利用RNA干扰技术抑制P2X_3受体基因表达治疗骨癌痛的实验研究
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摘要
P2X_3受体是ATP-门控性离子通道家族的一个亚型,在慢性疼痛的伤害性信息传递中起重要作用,由于P2X_3受体的分布仅限于感觉神经元,对该通道的处理就可能提供一个即镇痛作用强又无副作用的慢性疼痛的治疗方法。本研究在大鼠胫骨癌痛模型上研究P2X_3受体在骨癌痛发生机理中的作用,并利用RNA干扰技术以P2X_3受体为靶点探讨基因治疗慢性疼痛的可行性。
第一部分骨癌痛大鼠背根神经节中P2X_3受体基因表达及电流的变化
目的:制备大鼠骨癌痛模型,观察骨癌痛大鼠触诱发痛和热痛敏阈值的变化,检测骨癌痛大鼠背根神经节(DRG)中P2X_3受体mRNA转录水平、蛋白表达的变化及P2X_3受体ATP诱发电流的变化。方法①大鼠骨癌痛模型的建立及行为学测定:雌性SD大鼠18只,随机分为2组(n=9),即骨癌痛组:左侧胫骨内注射5ul(10~4/ul)walker256肉瘤细胞,右侧不予处理;对照组:左侧胫骨注射同剂量的生理盐水,右侧不予处理。接种肿瘤细胞后5d,10d,14d,21d观察感觉异常和痛觉过敏阈值的变化。感觉异常以Von Frey细丝触诱发痛阈值(g)表示;痛觉过敏阈值用CO_2激光刺激痛阈值(ms)表示。实验结束后观察有无淋巴结或其他脏器的转移。取双侧胫骨、肺做病理切片,HE染色,光学显微镜下观察肿瘤的生长及脏器的转移。以大鼠出现明显触诱发痛和热痛敏阈值降低,且病理切片证实肿瘤细胞生长作为癌痛模型成功指标。②骨癌痛大鼠DRG中P2X_3受体表达的变化:接种肿瘤细胞后21d,取骨癌痛组和对照组大鼠L_(4-6)DRG,RT-PCR方法检测P2X_3受体mRNA转录水平的变化,免疫印迹法检测P2X_3受体蛋白表达的变化。重复实验的电泳图谱即免疫印迹结果用荧光扫描分析仪分析,相对表达量根据各电泳区带的辉度和内参照β-actin的辉度之比来计算。③骨癌痛大鼠DRG中P2X_3受体诱发电流的变化:接种肿瘤细胞后21d,取骨癌痛组和对照组大鼠L_(4-6)DRG,急性分散,全细胞膜片钳记录方法比较喷射给予α,β-meATP 3μM后P2X_3受体诱发电流的变化。结果:①接种肿瘤细胞10d后,骨癌痛组大鼠触左侧诱发痛阈值和CO_2激光痛阈值开始降低(P＜0.05),此后疼痛症状进行性加重。接种14d,21d骨癌痛组大鼠左侧的触诱发痛阈值和CO_2激光痛阈值较右侧明显降低(P＜0.01);与同一时间点对照组左侧的触诱发痛阈值和CO_2激光痛阈值相比显著降低(P＜0.01)。观察期间对照组大鼠和骨癌痛组大鼠右侧触诱发痛阈值及CO_2激光热痛敏阈值均没有明显变化。大鼠接种细胞后21d行胫骨病理切片可见肿瘤组织在骨髓腔内浸润性生长,破坏骨质。②接种肿瘤细胞21d骨癌痛组大鼠DRG中P2X_3mRNA转录水平及蛋白表达较对照组均明显增强(P＜0.01)。③骨癌痛组大鼠P2X_3受体诱发电流峰值为1.4±0.23 nA(n=5),对照组大鼠为0.8±0.12 nA(n=5),二者相比有显著性差异(P＜0.01)。结论:胫骨内接种Walker256细胞可以成功制备骨癌性疼痛动物模型,用于癌症性疼痛的机制及治疗研究;大鼠骨癌痛模型中P2X_3受体转录水平及蛋白表达均上调,P2X_3受体电流增强,通道数目增加,提示该通道参与了骨癌痛的发生过程。
第二部分抑制P2X_3受体基因表达的慢病毒shRNA的构建
目的:本实验利用可转录产生siRNA的真核载体构建了若干针对P2X_3受体不同基因靶位的RNA干扰质粒,并筛选出其中特异性抑制效果最强的基因靶序列,构建慢病毒RNA干扰载体。方法:①根据RNA干扰的原理及siRNA的设计原则,设计3段长度为19bp的P2X_3基因特异性siRNA,编号分别为782,850,918,构建至pSR-GFP siRNA转录载体;同时,将P2X_3全长基因克隆至pEGFP-N1真核表达载体。用pEGFP-P2X_3质粒分别与pSR-782、pSR-850、pSR-918 RNA干扰质粒共转染293T细胞,转染36h后收集细胞并提取RNA,通过RT-PCR和免疫印迹检测P2X_3基因的转录和表达,筛选出抑制效果最佳的siRNA干扰质粒。②选择抑制效果最佳的850片段构建慢病毒颗粒,所用载体为PRNAT-u6.2载体,克隆位点为BamH1/Xho1。载体测序后进行病毒颗粒的大量包装和纯化,用定量PCR法测定病毒滴度。③用包装好的慢病毒颗粒感染海马神经元细胞,观测感染效率。④用包装好的慢病毒颗粒感染293T细胞,Immunblot检测P2x_3受体的表达水平。结果:①通过将siRNA质粒与表达P2X_3蛋白的pEGFP-P2X_3质粒共转293T细胞,从设计的3条siRNA序列中筛选获得具有最佳抑制效果的pSR-850 P2X_3 RNA干扰质粒。②将shRNA850片段克隆至PRNAT-u6.2慢病毒载体,经过病毒包装、纯化,测定出病毒滴度为5.3×10~8Tu/ml。③慢病毒颗粒可以有效感染海马神经元细胞,感染效率几乎为100%④慢病毒颗粒感染293T细胞后能显著抑制P2X_3蛋白的表达,感染复数(MOI)为2,5,10时,蛋白表达量分别为对照组的54%、28%、17%,而对GADPH表达没有明显抑制。结论:利用体外转录合成的P2X_3受体shRNA 850片段可以高效抑制P2X_3基因的表达,用慢病毒颗粒包装后可以高效率感染海马神经元细胞,证明慢病毒包装的shRNA可有效应用于哺乳动物神经元P2X_3受体基因沉默的研究。
第三部分鞘内注射慢病毒shRNA抑制P2x3受体基因表达对
大鼠骨癌痛的影响
目的:研究鞘内注射慢病毒包装的P2X_3受体shRNA对骨癌痛模型大鼠触诱发痛及热痛敏阈值的影响,对P2X_3mRNA转录水平及受体蛋白表达的影响,并观察慢病毒shRNA注射后的长期毒副作用。方法:①SD大鼠31只,随机分为5组,正常组和骨癌痛组每组5只,治疗组和阴性对照治疗组每组8只,另外5只正常大鼠鞘内注射shRNA850慢病毒颗粒用于观察慢病毒颗粒的毒副作用。正常组为正常大鼠;骨癌痛组为骨癌性疼痛大鼠,未做任何治疗;治疗组于癌痛模型10d,大鼠出现明显的痛觉过敏后鞘内注射shRNA850慢病毒颗粒10μl(5×10~6Tu);阴性对照治疗组于癌痛模型10d后鞘内注射阴性对照shRNA慢病毒颗粒10μl(5×10~6Tu)。在鞘内注射后3d、1w、2w、3w、4w、6w分别观察大鼠触诱发痛阈值和CO_2激光热痛敏阈值。②于第6w行为学测定结束后取L_(4～6)DRG,RT-PcR及Immunoblot检测P2X_3受体mRNA转录水平和蛋白的表达,方法同第一部分。③正常大鼠5只鞘内注射shRNA850慢病毒颗粒10μl(5×10~6Tu),6w后处死取脊髓、DRG、肺、肝、肾、心脏,HE染色,常规病理观察慢病毒颗粒的毒副作用。结果:①鞘内注射1w时,治疗组大鼠触诱发痛阈值和CO_2激光痛阈值较同一时间点骨癌痛组和阴性对照治疗组显著升高(P＜0.01),但仍低于正常组(P＜0.01);鞘内注射2w后治疗组大鼠触诱发痛阈值和CO2激光痛阈值与造模前正常痛阈值相比无显著性差异(P＞0.05),与同一时间点骨癌痛组及阴性对照治疗组相比均显著增高(P＜0.01),这种作用可持续至鞘内给药后6w。②鞘内注射shRNA850慢病毒颗粒,可显著降低治疗组大鼠DRG中P2X_3受体mRNA转录水平和蛋白表达(P＜0.01)。③鞘内注射shRNA850慢病毒颗粒对重要脏器在观察期内没有发现毒副作用。结论:鞘内注射shRNA850慢病毒颗粒在六周的观察期内可有效缓解骨癌痛大鼠的触诱发痛和痛觉过敏,沉默DRG中P2X_3基因的表达,同时观察期内重要脏器无病理改变。
P2X_3 receptor,a subtype of ATP-gated ion channel,is selectively expressed in sensory neurons and plays a critical role in mediating chronic pain by transmitting harmful information.Therefore,chronic pain treatment by blocking P2X_3 receptors,will become an effective and hopeful therapeutic method without side effects.This study was focused on observing the therapy effects of blocking P2X_3 receptor expression in rat bone cancer pain model by RNA interference and exploring the underlying mechanism of this treatment.Hopefully,all these data can provide hints for gene therapy of chornic pain targeted on P2X_3 receptor in the future.
PartⅠChanges of P2X_3 receptor expression and currents in a rat bone cancer pain model
Objective:To investigate the relationship of rat behavior and P2X_3 receptor expression in the rat bone cancer pain models induced by Walker 256 cells.
Methods:①Eighteen female SD rats were divided into two groups,each has nine.In bone cancer pain group,5ul(10~4/ul)walker256 mammary gland carcinoma cells were transplanted into left tibia of rats by intra-tibia injection.In control group,5ul normal salines were injected into tibia of rats.The changes of allodynia and thermal hyperalgesia were observed at stages of 5,10,14 and 21 days after injection by Von-Frey filaments and CO_2 laser.Metastasises were observed in various organs.HE staining pathologic slices of bilateral tibia,lung were applied to evaluate tumor growth and metastasis under microscope. Successful rat model was set up when allodynia and thermal hyperalgesia was induced as well as the tumor growth was demonstrated by pathologic slices.②Twenty-one days after tumor cells intra-inoculation,rats were sacrificed by decapitation,and the L4-6 dorsal root ganglias were dissected out and put on ice for total RNA extraction.Changes of P2X_3 gene mRNA transcription and protein level were determined by RT-PCR and Immunoblot.③ATP induced currents usingα,β-meATP spraying were recorded by whole-cell patch clamp on acute dissipated DRG cells.Results:①The threshold of mechanical allodynia and thermal hyperalgesia in left sides of bone cancer pain began to decrease 10 days after Walker 256 cells intra-tibia injection.The syndrome was progressively intensified.The mechanical allodynia and thermal hyperalgesia thresholds of right sides in cancer pain group rats and of both sides in control group rats were not changed during the observation period.Intra-tibial injections of walker256 cells produced a rapidly expanding tumor within the boundaries of the tibia, causing severe remodeling of the bone.②The mRNA level an protein level of P2X_3 gene were significantly higher in cancer pain group than that of the control group(P＜0.01).③The ATP induced currents were 1.44±0.23nA(n=5)in DRG cells derived from bone cancer pain rats,and were significantly higher than currents0.8±0.12nA(n=5)derived from control rats(P＜0.01).Conclusions:Rat bone cancer pain was successfully induced by Walker256 cells intra-tibia inoculation,and can be used to investigate mechanisms and therapy of bone cancer pain.Both of mRNA and protein level of P2X_3 receptor in bone cancer pain rats increase and induce P2X_3 currents.Therefore,these indicate that the P2X_3 dependent channel is potentially involved in the occurrence of bone cancer pain.
