time,the expression of IL-6 and TNF-αmRNA also significantly decreased(P<0.01). Intrathecal TLR4siRNA also could significantly reduce the expression of NF-κB p65 ,IL-6 and TNF-α.Conclusion TLR4/NF-κB signaling pathway mingt be involved in the bone cancer pain of the rat model. Part six: The role of TLR4/p38MAPK signaling pathway on the bone cancer pain of the rat modelObjective To observe the effects of intrathecal p38MAPK inhibitor SB203580 and TLR4siRNA on bone cancer pain in the rat model .Methods SB203580 (2μg/10μl ) or 5%DMSO 10μl was injected intrathecally to rats on the 9dayAI. Injections were given twice a day for three consecutive days. Nociceptive testing and tissue harvesting were carried out 6hALI. Combined the experiment samples in the quartus section, the expression of p38MAPK was detected with immunohistochemistry, western-blot and real time-PCR, and the expression of IL-1βand TNF-αwas detected with real time-PCR.Results Intrathecal SB203580 could alleviate rats’mechanical allodynia and spontaneous pain, and significantly reduce the expression of p38MAPK mRNA and protein in spinal cord of the rat model (P<0.01).At the same time,the expression of IL-1βand TNF-αmRNA also significantly decreased(P<0.01). Intrathecal TLR4siRNA aslo could significantly reduce the expression of p38MAPK , IL-1βand TNF-α.Conclusion TLR4/ p38MAPK signaling pathway may be involved in the bone cancer pain of the rat model.更多还原