背根神经节中嘌呤能受体P2X3R介导骨癌痛的表观调控机制研究

发布时间：2018-07-03 21:40

本文选题：骨癌 + 痛觉过敏　；参考：《苏州大学》2016年博士论文

【摘要】：原发性骨肿瘤或转移性肿瘤导致的骨癌痛是一种严重且顽固性疼痛,严重影响或损害了病人的功能状态,生活质量以及生存时间。目前,对骨疼痛的分子机制还是不明确,有效的临床治疗手段有限。因此,迫切需进一步探索骨癌痛发生发展的分子机制,以寻找新的治疗靶点。近年来的研究发现肿瘤对骨质的破坏以及酸中毒导致的炎症发生,肿瘤释放的产物,还有肿瘤引起的骨折、局部缺血、肿瘤入侵等导致的神经纤维损伤都可能是引起骨癌痛的原因。有不少研究报导嘌呤能受体P2X3R在炎性痛、神经病理性疼痛和内脏痛的发生发展中发挥着重要作用,但对其在骨癌痛中作用功能及其表达调控机制研究甚少。而在骨癌痛形成过程中,肿瘤细胞及基质细胞会分泌些促炎相关因子来敏化外周初级传入神经元,引起神经元中炎症相关因子表达升高,其中NF-κB作为一种重要炎症相关调控因子,广泛参与炎症,疼痛相关基因表达调控,但在骨癌痛形成过程中NF-κB是否参与调控P2X3R的表达仍然不清,因此,我们将从表观遗传的角度探讨P2X3R的调控特征及其NF-κB的作用特点,以寻求治疗骨癌痛的新靶点,为临床治疗骨癌痛提供理论依据。第一部分:研究P2X3R和NF-κB是否参与骨癌痛的发生发展1.研究目的(1)检测骨癌模型大鼠是否存在痛觉过敏及其相关电生理机制;(2)研究P2X3R是否参与骨癌痛的发生发展;(3)探讨NF-κB介导大鼠骨癌痛的作用机制。2.实验方法(1)实验研究对象为雌性成年SD大鼠。通过将大鼠乳腺癌Walker 256细胞(4×105个)注射到胫骨的骨髓腔内来建立骨癌痛大鼠模型。应用X射线成像及组织形态学方法检测胫骨形态学变化以验证模型建立是否成功,用von Frey filament(VFF)和热刺激仪分别检测机械痛阈和热痛阈,以及双足平衡仪检测双足平衡差异来评价骨癌诱导的痛觉过敏;(2)用Di I逆行示踪剂标记支配大鼠胫骨特异性DRG神经元;(3)用Real-time PCR和Western blotting方法检测大鼠胫骨相关的DRG中NF-κB家族成员Rel B,p52,p65 p-p65和RANKL,以及嘌呤受体P2X3R,P2X1R,P2X2R,CBS(硫化氢合成酶)的表达情况;(4)用全细胞膜片钳技术检测支配大鼠胫骨特异性DRG神经元的ATP电流;(5)检测嘌呤能受体抑制剂Suramin或P2X3R和P2X2/3R强效抑制剂A317491能否翻转骨癌大鼠痛敏来确定P2X3R是否参与骨癌痛的发生;(6)检测p65抑制剂PDTC或BAY-11-7082能否翻转骨癌大鼠痛敏来确定p65是否参与骨癌痛形成;(7)检测鞘内注射p65 sh RNA慢病毒是否降低胫骨特异性神经元兴奋性和骨癌痛敏行为,进一步确定p65是否参与骨癌痛的形成。3.实验结果(1)乳腺癌细胞注射两周后X射线成像和形态学观察到骨癌大鼠胫骨骨质发生明显破坏,胫骨骨髓腔中充满大量癌细胞;行为学检测显示骨癌大鼠出现明显的痛觉过敏;(2)荧光逆行示踪剂Di I标记的支配大鼠胫骨特异性神经元主要分布在腰段L2-L5这4个节段的DRG中;(3)电生理学检测发现,与对照组相比,肿瘤细胞注射两周后,骨癌大鼠胫骨特异性DRG神经元的ATP电流显著增强,ATP诱发的动作电位个数也显著增加;(4)癌细胞注射两周后大鼠胫骨相关的DRG中P2X3R表达显著上调。鞘内分别注射嘌呤能受体抑制剂Suramin,P2X3R和P2X2/3R强效抑制剂A317491能显著缓解骨癌大鼠的痛觉过敏,效果呈时间和剂量依赖性;(5)癌细胞注射两周后显著增加大鼠胫骨相关的DRG中p65及磷酸化p65的表达,用p65抑制剂PDTC或BAY-11-7082能显著减轻骨癌大鼠的痛觉过敏,缓解效果呈现时间和剂量依赖性;(6)鞘内注射p65 sh RNA慢病毒能显著缓解骨癌大鼠的痛敏行为,降低ATP诱发的电流,减少ATP诱发的动作电位个数。4.小结以上结果提示P2X3R和p65均参与了癌细胞诱导的骨癌大鼠痛觉过敏的发生发展,且P2X3R的功能受到了p65的调控。第二部分:研究NF-κB参与P2X3R表达调控的分子及表观遗传学机制1.研究目的(1)分析NF-κB p65和P2X3R两者表达的相关性,检测其是否共定位于大鼠胫骨特异性DRG神经元中;(2)检测p65特异性抑制剂或LV-p65 sh RNA沉默p65表达是否可以抑制P2X3R表达;(3)生物学软件预测p2x3r基因启动子区Cp G岛上可能存在的p65结合位点,并进一步用染色质免疫共沉淀法(Ch IP)进行验证;(4)检测p2x3r基因启动子区Cp G岛的DNA甲基化情况,探讨甲基化状态的改变对p65与其结合的影响;(5)初步探讨p2x3r基因启动子区Cp G岛甲基化状态发生改变的机制,即检测骨癌大鼠胫骨相关的DRG中主动去甲基化相关的蛋白MBD2,MBD4,Gadd45a,TDG以及被动去甲基化的DNA甲基化转移酶DNMT3a和DNMT3b表达变化情况。2.实验方法(1)应用统计学分析p65和P2X3R两者的表达是否存在相关性。用免疫荧光染色检测p65与P2X3R在胫骨特异性DRG神经元上的共定位情况;(2)检测p65抑制剂PDTC或p65 sh RNA慢病毒能否逆转P2X3R的高表达,以进一步验证p65是否调控P2X3R表达;(3)应用生物学软件Gene regulation(alibaba2)在线预测p2x3r基因启动子区Cp G岛上的p65结合位点,用染色质免疫沉淀法(Ch IP)进一步验证;(4)应用MSP(甲基化特异性PCR)和BSP(重亚硫酸氢盐测序PCR)检测大鼠胫骨相关的DRG中p2x3r基因启动子区的甲基化情况;(5)应用Real-time PCR方法检测大鼠胫骨相关的DRG中DNMT3a,DNMT3b,Gadd45a,MBD2,MBD4和TDG的表达情况。3.实验结果(1)在骨癌痛模型大鼠胫骨相关的DRG中p65和P2X3R两者表达呈相关性;p65与P2X3R共表达于大鼠胫骨特异性DRG神经元中;(2)p65抑制剂PDTC或p65 sh RNA慢病毒能显著抑制骨癌大鼠胫骨相关的DRG中P2X3R的表达;(3)在线软件预测p2x3r基因启动子区Cp G岛上存在5个p65结合位点,染色质免疫沉淀法(Ch IP)检测显示p65与Cp G岛上1,4和5这三个位点均有结合,且在第1位点上骨癌痛组结合显著增强;(4)骨癌痛大鼠胫骨相关的DRG中p2x3r基因启动子区Cp G岛发生明显的去甲基化;(5)骨癌大鼠胫骨相关的DRG中DNMT3a和DNMT3b表达水平显著降低,但Gadd45a,MBD2,MBD4和TDG的表达没有发生显著变化。4.小结癌细胞注射引起的大鼠胫骨相关DRG中的P2X3R高表达可能是由于其启动子区Cp G岛去甲基化增强进以及与p65结合增加导致的;p2x3r基因启动子区Cp G岛上去甲基化可能是由于DNMT3a和DNMT3b表达下降所致,这些结果提示DNA甲基化参与调控骨癌痛的发生发展。结论(1)癌细胞注射引起p2x3r基因启动子区DNA去甲基化增强以及与p65结合增加,导致P2X3R表达增加,ATP电流增强,从而产生骨癌大鼠痛觉过敏;(2)DNMT3A和DNMT3B表达下降可能是引起p2x3r基因启动子DNA去甲基化原因之一,提示表观调控参与骨癌痛发生发展;(3)P2X3R和p65参与骨癌痛的形成,提示P2X3R和p65可以作为治疗骨癌痛的潜在靶点。