PartⅡConstructing lentivirus expressing shRNA against P2X_3 receptor
Objectives:To synthesis shRNA for blocking P2X_3 receptor expression in vitro and select the best lentiviral construct.Methods:①Following siRNA design principles,three P2X_3 siRNAs,named 782,850,918,were synthesized and cloned into a vector(pSR-GFP vector)for siRNA expression.Full length P2X_3 cDNA was cloned in frame into pEGFP-N1,so the GFP-P2X_3 fusion protein could be expressed in mammalian cells,pEGFP-P2X_3 plasmids were co-transfected into 293T cells with P2X_3 RNAi vectors and the wild type vectors as control.Total cellular RNA was extracted at the time point of 36 hours post transfection,P2X_3 gene expression was analyzed by RT-PCR and Western-blot. The shRNA850 was shown to be the most efficient one and selected for the later work。②The shRNA850 construct was cloned into a lentiviral vector for siRNA expression.PRNAT-u6.2 was double digested with BamHⅠ/XhoⅠ,and ligated with DNA fragments encoding shRNA850.The ligation product was transformed into E.coli,recombinant plasmid was confirmed by sequencing.The recombinant vector was transfected into a lentiviral package cell line and the viral particles were obtained and purified.Viral titer was determined by real-time PCR.③Hippocampus neurons were infect by the lentiviral particles and the infection efficiency was evaluated.④The packaged lentiviral particles were applied to infect 293T cell,and the expression of P2X_3 was accessed by Immunoblot. Results:①Among three shRNAs,we saw the highest efficiency of No.850 blocking the expression of P2X_3 both at the level of mRNA and the protein vs pSR vector control(P＜0.01).②Lentiviral expression vector was constructed using shRNA850 and pRNAT-u6.2.The positive clone was applied to large scale viral preparation and the viral particles were purified,The titer was determined by real-time PCR and shown to be 5.3×10~8Tu/ml.③The lentivirus particles effectively infected hippocampus neurons and its infection efficiency was nearly 100%.④Expression of P2X_3 was blocked by the infection of recombinant lentiviral particles at MOI of 2,5,10 respectively.In control group,no significant difference of the GADPH expression was observed.Conclusions:Letiviral particles expressing P2X_3 shRNA could effectively infect neurons and block the expression of P2X_3.Therefore,this P2X_3 shRNA system is a powerful tool in our research wrok.
PartⅢEffects of intrathecal injection of lentivirus expressing
shRNA inhibiting P2X_3 gene on bone cancer pain rat
Objective:The aim of the study was to observe the effects of P2X_3 gene silenced by intrathecal injection of letiviral expressed shRNA on mechanical allodynia and thermal hyperalgesia and the expression of P2X_3 receptors in dorsal root ganglia of bone cancar pain rats.Moreover,side effects of intrathecal injection of letiviral expressed shRNA were also observed.Methods:Thirty-one SD rats were randomly divided into five groups,in normal group and cancer pain group each has five,in therapy group and placebo group each has eight,another five normal rats were used to observe side effects by intrathecal injection of letiviral expressed shRNA.Normal rats were in normal group,bone cancer pain rats without any treatment were in cancer pain group.In cancer pain group and placebo group,10 days after induction of cancer pain and when obvious hyperalgesia was observed, 10ul letivirus particles(5.3×l0~8Tu/ml)expressing shRNA850 or control shRNA were injected intrathecally into the rats separately.At various time points of 3d,7d, 2w,3 w,4 w,and 6 w after intrathecal injection,mechanical allodynia and thermal hyperalgesia values were determined.②Rats were sacrificed 6w after intrathecal injection and the L4-6 dorsal root ganglias were dissected on ice,P2X_3 receptor gene expressions in various groups were determined by RT-PCR and immunoblot separately.③Five normal rats receiving shRNA850 letivirus intrathecal injection were killed 6w after injection.Spine,DRG,lung,kidney,liver and heart were taken out and HE staining slices of these organs were making to obseve side effects of letivirus expressing shRNA850 after intrathecal injection.Results:①The mechanical allodynia and thermal hyperalgesia values of therapy group were increased significantly 1 week after 10ul(5×10~6Tu)letivirus particles containing shRNA850 were given compared with cancer pain group and placebo group(P＜0.01)at the same time point.Two weeks later,mechanical allodynia and thermal hyperalgesia values of therapy group showed no difference from that of the intact control group(P＞0.05)at the same time point.This therapeutic effect lasted 6w after intrathecal injection of letivirus particles containing shRNA850.②In concert with the findings,the expression of P2X_3 mRNA level and protein level were all decreased obviously in the therapy group compared with that of the cancer pain group and placebo control group(P＜0.01),while the expression of P2X_3 mRNA level and protein level in placebo group were same with the intact control group(P＞0.05).③We didn't see any side effects in normal rats after intrathecal injection of P2X_3 shRNAs expressing lentvirus throughout whole process.Conclusions:Under our observation,mechanical allodynia and thermal hyperalgesia of bone cancer pain can be alleviated for 6 weeks after intrathecal injection of P2X_3 shRNAs expressing lentvirus.Meanwhile we found this method can effectively silence the P2X_3 gene expression both at the protein and mRNA level.Moreover,we didn't see any side effects throughout whole process.
第一部分骨癌痛大鼠背根神经节中P2X_3受体基因表达及电流的变化
目的:制备大鼠骨癌痛模型,观察骨癌痛大鼠触诱发痛和热痛敏阈值的变化,检测骨癌痛大鼠背根神经节(DRG)中P2X_3受体mRNA转录水平、蛋白表达的变化及P2X_3受体ATP诱发电流的变化。方法①大鼠骨癌痛模型的建立及行为学测定:雌性SD大鼠18只,随机分为2组(n=9),即骨癌痛组:左侧胫骨内注射5ul(10~4/ul)walker256肉瘤细胞,右侧不予处理;对照组:左侧胫骨注射同剂量的生理盐水,右侧不予处理。接种肿瘤细胞后5d,10d,14d,21d观察感觉异常和痛觉过敏阈值的变化。感觉异常以Von Frey细丝触诱发痛阈值(g)表示;痛觉过敏阈值用CO_2激光刺激痛阈值(ms)表示。实验结束后观察有无淋巴结或其他脏器的转移。取双侧胫骨、肺做病理切片,HE染色,光学显微镜下观察肿瘤的生长及脏器的转移。以大鼠出现明显触诱发痛和热痛敏阈值降低,且病理切片证实肿瘤细胞生长作为癌痛模型成功指标。②骨癌痛大鼠DRG中P2X_3受体表达的变化:接种肿瘤细胞后21d,取骨癌痛组和对照组大鼠L_(4-6)DRG,RT-PCR方法检测P2X_3受体mRNA转录水平的变化,免疫印迹法检测P2X_3受体蛋白表达的变化。重复实验的电泳图谱即免疫印迹结果用荧光扫描分析仪分析,相对表达量根据各电泳区带的辉度和内参照β-actin的辉度之比来计算。③骨癌痛大鼠DRG中P2X_3受体诱发电流的变化:接种肿瘤细胞后21d,取骨癌痛组和对照组大鼠L_(4-6)DRG,急性分散,全细胞膜片钳记录方法比较喷射给予α,β-meATP 3μM后P2X_3受体诱发电流的变化。结果:①接种肿瘤细胞10d后,骨癌痛组大鼠触左侧诱发痛阈值和CO_2激光痛阈值开始降低(P＜0.05),此后疼痛症状进行性加重。接种14d,21d骨癌痛组大鼠左侧的触诱发痛阈值和CO_2激光痛阈值较右侧明显降低(P＜0.01);与同一时间点对照组左侧的触诱发痛阈值和CO_2激光痛阈值相比显著降低(P＜0.01)。观察期间对照组大鼠和骨癌痛组大鼠右侧触诱发痛阈值及CO_2激光热痛敏阈值均没有明显变化。大鼠接种细胞后21d行胫骨病理切片可见肿瘤组织在骨髓腔内浸润性生长,破坏骨质。②接种肿瘤细胞21d骨癌痛组大鼠DRG中P2X_3mRNA转录水平及蛋白表达较对照组均明显增强(P＜0.01)。③骨癌痛组大鼠P2X_3受体诱发电流峰值为1.4±0.23 nA(n=5),对照组大鼠为0.8±0.12 nA(n=5),二者相比有显著性差异(P＜0.01)。结论:胫骨内接种Walker256细胞可以成功制备骨癌性疼痛动物模型,用于癌症性疼痛的机制及治疗研究;大鼠骨癌痛模型中P2X_3受体转录水平及蛋白表达均上调,P2X_3受体电流增强,通道数目增加,提示该通道参与了骨癌痛的发生过程。
第二部分抑制P2X_3受体基因表达的慢病毒shRNA的构建
目的:本实验利用可转录产生siRNA的真核载体构建了若干针对P2X_3受体不同基因靶位的RNA干扰质粒,并筛选出其中特异性抑制效果最强的基因靶序列,构建慢病毒RNA干扰载体。方法:①根据RNA干扰的原理及siRNA的设计原则,设计3段长度为19bp的P2X_3基因特异性siRNA,编号分别为782,850,918,构建至pSR-GFP siRNA转录载体;同时,将P2X_3全长基因克隆至pEGFP-N1真核表达载体。用pEGFP-P2X_3质粒分别与pSR-782、pSR-850、pSR-918 RNA干扰质粒共转染293T细胞,转染36h后收集细胞并提取RNA,通过RT-PCR和免疫印迹检测P2X_3基因的转录和表达,筛选出抑制效果最佳的siRNA干扰质粒。②选择抑制效果最佳的850片段构建慢病毒颗粒,所用载体为PRNAT-u6.2载体,克隆位点为BamH1/Xho1。载体测序后进行病毒颗粒的大量包装和纯化,用定量PCR法测定病毒滴度。③用包装好的慢病毒颗粒感染海马神经元细胞,观测感染效率。④用包装好的慢病毒颗粒感染293T细胞,Immunblot检测P2x_3受体的表达水平。结果:①通过将siRNA质粒与表达P2X_3蛋白的pEGFP-P2X_3质粒共转293T细胞,从设计的3条siRNA序列中筛选获得具有最佳抑制效果的pSR-850 P2X_3 RNA干扰质粒。②将shRNA850片段克隆至PRNAT-u6.2慢病毒载体,经过病毒包装、纯化,测定出病毒滴度为5.3×10~8Tu/ml。③慢病毒颗粒可以有效感染海马神经元细胞,感染效率几乎为100%④慢病毒颗粒感染293T细胞后能显著抑制P2X_3蛋白的表达,感染复数(MOI)为2,5,10时,蛋白表达量分别为对照组的54%、28%、17%,而对GADPH表达没有明显抑制。结论:利用体外转录合成的P2X_3受体shRNA 850片段可以高效抑制P2X_3基因的表达,用慢病毒颗粒包装后可以高效率感染海马神经元细胞,证明慢病毒包装的shRNA可有效应用于哺乳动物神经元P2X_3受体基因沉默的研究。
第三部分鞘内注射慢病毒shRNA抑制P2x3受体基因表达对
大鼠骨癌痛的影响
目的:研究鞘内注射慢病毒包装的P2X_3受体shRNA对骨癌痛模型大鼠触诱发痛及热痛敏阈值的影响,对P2X_3mRNA转录水平及受体蛋白表达的影响,并观察慢病毒shRNA注射后的长期毒副作用。方法:①SD大鼠31只,随机分为5组,正常组和骨癌痛组每组5只,治疗组和阴性对照治疗组每组8只,另外5只正常大鼠鞘内注射shRNA850慢病毒颗粒用于观察慢病毒颗粒的毒副作用。正常组为正常大鼠;骨癌痛组为骨癌性疼痛大鼠,未做任何治疗;治疗组于癌痛模型10d,大鼠出现明显的痛觉过敏后鞘内注射shRNA850慢病毒颗粒10μl(5×10~6Tu);阴性对照治疗组于癌痛模型10d后鞘内注射阴性对照shRNA慢病毒颗粒10μl(5×10~6Tu)。在鞘内注射后3d、1w、2w、3w、4w、6w分别观察大鼠触诱发痛阈值和CO_2激光热痛敏阈值。②于第6w行为学测定结束后取L_(4～6)DRG,RT-PcR及Immunoblot检测P2X_3受体mRNA转录水平和蛋白的表达,方法同第一部分。③正常大鼠5只鞘内注射shRNA850慢病毒颗粒10μl(5×10~6Tu),6w后处死取脊髓、DRG、肺、肝、肾、心脏,HE染色,常规病理观察慢病毒颗粒的毒副作用。结果:①鞘内注射1w时,治疗组大鼠触诱发痛阈值和CO_2激光痛阈值较同一时间点骨癌痛组和阴性对照治疗组显著升高(P＜0.01),但仍低于正常组(P＜0.01);鞘内注射2w后治疗组大鼠触诱发痛阈值和CO2激光痛阈值与造模前正常痛阈值相比无显著性差异(P＞0.05),与同一时间点骨癌痛组及阴性对照治疗组相比均显著增高(P＜0.01),这种作用可持续至鞘内给药后6w。②鞘内注射shRNA850慢病毒颗粒,可显著降低治疗组大鼠DRG中P2X_3受体mRNA转录水平和蛋白表达(P＜0.01)。③鞘内注射shRNA850慢病毒颗粒对重要脏器在观察期内没有发现毒副作用。结论:鞘内注射shRNA850慢病毒颗粒在六周的观察期内可有效缓解骨癌痛大鼠的触诱发痛和痛觉过敏,沉默DRG中P2X_3基因的表达,同时观察期内重要脏器无病理改变。
