【作者】 姜雨鸽；

【导师】 张宏；

【作者基本信息】 中国人民解放军军医进修学院 ， 麻醉学， 2008， 博士

【摘要】 P2X₃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P2X₃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P2X₃受体在骨癌痛发生机理中的作用,并利用RNA干扰技术以P2X₃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P2X₃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P2X₃受体mRNA转录水平、蛋白表达的变化及P2X₃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（10⁴/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CO₂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P2X₃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_4-6DRG,RT-PCR方法检测P2X₃受体mRNA转录水平的变化,免疫印迹法检测P2X₃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P2X₃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_4-6DRG,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P2X₃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CO₂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CO₂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CO₂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CO₂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P2X₃mRNA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P2X₃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P2X₃受体转录水平及蛋白表达均上调,P2X₃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P2X₃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P2X₃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P2X₃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P2X₃全长基因克隆至pEGFP-N1真核表达载体。用pEGFP-P2X₃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P2X₃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P2x₃受体的表达水平。结果:①通过将siRNA质粒与表达P2X₃蛋白的pEGFP-P2X₃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 P2X₃ 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5.3×10⁸Tu/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P2X₃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P2X₃受体shRNA 850片段可以高效抑制P2X₃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P2X₃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P2X₃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P2X₃mRNA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（5×10⁶Tu）;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（5×10⁶Tu）。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CO₂激光热痛敏阈值。②于第6w行为学测定结束后取L_4～6DRG,RT-PcR及Immunoblot检测P2X₃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（5×10⁶Tu）,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CO₂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P2X₃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P2X₃基因的表达,同时观察期内重要脏器无病理改变。更多还原

【Abstract】 P2X₃ 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 P2X₃ receptors,will become an effective and hopeful therapeutic method without side effects.This study was focused on observing the therapy effects of blocking P2X₃ 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 P2X₃ receptor in the future.PartⅠChanges of P2X₃ receptor expression and currents in a rat bone cancer pain modelObjective:To investigate the relationship of rat behavior and P2X₃ 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（10⁴/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 CO₂ 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 P2X₃ 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 P2X₃ 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 P2X₃ receptor in bone cancer pain rats increase and induce P2X₃ currents.Therefore,these indicate that the P2X₃ dependent channel is potentially involved in the occurrence of bone cancer pain.PartⅡConstructing lentivirus expressing shRNA against P2X₃ receptorObjectives:To synthesis shRNA for blocking P2X₃ receptor expression in vitro and select the best lentiviral construct.Methods:①Following siRNA design principles,three P2X₃ siRNAs,named 782,850,918,were synthesized and cloned into a vector（pSR-GFP vector）for siRNA expression.Full length P2X₃ cDNA was cloned in frame into pEGFP-N1,so the GFP-P2X₃ fusion protein could be expressed in mammalian cells,pEGFP-P2X₃ plasmids were co-transfected into 293T cells with P2X₃ RNAi vectors and the wild type vectors as control.Total cellular RNA was extracted at the time point of 36 hours post transfection,P2X₃ 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 P2X₃ was accessed by Immunoblot. Results:①Among three shRNAs,we saw the highest efficiency of No.850 blocking the expression of P2X₃ 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 5.3×10⁸Tu/ml.③The lentivirus particles effectively infected hippocampus neurons and its infection efficiency was nearly 100%.④Expression of P2X₃ 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 P2X₃ shRNA could effectively infect neurons and block the expression of P2X₃.Therefore,this P2X₃ shRNA system is a powerful tool in our research wrok. PartⅢEffects of intrathecal injection of lentivirus expressingshRNA inhibiting P2X₃ gene on bone cancer pain ratObjective:The aim of the study was to observe the effects of P2X₃ gene silenced by intrathecal injection of letiviral expressed shRNA on mechanical allodynia and thermal hyperalgesia and the expression of P2X₃ 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×l0⁸Tu/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,P2X₃ 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（5×10⁶Tu）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 P2X₃ 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 P2X₃ 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 P2X₃ 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 P2X₃ shRNAs expressing lentvirus.Meanwhile we found this method can effectively silence the P2X₃ gene expression both at the protein and mRNA level.Moreover,we didn’t see any side effects throughout whole process.更多还原