[Abstract]:Bone cancer pain caused by primary bone tumor or metastatic tumor is a serious and stubborn pain, which seriously affects or damages the functional status, life quality and life time of the patients. At present, the molecular mechanism of bone pain is still unclear and effective clinical treatment is limited. Therefore, it is urgent to further explore the occurrence of bone cancer pain. The molecular mechanism of the exhibition is to find new therapeutic targets. Recent studies have found that the destruction of the bone and the inflammation caused by acidosis, the products released by the tumor, the fracture caused by the tumor, the local ischemia, the invasion of the tumor and so on can cause the pain of bone cancer. Methotrexate receptor P2X3R plays an important role in the development of inflammatory pain, neuropathic pain and visceral pain, but there is little study on its function and expression regulation mechanism in bone cancer pain. In the process of bone cancer pain formation, tumor cells and stromal cells secrete some proinflammatory factors to sensitize the peripheral primary afferent nerve. The expression of inflammatory related factors in neurons is increased, and NF- kappa B is an important regulatory factor of inflammation, and it is widely involved in inflammation and regulation of the expression of pain related genes. However, whether NF- kappa B is involved in the regulation of P2X3R is still unclear during the process of bone cancer pain. Therefore, we will discuss the regulation of P2X3R from the epigenetic angle. Characteristics and the characteristics of NF- kappa B to seek new targets for the treatment of bone cancer pain and provide a theoretical basis for clinical treatment of bone cancer pain. Part 1: study the 1. research purposes of whether P2X3R and NF- kappa B participate in the development of bone cancer pain (1) detection of hyperalgesia and related electrophysiological mechanisms in the rat model of bone cancer; (2) the study of whether P2X3R is a reference. And the development of bone cancer pain; (3) to explore the mechanism of NF- kappa B mediated rat bone cancer pain mechanism.2. experimental method (1) the experimental study subjects were female adult rats. The rat model of bone cancer pain was established by injecting Walker 256 cells (4 x 105) into the bone marrow cavity of the tibia of the rat breast cancer. The X ray imaging and histomorphological examination were used. The morphological changes of the tibia were measured to verify the success of the model. Von Frey filament (VFF) and TSPS were used to detect the mechanical pain threshold and thermal