P2X_3 receptor,a subtype of ATP-gated ion channel,is selectively expressed in sensory neurons and plays a critical role in mediating chronic pain by transmitting harmful information.Therefore,chronic pain treatment by blocking P2X_3 receptors,will become an effective and hopeful therapeutic method without side effects.This study was focused on observing the therapy effects of blocking P2X_3 receptor expression in rat bone cancer pain model by RNA interference and exploring the underlying mechanism of this treatment.Hopefully,all these data can provide hints for gene therapy of chornic pain targeted on P2X_3 receptor in the future.
PartⅠChanges of P2X_3 receptor expression and currents in a rat bone cancer pain model
Objective:To investigate the relationship of rat behavior and P2X_3 receptor expression in the rat bone cancer pain models induced by Walker 256 cells.
Methods:①Eighteen female SD rats were divided into two groups,each has nine.In bone cancer pain group,5ul(10~4/ul)walker256 mammary gland carcinoma cells were transplanted into left tibia of rats by intra-tibia injection.In control group,5ul normal salines were injected into tibia of rats.The changes of allodynia and thermal hyperalgesia were observed at stages of 5,10,14 and 21 days after injection by Von-Frey filaments and CO_2 laser.Metastasises were observed in various organs.HE staining pathologic slices of bilateral tibia,lung were applied to evaluate tumor growth and metastasis under microscope. Successful rat model was set up when allodynia and thermal hyperalgesia was induced as well as the tumor growth was demonstrated by pathologic slices.②Twenty-one days after tumor cells intra-inoculation,rats were sacrificed by decapitation,and the L4-6 dorsal root ganglias were dissected out and put on ice for total RNA extraction.Changes of P2X_3 gene mRNA transcription and protein level were determined by RT-PCR and Immunoblot.③ATP induced currents usingα,β-meATP spraying were recorded by whole-cell patch clamp on acute dissipated DRG cells.Results:①The threshold of mechanical allodynia and thermal hyperalgesia in left sides of bone cancer pain began to decrease 10 days after Walker 256 cells intra-tibia injection.The syndrome was progressively intensified.The mechanical allodynia and thermal hyperalgesia thresholds of right sides in cancer pain group rats and of both sides in control group rats were not changed during the observation period.Intra-tibial injections of walker256 cells produced a rapidly expanding tumor within the boundaries of the tibia, causing severe remodeling of the bone.②The mRNA level an protein level of P2X_3 gene were significantly higher in cancer pain group than that of the control group(P＜0.01).③The ATP induced currents were 1.44±0.23nA(n=5)in DRG cells derived from bone cancer pain rats,and were significantly higher than currents0.8±0.12nA(n=5)derived from control rats(P＜0.01).Conclusions:Rat bone cancer pain was successfully induced by Walker256 cells intra-tibia inoculation,and can be used to investigate mechanisms and therapy of bone cancer pain.Both of mRNA and protein level of P2X_3 receptor in bone cancer pain rats increase and induce P2X_3 currents.Therefore,these indicate that the P2X_3 dependent channel is potentially involved in the occurrence of bone cancer pain.
PartⅡConstructing lentivirus expressing shRNA against P2X_3 receptor
Objectives:To synthesis shRNA for blocking P2X_3 receptor expression in vitro and select the best lentiviral construct.Methods:①Following siRNA design principles,three P2X_3 siRNAs,named 782,850,918,were synthesized and cloned into a vector(pSR-GFP vector)for siRNA expression.Full length P2X_3 cDNA was cloned in frame into pEGFP-N1,so the GFP-P2X_3 fusion protein could be expressed in mammalian cells,pEGFP-P2X_3 plasmids were co-transfected into 293T cells with P2X_3 RNAi vectors and the wild type vectors as control.Total cellular RNA was extracted at the time point of 36 hours post transfection,P2X_3 gene expression was analyzed by RT-PCR and Western-blot. The shRNA850 was shown to be the most efficient one and selected for the later work。②The shRNA850 construct was cloned into a lentiviral vector for siRNA expression.PRNAT-u6.2 was double digested with BamHⅠ/XhoⅠ,and ligated with DNA fragments encoding shRNA850.The ligation product was transformed into E.coli,recombinant plasmid was confirmed by sequencing.The recombinant vector was transfected into a lentiviral package cell line and the viral particles were obtained and purified.Viral titer was determined by real-time PCR.③Hippocampus neurons were infect by the lentiviral particles and the infection efficiency was evaluated.④The packaged lentiviral particles were applied to infect 293T cell,and the expression of P2X_3 was accessed by Immunoblot. Results:①Among three shRNAs,we saw the highest efficiency of No.850 blocking the expression of P2X_3 both at the level of mRNA and the protein vs pSR vector control(P＜0.01).②Lentiviral expression vector was constructed using shRNA850 and pRNAT-u6.2.The positive clone was applied to large scale viral preparation and the viral particles were purified,The titer was determined by real-time PCR and shown to be 5.3×10~8Tu/ml.③The lentivirus particles effectively infected hippocampus neurons and its infection efficiency was nearly 100%.④Expression of P2X_3 was blocked by the infection of recombinant lentiviral particles at MOI of 2,5,10 respectively.In control group,no significant difference of the GADPH expression was observed.Conclusions:Letiviral particles expressing P2X_3 shRNA could effectively infect neurons and block the expression of P2X_3.Therefore,this P2X_3 shRNA system is a powerful tool in our research wrok.
PartⅢEffects of intrathecal injection of lentivirus expressing
shRNA inhibiting P2X_3 gene on bone cancer pain rat
Objective:The aim of the study was to observe the effects of P2X_3 gene silenced by intrathecal injection of letiviral expressed shRNA on mechanical allodynia and thermal hyperalgesia and the expression of P2X_3 receptors in dorsal root ganglia of bone cancar pain rats.Moreover,side effects of intrathecal injection of letiviral expressed shRNA were also observed.Methods:Thirty-one SD rats were randomly divided into five groups,in normal group and cancer pain group each has five,in therapy group and placebo group each has eight,another five normal rats were used to observe side effects by intrathecal injection of letiviral expressed shRNA.Normal rats were in normal group,bone cancer pain rats without any treatment were in cancer pain group.In cancer pain group and placebo group,10 days after induction of cancer pain and when obvious hyperalgesia was observed, 10ul letivirus particles(5.3×l0~8Tu/ml)expressing shRNA850 or control shRNA were injected intrathecally into the rats separately.At various time points of 3d,7d, 2w,3 w,4 w,and 6 w after intrathecal injection,mechanical allodynia and thermal hyperalgesia values were determined.②Rats were sacrificed 6w after intrathecal injection and the L4-6 dorsal root ganglias were dissected on ice,P2X_3 receptor gene expressions in various groups were determined by RT-PCR and immunoblot separately.③Five normal rats receiving shRNA850 letivirus intrathecal injection were killed 6w after injection.Spine,DRG,lung,kidney,liver and heart were taken out and HE staining slices of these organs were making to obseve side effects of letivirus expressing shRNA850 after intrathecal injection.Results:①The mechanical allodynia and thermal hyperalgesia values of therapy group were increased significantly 1 week after 10ul(5×10~6Tu)letivirus particles containing shRNA850 were given compared with cancer pain group and placebo group(P＜0.01)at the same time point.Two weeks later,mechanical allodynia and thermal hyperalgesia values of therapy group showed no difference from that of the intact control group(P＞0.05)at the same time point.This therapeutic effect lasted 6w after intrathecal injection of letivirus particles containing shRNA850.②In concert with the findings,the expression of P2X_3 mRNA level and protein level were all decreased obviously in the therapy group compared with that of the cancer pain group and placebo control group(P＜0.01),while the expression of P2X_3 mRNA level and protein level in placebo group were same with the intact control group(P＞0.05).③We didn't see any side effects in normal rats after intrathecal injection of P2X_3 shRNAs expressing lentvirus throughout whole process.Conclusions:Under our observation,mechanical allodynia and thermal hyperalgesia of bone cancer pain can be alleviated for 6 weeks after intrathecal injection of P2X_3 shRNAs expressing lentvirus.Meanwhile we found this method can effectively silence the P2X_3 gene expression both at the protein and mRNA level.Moreover,we didn't see any side effects throughout whole process.
引文
[1]Thurlimann B,de Stoutoutz ND.Causes and treatment of bone pain of malignant origin.Drugs.1996,51(3):383-398.
[2]Schwei MJ,Honore P,Rogers SD,et al.Neurochemical and cellular recorganization of the spinal cord in a marine model of bone cancer pain.J Neurosci.1999,19:10886-10897.
[3]Medhurst S J,Walker K,Bowes M,et al.A rat model of bone cancer pain.Pain.2002,96:129-140.
[4]Liu XJ,Salter MW.Purines and pain mechanisms:recent.Curr Opin Investig Drugs.2005,6(1):65-75.
[5]Bumstock G.Release of vasoactive substances from endothelial cells by shear stress and purinergic mechano-sensory transduction.J Anatomy.1999,194:335-342.
[6]邓伦斌,姚磊,韩济生.神经源性痛诱发大鼠背根神经节细胞电生理学变化.针刺研究.2001,26(3):228.
[7]Chen CC,Akopian AN,Sivilotti L,et al.A P2X purinoceptor expressed by a subset of sensory neurons.Nature.1995,377(6548):428-31.
[8]Bradbury E J,Burnstock G,McMahon SB.The expression of P2X_3purinoreceptors in sensory neurons:effects of axotomy and glial-derived neurotrophic factor.Mol Cell Neurosci.1998,12(4-5):256-68.
[9]Tsuda M,Koizumi S,Kita A,et al.Mechanical allodynia caused by intraplantar injection of P2X receptor agonist in rats:involvement of heteromeric P2X2/3 receptor signaling in capsaicin-insensitive primary afferent neurons.J Neurosci.2000,20:RC90,1-5.
[10]Cockayne DA,Hamilton SG,Zhu QM,et al.Urinary bladder hyporeflexia and reduced pain-related behaviour in P2X_3-deficient mice.Nature.2000,407:1011-1015.
[11] Oliveira MC, Parada CA, Veiga MC,et al. Evidence for the involvement of endogenous ATP and P2X receptors in TMJ pain. Eur J Pain.2005,9(1):87-93.
[12] McGaraughty S, Jarvis MF. Antinociceptive properties of a non-nucleotide P2X3 /P2X2/3 receptor antagonist.Drug News Perspect. 2005 , 18(8):501-507.
[13] Tomari Y, Zamore PD. Perspective: machines for RNAi. Genes Dev.2005,19(5):517-29.
[14] Paddison PJ, Caudy AA, Bernstein E, et al. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes & Dev.2002,16:948-958.
[15] Sontheimer EJ ,Carthew RW. Silence from within : endogenous siRNAs and miRNAs. Cell.2005 ,122(1) :9 - 12.
[16] Gu Y, Xu Y, Li GW,et al.Remote nerve injection of mu opioid receptor adeno-associated viral vector increases antinociception of intrathecal morphine. J Pain. 2005,6(7):447-54.
[17] Prasad M, Fearon IM, Zhang M,et al.Expression of P2X2 and P2X3 receptor subunits in rat carotid body afferent neurones: role in chemo-sensory signalling.J Physiol. 2001 Dec 15,537(Pt 3):667-77.
[18] Dornelas CA, Almeida PR, Nascimento GL, et al. Experimental model of Walker 256 carcinosarcoma in rats bladder. Acta Cir Bras. 2006, 21(1):38-42.
[19] Buffon A, Ribeiro VB, Schanoski AS, et al. Diminution in adenine nucleotide hydrolysis by platelets and serum from rats submitted to Walker 256 tumour. Mol Cell Biochem. 2006,281(1-2):189-195.
[20] Miyazaki M, Shimoda T, Itoh H, et al. Enhancement of cytotoxicity of doxorubicin by verapamil in the hepatic artery infusion for liver tumors in rats. Cancer. 1993,72(2):349-354.
[21] Luo C,Jiang Y,Liu Y,et al. Experimental study on mechanism and rarity of metastases in skeletal muscle.Chin Med J(Engl).2002,115:1645-1649.
[22]Muta M,Matsumoto G,Nakashima E,et al.Mechanical analysis of tumor growth regression by the cyclooxygenase-2 inhibitor,DFU,in a Walker256rat tumor model:importance of monocyte chemoattractant protein-1modulation.Clin Cancer Res.2006,12(1):264-272
[23]Kostenuik P J,Orr FW,Suyama K,et al.Increased growth rate and tumor burden of spontaneously metastatic Walker 256 cancer cells in the skeleton of bisphosphonate-treated rats.Cancer Res.1993,53(22):5452-5457.
[24]杜恒,崔刚,张增铁等.氯化镓对Walket256癌骨侵袭的抑制作用.西安交通大学学报(医学版),2004,25(4):369-371.
[25]刘艳红,张宏,徐龙河.大鼠骨癌痛模型的制备及电压依赖性钠通道Na1.8在其背根神经节的表达研究.解放军医学杂志,2007,32(4)319-322.
[26]Manthyh PW,Clohisy DR,Koltzenburg M,et al.Molecular mechanisms of cancer pain.Nature Rev(cancer).2002,2(3):201-209.
[27]Martin W J,Liu H,Wang H,et al.Inflammation-induce up-regulation of protein kinase Cgamma immunoreactivity in rat spinal cord correlates with enhanced nociceptive processing.Neuroscience.1999,88(4):1267-74.
[28]Calza L,Pozza M,Zanni M,et al.Peptide lasticity in primary sensory neurons and spinal cord during djuvant-induced arthritis in the rat:an immunocytochemical and in situ hybridization study.Neuroscience.1998Jan,82(2):575-89.
[29]Simonet WS,Lacey DL,Dunstan CR,et al.Osteoprotegerin:a novel secreted protein involved in the regulation of bone density.Cell.1997,89(2):309-319.
[30]Kong YY,Yoshida H,Sarosi I,et al.OPGL is a key regulator of osteoclastogenesis,lymphocyte development and lymphnode organo -genesis.Nature.1999,397(6717):315-323.
[31] Luger NM, Honore P, Sabino MA, et al. Osteoprotegerin diminishes advanced bone cancer pain. Cancer Res. 2001,61(10):4038-4047.
[32] Bekker PJ, Holloway D, Nakanishi A,etal. The effect of a single dose of osteoprotegerin in postmenopausal women. J Bone Miner Res. 2001,16(2):348-360.
[33] Coleman RE.How can we improve the treatment of bone metastases further? Curr Opin Oncol. 1998,10( Suppl 1):S7-13.
[34] Toyras A, Ollikainen J, Taskinen M, et al. Inhibition of mevalonate pathway is involved in alendronate-induced cell growth inhibition, but not in cytokine secretion from macrophages in vitro.Eur J Pharm Sci. 2003 ,19(4): 223-230.
[35] Sevcik MA, Luger NM, Mach DB, et al . Bone cancer pain: the effects of the bisphosphonate alendronate on pain, skeletal remodeling, tumor growth and tumor necrosis.Pain. 2004 ,111(1-2): 169-180.
[36] Peters CM, Lindsay TH, Pomonis JD, et al . Endothelin and the tumorigenic component of bone cancer pain. Neuroscience. 2004,126(4): 1043-1052.
[37] Sabino MA, Ghilardi JR, Jongen JL,et al. Simultaneous reduction in cancer pain, bone destruction, and tumor growth by selective inhibition of cyclooxygenase-2.Cancer Res. 2002,62(24):7343-7349.
[38] Li X, Tomita M, Pilbeam CC,et al .Prostaglandin receptor EP2 mediates PGE2 stimulated hypercalcemia in mice in ivo.Prostaglandins Other Lipid Mediat. 2002,67(3-4):173-180.
[39] Honore P, Rogers SD, Schwei MJ, et al.Murine models of inflammatory neuropathic and cancer pain each generates a unique set of neurochemical changes in the spinal cord and sensorv neurons. Neuroscience, 2000, 98:585-598.
[40] Wacnick PW, Eikmeier TJ, Ruggles TR, et al. Functional interactions between tumor and peripheral nerve: morphology, algogen identification, and behavioral characterization of a new murive model of cancer pain.J Neurosci,2001,21:9355-9366.
[41]Colburn RW,DeLeoJA,Rickman AJ,etal.Dissociation of microglial activation and neuropathic pain behaviors following peripheral nerve injury in the rat.J Neuroimmunol,1997,79:163-175.
[42]Schwei MJ,Honore P,Rogers SD,et al.Neurochemical and cellular reorganization of the spinal cord in a murine model of bone cancer pain.J Neurosci,1999,19:10886-10897.
[43]Xu GY,Huang LY.Peripheral inflammation sensitizes P2X receptor -mediated responses in rat dorsal root ganglion neurons.J Neurosci.2002,22(1):93-102.
[44]Wildman SS,King BF,Burnstock G.Potentiation of ATP-responses at a recombinant P2X2 receptor by neurotransmitters and related substances.Br J Pharmacol.1997,120:221-224.
[45]Prostaglandin E2 potentiation of P2X3 receptor mediated currents in dorsal root ganglion neurons Molecular Pain 2007,3:22.
[46]赵士洁,殷光甫,谷远征,等.中国组织化学与细胞化学杂志,2006.15:91-95.
[47]Fire A,Xu S,Montgomery MK,et al.Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.Nature.1998,391(6669):806-811.
[48]孟国良,汤富酬,张俊争.小鼠胚胎干细胞中RNA干涉现象.生物化学与生物物理学报,2003,35(3):238-246.
[49]雷迎峰,薛小平,尹文.RNAi研究进展.国外医学(分子生物学分册),2002,24(4):196-199.
[50]Brummelkamp TR,Bernards R,Agami R.Stable suppression of tumorigenicity by virus-mediated RNA interference.Cancer Cell.2002,2(3):243-247.
[51]Elbashir SM,Harborth J,Lendeckel W,et al.Duplexes of 21- nucleotide RNAs mediate RNA interference in cultured mammalian cells.Nature.2001,411(6836):494-498.
[52] Lin SL, Chuong CM, Ying SY A Novel mRNA-cDNA interference Phenomenon for silencing bcl-2 expression in human LNCaP cells.Biochem Biophys Res Commun. 2001,281 (3):639-644.
[53] Scherr M, Morgan MA, Eder M. Gene silencing mediated by small interfering RNAs in mammalian cells.Curr Med Chem. 2003, 10(3) :245-256.
[54] Amarzguioui M, Rossi JJ, Kim D.Approaches for chemically synthesized siRNA and vector-mediated RNAi.FEBS Lett. 2005,579(26):5974-5981.
[55] Kitabwalla M, Ruprecht RM. RNA interference~a new weapon against HIV and beyond. N Engl J Med. 2002 ,24,347(17):1364-1367.
[56] Lois C, Hong EJ, Pease S, et al. Germline transmission and tissue specific expression of transgenes delivered by lentiviral vectors. Science, 2002,295:868-872.
[57] Pfeifer A, Ikawa M, Dayn Y, etal. Transgenes is by lentiviral vectors: Lack Of gene silencing in mammalian embryonicstem cells and preimplantati on embryos. Proc Natl Acad Sci USA, 2002,99:2140-2145.
[58] Lai Z, Brady RO. Gene transfer into the central nervous system in vivo using a recombinant lentivirus vector. J Neurosci Res, 2002,67:363-371.
[59] Zufferey R. Self in activating lentivirus vector for safe and efficient in vivo gene delivery. J Virol, 1998, 72:9873-9880.
[60] Yu X, Zhan X, Cheng L, et al. Lentiviral vectors with two independent Internal promoters transfer high level expression of multiple transgenes to human hematopoietic stem progenitor cells. Molecular Therapy, 2003,7:827-838.
[61] Trono D, Lentiviral vectors: turning a deadly foe Into a therapic agent.Gene Ther, 2000,7:20-23.
[62] Steffens S, Tebbets J, Kramm CM, et al. Transduction of human glial and neuronal tumor cells with different lentivirus vector pseudotypes.J Neurooncol.2004,70:281-288.
[63]Iwakuma T,Cui Y,Chang LJ.Self-inactivating lentiviral vectors with U3and U5 modifications.Virology.1999,261(1):120-132.
[64]Kafri T,van Praag H,Gage FH,Verma IM.Lentiviral vectors:regulated gene expression.Mol Ther.2000,1(6):516-521.
[65]Jacque JM,Triques K,Stevenson M.Modulation of HIV-1 replication by RNA interference.Nature.2002,418(6896):435-438.
[66]Crittenden JR,Heidersbach A,McManus MT Lentiviral strategies for RNAi knockdown of neuronal genes.Curt Protoc Neurosci.2007Apr;Chapter 5:Unit 5.26.
[67]Agrawal N,Dasaradhi PV,Mohmmed A,et al.RNA interference:biology,mechanism,and applications.Microbiol Mol Biol Rev.2003,67(4):657-685.
[68]Holen T,Amarzguioui M,Wiiger MT,et al.Positional effects of short Interfering RNAs targetting the human coagulation trigger tissue factor.Nucleic Acid Res.2002,30:1757-1766.
[69]Miyagishi M,Taira K.U6 promoter-driven siRNAs with four uridine 3'Overhangs efficiently suppress targeted gene expression in mammalian cells.Nat Biotechnol.2002,20(5):497-500.
[70]Pedro Gonzalez-Alegre,1 Nicole Bode,1 Beverly L.Davidson,1,2 and Henry L.Paulsonl Silencing Primary Dystonia:Lentiviral-Mediated RNA Interference Therapy for DYT1 Dystonia.J Neurosci.2005.25(45):10502-10509.
[71]Poggeler S,Masloff S,Hoff B,Mayrhofer S,Kuck U.Versatile EGFP Reporter plasmids for cellular localization of recombinant gene products in filamentous fungi.Curr Genet.2003,43(1):54-61.
[72]唐甩恩,周文华,顾钧,等.脊髓鞘内给药改良法-腰骶部埋管及直接注射.上海实验动物科学.2003,23(1):53-54.
[73] Phillips DM. JCAHO pain management standards are unveiled. Joint Commission on Accreditation of ealthcare Organizations. JAMA.2000,284(4):428-429.
[74] Zimmermann TS, Lee ACH, Akinc A, etal. RNAi mediated gene silencing in non-human primates. Nature,2006,441(7089): 111-114.
[75] Fire A, Xu S, Montgomery MK, etal. Potent and specific genetic interference by double-stranded RNA in Caenothabditis elegans.Nature, 1998, 91(6669): 806 -811.
[76] Zhang L,Yang N, Hadley A,et al.Vector-based RNAi, a novel tool for isoform-specific knock-down of VEGF and anti-angiogenesis gene therapy of cancer. Biochem Biophys Res Commun. 2003, 303(4):1169-1178.
[77] Wilda M,FuchsU,WossmannW,etal.Killing of leukemic cells with a BCR/ABL fusion gene by RNA interference(RNAi) .Oneogene.2002,21:5716-5724.
[78] JacqueJM,TriquesK,StevensonM. Modulation of HIV-1 replieation by RNA interference.Nature,2002,418:435-438.
[79] Makinen PI, Koponen JK, Karkkainen AM, et al. Stable RNA interference: comparison of U6 and H1 promoters in endothelial cells and in mouse brain.J Gene Med. 2006,8(4):433-441.
[80] Nash KL, Alexander GJ, Lever AM.Inhibition of hepatitis B virus by Lentiviral vector delivered antisense RNA and hammerhead ribozymes. J Viral Hepat. 2005 ,12(4):346-356.
[81] Naldini L, Blomer U, Gage FH, Trono D, Verma IM. Efficient transfer,integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector.Proc Natl Acad Sci U S A.1996,93(21):11382-11388.
[82] ChangLJ,ZaissAK.Self-inactivating lentiviral vectors and a sensitive Cre-loxp reportersystem.Methods Mol Med,2003,76:367-382.
[83] Wong LF, Goodhead L, Prat C .et al. Lentivirus-mediated gene transfer to the central nervous system: therapeutic And research applications. Hum Gene Ther. 2006 ,17(1):1-9.
[84] ZuffereyR,DullT,MandelRJ,etal.Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery.J Virol, 1998,72:9873-9880.
[85] Jakobsson J,Georgievska B,Ericson C,etal.Lesion-dependent regulation of transgene expression in the rat brain using a human glial fibrillary acidicprotein-lentiviralvector.Eur J Neurosci. 2004,19(3):761-765.
1. Catterall WA. From ionic currents to molecular mechanisms: the structure and function of voltage gated sodium channels.Neuron,2000,26(1):13-25.
2. Black A, Dib-Hajj S, McNabola K, et al. Spinal sensory neurons express multiple sodium channel a subunit mRNAs. Brain Res Mol Brain Res ,1996,43:117-131.
3. Djouhri L, Newton R, Levinson SR, et al. Sensory and electrophysiological properties of guinea-pig sensory neurons expressing Nav 1.7 (PN1) Na~+ channel alpha subunit protein J Physiol, 2003, 546:565-576.
4. Cummins TR, Howe JR, Waxman SG. Slow closed-state inactivation: a novel mechanism underlying ramp currents in cells expressing the hNE/PNl sodium channel.J Neurosci, 1998,18: 9607-9619.
5. Yang Y,Wang Y, Li S ,et al, Mutations in SCN9A, encoding a sodium channel alpha subunit, in patients with primary erythermalgia. J Med Genet, 2004, 41:171-174.
6. Dib-Hajj SD, Rush AM, Cummins TR, et al Gain-of-function mutation in Na_v1.7 in familial erythromelalgia induces bursting of sensory neurons .Brain,2005, 128:1847-1854.
7. Han C, Rush AM, Dib-Hajj SD, et al, Sporadic onset of erythermalgia: a gain- of- function mutation in Na(v)1.7. Ann Neurol,2006, 59: 553-558.
8. Cummins TR, Dib-Haj SD, Waxman SG, Electrophysiological properties of mutant Na_v1.7 sodium channels in a painful inherited neuropathy, J Neurosci 2004,24: 8232-8236.
9. Harty TP, Dib-Hajj SD, Tyrrell L,et al. Na(V)1.7 mutant A863P in erythromelalgia: effects of altered activation and steady-state inactivation on excitability of nociceptive dorsal root ganglion neurons, J Neurosci ,2006:26 12566-12575.
10. Fertleman CR, Baker MD, Parker KA, et al SCN9A mutations in paroxysmal extreme pain disorder: allelic variants underlie distinct channel defects and phenotypes[J]. Neuron ,2006, 52 : 767-774.
11. Hong S, Morrow TJ, Paulson PE, et al. Early painful diabetic neuropathy is associated with differential changes in tetrodotoxin-sensitive and -resistant sodium channels in dorsal root ganglion neurons in the rat[J].J Biol Chem ,2004,279: 29341-29350
12. Black JA, Liu S, Tanaka M,et al. Changes in the expression of tetrodotoxin -sensitive sodium channels within dorsal root ganglia neurons in inflammatory pain[J]. Pain , 2004, 108: 237-247.
13. Yeomans DC, Levinson SR, Peters MC, et al. Decrease in inflammatory hyperalgesia by herpes vector-mediated knockdown of Na_v1.7 sodium channels in primary afferents[J].Hum Gene Ther, 2005,16:271-277.
14. Nassar MA, Stirling LC, Forlani G, et al. Nociceptor-specific gene deletion reveals a major role for Na_v1.7 (PN1) in acute and inflammatory pain[J]. Proc NatlAcadSci USA , 2004,101: 12706-12711.
15. Nassar MA, ALevato, Stirling LC, et al. Neuropathic pain develops normally in mice lacking both Na_vl.7 and Na_v1.8[J]. Mol Pain,2005, 1: 24.
16. Goldberg Y, Macfarlane J, Macdonald M, et al.Loss-of-function mutations in the Na(v)1.7 gene underlie congenital indifference to pain in multiple human populations[J].Clin Genet, 2007, 71:311-319.
17. Goldin AL,Barchi RL,Caldwell JH,et al. Nomenclature of voltage-gated sodium channels[J]. Neuron ,2000,28(2):365-368.
18. Akopian AN, Souslova V, England S, et al., The tetrodotoxin-resistant sodium channel SNS has a specialized function in pain pathways[J]. Nat Neumsci.1999,2: 541-548.
19. Joshi SK, Mikusa JP, Hernandez G, et al., Involvement of the TTX-resistant sodium channel Na_v1.8 in inflammatory and neuropathic, but not post-operative, pain state[J]. Pain . 2006,123:75-82.
20. Abe M, KuriharaT,HanW,etal.Changes in expression of voltage-dependent ion channel subunit in dorsal root ganglia of rat with radicular injury and pain[J].Spine.2002,27(l):1517-1524.
21. Lai J, Hunter JC, Ossipov MH, et al. Blockade of neuropathic pain by antisense targeting of etrodotoxin-resistantsodium channels in sensory neurons[J].Methods Enzymol. 2000,314: 201-213.
22. Yiangou Y, Birch R, SangameWalkeraran L, et al. SNS/PN3 and SNS2/NaN sodium channel-like immunoreactivity in human adult and neonate injured sensory nerves .FEBS Lett, 2000, 467(2-3):249-252.
23. Roza C, Laird JM, Souslova V,et al. The tetrodotoxin-resistant Na+ channel Na_v1.8 is essential for the expression of spontaneous activity in damaged sensory axons of mice[J]. J Physiol, 2003, 550:921-926.
24. Dong XW, Goregoaker S, Engler H, et al. Small interfering RNA-mediated selective knockdown of Na(V)1.8 tetrodotoxin-resistant sodium channel reverses mechanical allodynia in neuropathic rats[J].Neuroscience,2007,146:812-821.
25. Nassar MA, Levato A, Stirling LC,et al. Wood, Neuropathic pain develops normally in mice lacking both Na_v1.7 and Na_v1.8[J].Mol Pain, 2005, 1: 24.
26. Ekberg J, Jayamanne A, Vaughan CW, et al. muO-conotoxin MrVIB selectively blocks Na_v1.8 sensory neuron specific sodium channels and chronic pain behavior without motor deficits[J]. Proc Natl Acad Sci USA,2006,103:17030-17035.
27. Jarvis MF, Honore P, Shieh CC, et al.A-803467, a potent and selective Na_v1.8 sodium channel blocker, attenuates neuropathic and inflammatory pain in the rat[J].Proc Natl Acad Sci USA, 2007,104(20):8520-8525.
28. Herzog RI, Cummins TR , Waxman SG. Persistent TTX-resistant Na+ current affects resting potential and response to depolarization in simulated spinal sensory neurons[J].J Neurophysiol, 2001, 86:1351-1364.
29. Priest BT, Murphy BA, Lindia JA, et al Contribution of the tetrodotoxin-resistant voltage-gated sodium channel NaV1.9 to sensory transmission and nociceptive behavior [J].Proc Natl Acad Sci USA, 2005, 102: 9382-9387.
30. Amaya F, Wang H, Costigan M, et al.The voltage-gated sodium channel Na(v)1.9 is an effector of peripheral inflammatory pain hypersensitivity[J]. J Neurosci,2006,26: 12852-12860.
31. Blum R, Kafitz KW, Konnerth . Neurotrophin-evoked depolarization requires the sodium channel Na(V)1.9[J] Nature,2002,419:687-693.
32. Davis JB, Gray J,Gunthorpe MJ, et al. Vanilloid receptor-1 is essential for inflamma-tory thermal hyperalgesia[J]. Nature,2000,405(6783): 183-187.
33. Hong S, Wiley JW. Early painful diabetic neuropathy is associated with differential changes in the expression and function of vanilloid receptor 1 [J].J Biol Chem, 2005,280: 618-627.
34. Christoph T, Grunweller A, Mika J,et al. Silencing of vanilloid receptor TRPVl by RNAi reduces neuropathic and visceral pain in vivo[J].Biochem Biophys Res Commun,2006, 350:238-243.
35. Bolcskei K, Helyes Z, Szabo A,et al. Investigation of the role of TRPV1 receptors in acute and chronic nociceptive processes using gene-deficient mice[J]. Pain, 2005,117:368-376.
36. Christoph T, Gillen C, Mika J,et al. Antinociceptive effect of antisense oligonucleotides against the vanilloid receptor VR1/TRPV1[J]. Neurochem.Int. 2007,50(1):281-90.
37. Sterner O, Szallasi A. Novel natural vanilloid receptor agonists: new therapeutic targets for drug development J].Trends Pharmacol Sci,1999;20(11):459-465.
38. Urban I, Campbell EA, Panesar M, et al. In vivo Pharmacology of SDZ 249-665, a novel,non-pungent capsaicin analogue[J]. Pain,2000,89(1):65-74.
39. Peng CH, Kuo HC.Multiple intravesical instillations of low-dose resiniferatoxin in the treatment of refractory interstitial cystitis[J].Urol Int,2007;78(1):78-81.
40. Krause JE, Chenard BL ,Cortright DN. Transient receptor potential ion channels as targets for the discovery of pain therapeutics[J]. Curr Opin Investig. Drugs, 2005, 6:48-57.
41. Christoph T, Grunweller A, Mika J, et al. Silencing of vanilloid receptor TRPV1 by RNAi reduces neuropathic and visceral pain in vivo[J]. Biochem Biophys Res Commun,2006, 350: 238-243.
42. Christoph T, Gillen C, Mika J, et al. Antinociceptive effect of antisense oligonucleotides against the vanilloid receptor VR1/TRPV1[J]. Neurochem Int,2007,50: 281-290.
43. Story GM, Peier AM, Reeve AJ, et al. ANKTM1, a TRP like channel expressed in nociceptive neuron, is activated by cold temperature[J]. Cell,2003,112:819-829.
44. Kobayashi K, Fukuoka T, Obata K, Yamanaka H, Dai Y, Tokunaga A and Noguchi K. Distinct expression of TRPM8, TRPA1, and TRPV1 mRNAs in rat primary afferent neurons with adelta/c-fibers and colocalization with trk receptors[J]. J. Comp. Neurol, 2005,493:596-606.
45. Obata H, Katsura T, Mizushima H, Yamanaka K, Kobayashi Y, Dai T,Fukuoka A, Tokunaga M, Tominaga and K. Noguchi, TRPA1 induced in sensory neurons contributes to cold hyperalgesia after inflammation and nerve injury[J]. J. Clin. Invest,2005, 115: 2393-2401.
46. Katsura H, Obata K, Mizushima T, et al. Antisense knock down of TRPA1, but not TRPM8, alleviates cold hyperalgesia after spinal nerve ligation in rats[J].Exp. Neurol. 2006,200: 112-123.
47. Mckemy DD, Neuhausser WM, Julius D. Identification of a cold receptor reveals a general role for TRP channels in thermosensation[J]. Nature,2002,416:52-58.
48. Proudfoot CJ, Garry EM, Cottrell DF, et al. Analgesia mediated by the TRPM8 cold receptor in chronic neuropathic pain[J]. Curr. Biol, 2006,16:1591-1605.
49. Tsuzuki K, A Ase, Seguela P, et al. TNP ATP resistant P_2X ionic currents on the central terminals and somata of rat primary sensory neurons[J]. J Neurophysiol, 2003,89(6):3235- 3242.
50. SouslovaV,CesareP,DingY,etal.Warm-codingdeficitsandaberrantinflammatory paininmicelackingP2X3receptors[J].Nature,2000,407(6807): 1015-1017.
51. Dorn G, Patel S, Wotherspoon G, et al. siRNA relieves chronic neuropathic pain [J].Nucleic Acids Res, 2004; 32(5):e49.
52. McGaraughty S,Jarvis MF. Antinociceptive properties of a non-nucleotide P2X3/ P2X2/3 receptor antagonist[J].DrugNewsPerspect,2005,18(8):501-507.
[2]Schwei MJ,Honore P,Rogers SD,et al.Neurochemical and cellular recorganization of the spinal cord in a marine model of bone cancer pain.J Neurosci.1999,19:10886-10897.
[3]Medhurst S J,Walker K,Bowes M,et al.A rat model of bone cancer pain.Pain.2002,96:129-140.
[4]Liu XJ,Salter MW.Purines and pain mechanisms:recent.Curr Opin Investig Drugs.2005,6(1):65-75.
[5]Bumstock G.Release of vasoactive substances from endothelial cells by shear stress and purinergic mechano-sensory transduction.J Anatomy.1999,194:335-342.
[6]邓伦斌,姚磊,韩济生.神经源性痛诱发大鼠背根神经节细胞电生理学变化.针刺研究.2001,26(3):228.
[7]Chen CC,Akopian AN,Sivilotti L,et al.A P2X purinoceptor expressed by a subset of sensory neurons.Nature.1995,377(6548):428-31.
[8]Bradbury E J,Burnstock G,McMahon SB.The expression of P2X_3purinoreceptors in sensory neurons:effects of axotomy and glial-derived neurotrophic factor.Mol Cell Neurosci.1998,12(4-5):256-68.
[9]Tsuda M,Koizumi S,Kita A,et al.Mechanical allodynia caused by intraplantar injection of P2X receptor agonist in rats:involvement of heteromeric P2X2/3 receptor signaling in capsaicin-insensitive primary afferent neurons.J Neurosci.2000,20:RC90,1-5.
[10]Cockayne DA,Hamilton SG,Zhu QM,et al.Urinary bladder hyporeflexia and reduced pain-related behaviour in P2X_3-deficient mice.Nature.2000,407:1011-1015.
[11] Oliveira MC, Parada CA, Veiga MC,et al. Evidence for the involvement of endogenous ATP and P2X receptors in TMJ pain. Eur J Pain.2005,9(1):87-93.
[12] McGaraughty S, Jarvis MF. Antinociceptive properties of a non-nucleotide P2X3 /P2X2/3 receptor antagonist.Drug News Perspect. 2005 , 18(8):501-507.
[13] Tomari Y, Zamore PD. Perspective: machines for RNAi. Genes Dev.2005,19(5):517-29.
[14] Paddison PJ, Caudy AA, Bernstein E, et al. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes & Dev.2002,16:948-958.
[15] Sontheimer EJ ,Carthew RW. Silence from within : endogenous siRNAs and miRNAs. Cell.2005 ,122(1) :9 - 12.
[16] Gu Y, Xu Y, Li GW,et al.Remote nerve injection of mu opioid receptor adeno-associated viral vector increases antinociception of intrathecal morphine. J Pain. 2005,6(7):447-54.
[17] Prasad M, Fearon IM, Zhang M,et al.Expression of P2X2 and P2X3 receptor subunits in rat carotid body afferent neurones: role in chemo-sensory signalling.J Physiol. 2001 Dec 15,537(Pt 3):667-77.
[18] Dornelas CA, Almeida PR, Nascimento GL, et al. Experimental model of Walker 256 carcinosarcoma in rats bladder. Acta Cir Bras. 2006, 21(1):38-42.
[19] Buffon A, Ribeiro VB, Schanoski AS, et al. Diminution in adenine nucleotide hydrolysis by platelets and serum from rats submitted to Walker 256 tumour. Mol Cell Biochem. 2006,281(1-2):189-195.
[20] Miyazaki M, Shimoda T, Itoh H, et al. Enhancement of cytotoxicity of doxorubicin by verapamil in the hepatic artery infusion for liver tumors in rats. Cancer. 1993,72(2):349-354.
[21] Luo C,Jiang Y,Liu Y,et al. Experimental study on mechanism and rarity of metastases in skeletal muscle.Chin Med J(Engl).2002,115:1645-1649.
[22]Muta M,Matsumoto G,Nakashima E,et al.Mechanical analysis of tumor growth regression by the cyclooxygenase-2 inhibitor,DFU,in a Walker256rat tumor model:importance of monocyte chemoattractant protein-1modulation.Clin Cancer Res.2006,12(1):264-272
[23]Kostenuik P J,Orr FW,Suyama K,et al.Increased growth rate and tumor burden of spontaneously metastatic Walker 256 cancer cells in the skeleton of bisphosphonate-treated rats.Cancer Res.1993,53(22):5452-5457.
[24]杜恒,崔刚,张增铁等.氯化镓对Walket256癌骨侵袭的抑制作用.西安交通大学学报(医学版),2004,25(4):369-371.
[25]刘艳红,张宏,徐龙河.大鼠骨癌痛模型的制备及电压依赖性钠通道Na1.8在其背根神经节的表达研究.解放军医学杂志,2007,32(4)319-322.
[26]Manthyh PW,Clohisy DR,Koltzenburg M,et al.Molecular mechanisms of cancer pain.Nature Rev(cancer).2002,2(3):201-209.
[27]Martin W J,Liu H,Wang H,et al.Inflammation-induce up-regulation of protein kinase Cgamma immunoreactivity in rat spinal cord correlates with enhanced nociceptive processing.Neuroscience.1999,88(4):1267-74.
[28]Calza L,Pozza M,Zanni M,et al.Peptide lasticity in primary sensory neurons and spinal cord during djuvant-induced arthritis in the rat:an immunocytochemical and in situ hybridization study.Neuroscience.1998Jan,82(2):575-89.
[29]Simonet WS,Lacey DL,Dunstan CR,et al.Osteoprotegerin:a novel secreted protein involved in the regulation of bone density.Cell.1997,89(2):309-319.
[30]Kong YY,Yoshida H,Sarosi I,et al.OPGL is a key regulator of osteoclastogenesis,lymphocyte development and lymphnode organo -genesis.Nature.1999,397(6717):315-323.
[31] Luger NM, Honore P, Sabino MA, et al. Osteoprotegerin diminishes advanced bone cancer pain. Cancer Res. 2001,61(10):4038-4047.
[32] Bekker PJ, Holloway D, Nakanishi A,etal. The effect of a single dose of osteoprotegerin in postmenopausal women. J Bone Miner Res. 2001,16(2):348-360.
[33] Coleman RE.How can we improve the treatment of bone metastases further? Curr Opin Oncol. 1998,10( Suppl 1):S7-13.
[34] Toyras A, Ollikainen J, Taskinen M, et al. Inhibition of mevalonate pathway is involved in alendronate-induced cell growth inhibition, but not in cytokine secretion from macrophages in vitro.Eur J Pharm Sci. 2003 ,19(4): 223-230.
[35] Sevcik MA, Luger NM, Mach DB, et al . Bone cancer pain: the effects of the bisphosphonate alendronate on pain, skeletal remodeling, tumor growth and tumor necrosis.Pain. 2004 ,111(1-2): 169-180.
[36] Peters CM, Lindsay TH, Pomonis JD, et al . Endothelin and the tumorigenic component of bone cancer pain. Neuroscience. 2004,126(4): 1043-1052.
[37] Sabino MA, Ghilardi JR, Jongen JL,et al. Simultaneous reduction in cancer pain, bone destruction, and tumor growth by selective inhibition of cyclooxygenase-2.Cancer Res. 2002,62(24):7343-7349.
[38] Li X, Tomita M, Pilbeam CC,et al .Prostaglandin receptor EP2 mediates PGE2 stimulated hypercalcemia in mice in ivo.Prostaglandins Other Lipid Mediat. 2002,67(3-4):173-180.
[39] Honore P, Rogers SD, Schwei MJ, et al.Murine models of inflammatory neuropathic and cancer pain each generates a unique set of neurochemical changes in the spinal cord and sensorv neurons. Neuroscience, 2000, 98:585-598.
[40] Wacnick PW, Eikmeier TJ, Ruggles TR, et al. Functional interactions between tumor and peripheral nerve: morphology, algogen identification, and behavioral characterization of a new murive model of cancer pain.J Neurosci,2001,21:9355-9366.
[41]Colburn RW,DeLeoJA,Rickman AJ,etal.Dissociation of microglial activation and neuropathic pain behaviors following peripheral nerve injury in the rat.J Neuroimmunol,1997,79:163-175.
[42]Schwei MJ,Honore P,Rogers SD,et al.Neurochemical and cellular reorganization of the spinal cord in a murine model of bone cancer pain.J Neurosci,1999,19:10886-10897.
[43]Xu GY,Huang LY.Peripheral inflammation sensitizes P2X receptor -mediated responses in rat dorsal root ganglion neurons.J Neurosci.2002,22(1):93-102.
[44]Wildman SS,King BF,Burnstock G.Potentiation of ATP-responses at a recombinant P2X2 receptor by neurotransmitters and related substances.Br J Pharmacol.1997,120:221-224.
[45]Prostaglandin E2 potentiation of P2X3 receptor mediated currents in dorsal root ganglion neurons Molecular Pain 2007,3:22.
[46]赵士洁,殷光甫,谷远征,等.中国组织化学与细胞化学杂志,2006.15:91-95.
[47]Fire A,Xu S,Montgomery MK,et al.Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.Nature.1998,391(6669):806-811.
[48]孟国良,汤富酬,张俊争.小鼠胚胎干细胞中RNA干涉现象.生物化学与生物物理学报,2003,35(3):238-246.
[49]雷迎峰,薛小平,尹文.RNAi研究进展.国外医学(分子生物学分册),2002,24(4):196-199.
[50]Brummelkamp TR,Bernards R,Agami R.Stable suppression of tumorigenicity by virus-mediated RNA interference.Cancer Cell.2002,2(3):243-247.
[51]Elbashir SM,Harborth J,Lendeckel W,et al.Duplexes of 21- nucleotide RNAs mediate RNA interference in cultured mammalian cells.Nature.2001,411(6836):494-498.
[52] Lin SL, Chuong CM, Ying SY A Novel mRNA-cDNA interference Phenomenon for silencing bcl-2 expression in human LNCaP cells.Biochem Biophys Res Commun. 2001,281 (3):639-644.
[53] Scherr M, Morgan MA, Eder M. Gene silencing mediated by small interfering RNAs in mammalian cells.Curr Med Chem. 2003, 10(3) :245-256.
[54] Amarzguioui M, Rossi JJ, Kim D.Approaches for chemically synthesized siRNA and vector-mediated RNAi.FEBS Lett. 2005,579(26):5974-5981.
[55] Kitabwalla M, Ruprecht RM. RNA interference~a new weapon against HIV and beyond. N Engl J Med. 2002 ,24,347(17):1364-1367.
[56] Lois C, Hong EJ, Pease S, et al. Germline transmission and tissue specific expression of transgenes delivered by lentiviral vectors. Science, 2002,295:868-872.
[57] Pfeifer A, Ikawa M, Dayn Y, etal. Transgenes is by lentiviral vectors: Lack Of gene silencing in mammalian embryonicstem cells and preimplantati on embryos. Proc Natl Acad Sci USA, 2002,99:2140-2145.
[58] Lai Z, Brady RO. Gene transfer into the central nervous system in vivo using a recombinant lentivirus vector. J Neurosci Res, 2002,67:363-371.
[59] Zufferey R. Self in activating lentivirus vector for safe and efficient in vivo gene delivery. J Virol, 1998, 72:9873-9880.
[60] Yu X, Zhan X, Cheng L, et al. Lentiviral vectors with two independent Internal promoters transfer high level expression of multiple transgenes to human hematopoietic stem progenitor cells. Molecular Therapy, 2003,7:827-838.
[61] Trono D, Lentiviral vectors: turning a deadly foe Into a therapic agent.Gene Ther, 2000,7:20-23.
[62] Steffens S, Tebbets J, Kramm CM, et al. Transduction of human glial and neuronal tumor cells with different lentivirus vector pseudotypes.J Neurooncol.2004,70:281-288.
[63]Iwakuma T,Cui Y,Chang LJ.Self-inactivating lentiviral vectors with U3and U5 modifications.Virology.1999,261(1):120-132.
[64]Kafri T,van Praag H,Gage FH,Verma IM.Lentiviral vectors:regulated gene expression.Mol Ther.2000,1(6):516-521.
[65]Jacque JM,Triques K,Stevenson M.Modulation of HIV-1 replication by RNA interference.Nature.2002,418(6896):435-438.
[66]Crittenden JR,Heidersbach A,McManus MT Lentiviral strategies for RNAi knockdown of neuronal genes.Curt Protoc Neurosci.2007Apr;Chapter 5:Unit 5.26.
[67]Agrawal N,Dasaradhi PV,Mohmmed A,et al.RNA interference:biology,mechanism,and applications.Microbiol Mol Biol Rev.2003,67(4):657-685.
[68]Holen T,Amarzguioui M,Wiiger MT,et al.Positional effects of short Interfering RNAs targetting the human coagulation trigger tissue factor.Nucleic Acid Res.2002,30:1757-1766.
[69]Miyagishi M,Taira K.U6 promoter-driven siRNAs with four uridine 3'Overhangs efficiently suppress targeted gene expression in mammalian cells.Nat Biotechnol.2002,20(5):497-500.
[70]Pedro Gonzalez-Alegre,1 Nicole Bode,1 Beverly L.Davidson,1,2 and Henry L.Paulsonl Silencing Primary Dystonia:Lentiviral-Mediated RNA Interference Therapy for DYT1 Dystonia.J Neurosci.2005.25(45):10502-10509.
[71]Poggeler S,Masloff S,Hoff B,Mayrhofer S,Kuck U.Versatile EGFP Reporter plasmids for cellular localization of recombinant gene products in filamentous fungi.Curr Genet.2003,43(1):54-61.
[72]唐甩恩,周文华,顾钧,等.脊髓鞘内给药改良法-腰骶部埋管及直接注射.上海实验动物科学.2003,23(1):53-54.
[73] Phillips DM. JCAHO pain management standards are unveiled. Joint Commission on Accreditation of ealthcare Organizations. JAMA.2000,284(4):428-429.
[74] Zimmermann TS, Lee ACH, Akinc A, etal. RNAi mediated gene silencing in non-human primates. Nature,2006,441(7089): 111-114.
[75] Fire A, Xu S, Montgomery MK, etal. Potent and specific genetic interference by double-stranded RNA in Caenothabditis elegans.Nature, 1998, 91(6669): 806 -811.
[76] Zhang L,Yang N, Hadley A,et al.Vector-based RNAi, a novel tool for isoform-specific knock-down of VEGF and anti-angiogenesis gene therapy of cancer. Biochem Biophys Res Commun. 2003, 303(4):1169-1178.
[77] Wilda M,FuchsU,WossmannW,etal.Killing of leukemic cells with a BCR/ABL fusion gene by RNA interference(RNAi) .Oneogene.2002,21:5716-5724.
[78] JacqueJM,TriquesK,StevensonM. Modulation of HIV-1 replieation by RNA interference.Nature,2002,418:435-438.
[79] Makinen PI, Koponen JK, Karkkainen AM, et al. Stable RNA interference: comparison of U6 and H1 promoters in endothelial cells and in mouse brain.J Gene Med. 2006,8(4):433-441.
[80] Nash KL, Alexander GJ, Lever AM.Inhibition of hepatitis B virus by Lentiviral vector delivered antisense RNA and hammerhead ribozymes. J Viral Hepat. 2005 ,12(4):346-356.
[81] Naldini L, Blomer U, Gage FH, Trono D, Verma IM. Efficient transfer,integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector.Proc Natl Acad Sci U S A.1996,93(21):11382-11388.
[82] ChangLJ,ZaissAK.Self-inactivating lentiviral vectors and a sensitive Cre-loxp reportersystem.Methods Mol Med,2003,76:367-382.
[83] Wong LF, Goodhead L, Prat C .et al. Lentivirus-mediated gene transfer to the central nervous system: therapeutic And research applications. Hum Gene Ther. 2006 ,17(1):1-9.
[84] ZuffereyR,DullT,MandelRJ,etal.Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery.J Virol, 1998,72:9873-9880.
[85] Jakobsson J,Georgievska B,Ericson C,etal.Lesion-dependent regulation of transgene expression in the rat brain using a human glial fibrillary acidicprotein-lentiviralvector.Eur J Neurosci. 2004,19(3):761-765.
1. Catterall WA. From ionic currents to molecular mechanisms: the structure and function of voltage gated sodium channels.Neuron,2000,26(1):13-25.
2. Black A, Dib-Hajj S, McNabola K, et al. Spinal sensory neurons express multiple sodium channel a subunit mRNAs. Brain Res Mol Brain Res ,1996,43:117-131.
3. Djouhri L, Newton R, Levinson SR, et al. Sensory and electrophysiological properties of guinea-pig sensory neurons expressing Nav 1.7 (PN1) Na~+ channel alpha subunit protein J Physiol, 2003, 546:565-576.
4. Cummins TR, Howe JR, Waxman SG. Slow closed-state inactivation: a novel mechanism underlying ramp currents in cells expressing the hNE/PNl sodium channel.J Neurosci, 1998,18: 9607-9619.
5. Yang Y,Wang Y, Li S ,et al, Mutations in SCN9A, encoding a sodium channel alpha subunit, in patients with primary erythermalgia. J Med Genet, 2004, 41:171-174.
6. Dib-Hajj SD, Rush AM, Cummins TR, et al Gain-of-function mutation in Na_v1.7 in familial erythromelalgia induces bursting of sensory neurons .Brain,2005, 128:1847-1854.
7. Han C, Rush AM, Dib-Hajj SD, et al, Sporadic onset of erythermalgia: a gain- of- function mutation in Na(v)1.7. Ann Neurol,2006, 59: 553-558.
8. Cummins TR, Dib-Haj SD, Waxman SG, Electrophysiological properties of mutant Na_v1.7 sodium channels in a painful inherited neuropathy, J Neurosci 2004,24: 8232-8236.
9. Harty TP, Dib-Hajj SD, Tyrrell L,et al. Na(V)1.7 mutant A863P in erythromelalgia: effects of altered activation and steady-state inactivation on excitability of nociceptive dorsal root ganglion neurons, J Neurosci ,2006:26 12566-12575.
10. Fertleman CR, Baker MD, Parker KA, et al SCN9A mutations in paroxysmal extreme pain disorder: allelic variants underlie distinct channel defects and phenotypes[J]. Neuron ,2006, 52 : 767-774.
11. Hong S, Morrow TJ, Paulson PE, et al. Early painful diabetic neuropathy is associated with differential changes in tetrodotoxin-sensitive and -resistant sodium channels in dorsal root ganglion neurons in the rat[J].J Biol Chem ,2004,279: 29341-29350
12. Black JA, Liu S, Tanaka M,et al. Changes in the expression of tetrodotoxin -sensitive sodium channels within dorsal root ganglia neurons in inflammatory pain[J]. Pain , 2004, 108: 237-247.
13. Yeomans DC, Levinson SR, Peters MC, et al. Decrease in inflammatory hyperalgesia by herpes vector-mediated knockdown of Na_v1.7 sodium channels in primary afferents[J].Hum Gene Ther, 2005,16:271-277.
14. Nassar MA, Stirling LC, Forlani G, et al. Nociceptor-specific gene deletion reveals a major role for Na_v1.7 (PN1) in acute and inflammatory pain[J]. Proc NatlAcadSci USA , 2004,101: 12706-12711.
15. Nassar MA, ALevato, Stirling LC, et al. Neuropathic pain develops normally in mice lacking both Na_vl.7 and Na_v1.8[J]. Mol Pain,2005, 1: 24.
16. Goldberg Y, Macfarlane J, Macdonald M, et al.Loss-of-function mutations in the Na(v)1.7 gene underlie congenital indifference to pain in multiple human populations[J].Clin Genet, 2007, 71:311-319.
17. Goldin AL,Barchi RL,Caldwell JH,et al. Nomenclature of voltage-gated sodium channels[J]. Neuron ,2000,28(2):365-368.
18. Akopian AN, Souslova V, England S, et al., The tetrodotoxin-resistant sodium channel SNS has a specialized function in pain pathways[J]. Nat Neumsci.1999,2: 541-548.
19. Joshi SK, Mikusa JP, Hernandez G, et al., Involvement of the TTX-resistant sodium channel Na_v1.8 in inflammatory and neuropathic, but not post-operative, pain state[J]. Pain . 2006,123:75-82.
20. Abe M, KuriharaT,HanW,etal.Changes in expression of voltage-dependent ion channel subunit in dorsal root ganglia of rat with radicular injury and pain[J].Spine.2002,27(l):1517-1524.
21. Lai J, Hunter JC, Ossipov MH, et al. Blockade of neuropathic pain by antisense targeting of etrodotoxin-resistantsodium channels in sensory neurons[J].Methods Enzymol. 2000,314: 201-213.
22. Yiangou Y, Birch R, SangameWalkeraran L, et al. SNS/PN3 and SNS2/NaN sodium channel-like immunoreactivity in human adult and neonate injured sensory nerves .FEBS Lett, 2000, 467(2-3):249-252.
23. Roza C, Laird JM, Souslova V,et al. The tetrodotoxin-resistant Na+ channel Na_v1.8 is essential for the expression of spontaneous activity in damaged sensory axons of mice[J]. J Physiol, 2003, 550:921-926.
24. Dong XW, Goregoaker S, Engler H, et al. Small interfering RNA-mediated selective knockdown of Na(V)1.8 tetrodotoxin-resistant sodium channel reverses mechanical allodynia in neuropathic rats[J].Neuroscience,2007,146:812-821.
25. Nassar MA, Levato A, Stirling LC,et al. Wood, Neuropathic pain develops normally in mice lacking both Na_v1.7 and Na_v1.8[J].Mol Pain, 2005, 1: 24.
26. Ekberg J, Jayamanne A, Vaughan CW, et al. muO-conotoxin MrVIB selectively blocks Na_v1.8 sensory neuron specific sodium channels and chronic pain behavior without motor deficits[J]. Proc Natl Acad Sci USA,2006,103:17030-17035.
27. Jarvis MF, Honore P, Shieh CC, et al.A-803467, a potent and selective Na_v1.8 sodium channel blocker, attenuates neuropathic and inflammatory pain in the rat[J].Proc Natl Acad Sci USA, 2007,104(20):8520-8525.
28. Herzog RI, Cummins TR , Waxman SG. Persistent TTX-resistant Na+ current affects resting potential and response to depolarization in simulated spinal sensory neurons[J].J Neurophysiol, 2001, 86:1351-1364.
29. Priest BT, Murphy BA, Lindia JA, et al Contribution of the tetrodotoxin-resistant voltage-gated sodium channel NaV1.9 to sensory transmission and nociceptive behavior [J].Proc Natl Acad Sci USA, 2005, 102: 9382-9387.
30. Amaya F, Wang H, Costigan M, et al.The voltage-gated sodium channel Na(v)1.9 is an effector of peripheral inflammatory pain hypersensitivity[J]. J Neurosci,2006,26: 12852-12860.
31. Blum R, Kafitz KW, Konnerth . Neurotrophin-evoked depolarization requires the sodium channel Na(V)1.9[J] Nature,2002,419:687-693.
32. Davis JB, Gray J,Gunthorpe MJ, et al. Vanilloid receptor-1 is essential for inflamma-tory thermal hyperalgesia[J]. Nature,2000,405(6783): 183-187.
33. Hong S, Wiley JW. Early painful diabetic neuropathy is associated with differential changes in the expression and function of vanilloid receptor 1 [J].J Biol Chem, 2005,280: 618-627.
34. Christoph T, Grunweller A, Mika J,et al. Silencing of vanilloid receptor TRPVl by RNAi reduces neuropathic and visceral pain in vivo[J].Biochem Biophys Res Commun,2006, 350:238-243.
35. Bolcskei K, Helyes Z, Szabo A,et al. Investigation of the role of TRPV1 receptors in acute and chronic nociceptive processes using gene-deficient mice[J]. Pain, 2005,117:368-376.
36. Christoph T, Gillen C, Mika J,et al. Antinociceptive effect of antisense oligonucleotides against the vanilloid receptor VR1/TRPV1[J]. Neurochem.Int. 2007,50(1):281-90.
37. Sterner O, Szallasi A. Novel natural vanilloid receptor agonists: new therapeutic targets for drug development J].Trends Pharmacol Sci,1999;20(11):459-465.
38. Urban I, Campbell EA, Panesar M, et al. In vivo Pharmacology of SDZ 249-665, a novel,non-pungent capsaicin analogue[J]. Pain,2000,89(1):65-74.
39. Peng CH, Kuo HC.Multiple intravesical instillations of low-dose resiniferatoxin in the treatment of refractory interstitial cystitis[J].Urol Int,2007;78(1):78-81.
40. Krause JE, Chenard BL ,Cortright DN. Transient receptor potential ion channels as targets for the discovery of pain therapeutics[J]. Curr Opin Investig. Drugs, 2005, 6:48-57.
41. Christoph T, Grunweller A, Mika J, et al. Silencing of vanilloid receptor TRPV1 by RNAi reduces neuropathic and visceral pain in vivo[J]. Biochem Biophys Res Commun,2006, 350: 238-243.
42. Christoph T, Gillen C, Mika J, et al. Antinociceptive effect of antisense oligonucleotides against the vanilloid receptor VR1/TRPV1[J]. Neurochem Int,2007,50: 281-290.
43. Story GM, Peier AM, Reeve AJ, et al. ANKTM1, a TRP like channel expressed in nociceptive neuron, is activated by cold temperature[J]. Cell,2003,112:819-829.
44. Kobayashi K, Fukuoka T, Obata K, Yamanaka H, Dai Y, Tokunaga A and Noguchi K. Distinct expression of TRPM8, TRPA1, and TRPV1 mRNAs in rat primary afferent neurons with adelta/c-fibers and colocalization with trk receptors[J]. J. Comp. Neurol, 2005,493:596-606.
45. Obata H, Katsura T, Mizushima H, Yamanaka K, Kobayashi Y, Dai T,Fukuoka A, Tokunaga M, Tominaga and K. Noguchi, TRPA1 induced in sensory neurons contributes to cold hyperalgesia after inflammation and nerve injury[J]. J. Clin. Invest,2005, 115: 2393-2401.
46. Katsura H, Obata K, Mizushima T, et al. Antisense knock down of TRPA1, but not TRPM8, alleviates cold hyperalgesia after spinal nerve ligation in rats[J].Exp. Neurol. 2006,200: 112-123.
47. Mckemy DD, Neuhausser WM, Julius D. Identification of a cold receptor reveals a general role for TRP channels in thermosensation[J]. Nature,2002,416:52-58.
48. Proudfoot CJ, Garry EM, Cottrell DF, et al. Analgesia mediated by the TRPM8 cold receptor in chronic neuropathic pain[J]. Curr. Biol, 2006,16:1591-1605.
49. Tsuzuki K, A Ase, Seguela P, et al. TNP ATP resistant P_2X ionic currents on the central terminals and somata of rat primary sensory neurons[J]. J Neurophysiol, 2003,89(6):3235- 3242.
50. SouslovaV,CesareP,DingY,etal.Warm-codingdeficitsandaberrantinflammatory paininmicelackingP2X3receptors[J].Nature,2000,407(6807): 1015-1017.
51. Dorn G, Patel S, Wotherspoon G, et al. siRNA relieves chronic neuropathic pain [J].Nucleic Acids Res, 2004; 32(5):e49.
52. McGaraughty S,Jarvis MF. Antinociceptive properties of a non-nucleotide P2X3/ P2X2/3 receptor antagonist[J].DrugNewsPerspect,2005,18(8):501-507.