pain threshold, and the bipedal balance instrument was used to detect the difference of bipedal balance to evaluate the hyperalgesia induced by bone cancer. (2) the Di I retrograde tracer was used to control the specific DRG neurons of the tibia in rats; (3) Real- Time PCR and Western blotting methods were used to detect the expression of Rel B, p52, p65 p-p65, and purine receptor of the members of the NF- kappa B family associated with the tibia of the rat tibia; (4) detection of the current of the rat tibial specific neurons by the whole cell patch clamp technique; (5) the detection of the purinergic receptor. Whether inhibitor Suramin or P2X3R and P2X2/3R strong inhibitor A317491 can reverse the pain sensitivity of bone cancer rats to determine whether P2X3R participates in the occurrence of bone cancer pain; (6) detection of p65 inhibitor PDTC or BAY-11-7082 can reverse the pain sensitivity of bone cancer rats to determine whether p65 participates in the formation of bone cancer pain; (7) detection of intrathecal p65 sh RNA lentivirus reduces tibia Specific neuronal excitability and bone cancer pain sensitivity, further determine whether p65 participates in the formation of bone cancer pain in.3. experimental results (1) after two weeks of injection of breast cancer cells, X ray imaging and morphological observation of bone carcinomas in bone cancer rats are obviously damaged, and the bone marrow cavity of the tibia is filled with a large number of cancer cells; behavior detection shows that the bone cancer rats are produced. There were obvious hyperalgesia; (2) the tibial specific neurons dominated by Di I marked by fluorescence retrograde tracer were mainly distributed in the DRG of the 4 segments of the lumbar segment L2-L5. (3) electrophysiological tests found that the ATP current of the tibial specific DRG neurons of the bone cancer rats increased significantly after two weeks of injection of the tumor cells, and the ATP induced by ATP. The number of action potential increased significantly; (4) the expression of P2X3R in the tibial related DRG of rats was significantly up-regulated after two weeks of injection of cancer cells. Intrathecal injection of purinergic receptor inhibitor Suramin, P2X3R and P2X2/3R strong inhibitor A317491 could significantly alleviate the hyperalgesia of bone cancer rats. The effect was time and dose dependent; (5) two cancer cells were injected. The expression of p65 and phosphorylated p65 in the tibial related DRG was significantly increased after the week, and the p65 inhibitor PDTC or BAY-11-7082 could significantly reduce the hyperalgesia of the bone cancer rats. The remission effect showed time and dose dependence. (6) intrathecal p65 sh RNA lentivirus could significantly alleviate the pain sensitivity of the bone cancer rats, reduce the current induced by ATP, and reduce the current induced by ATP. The results of a few ATP induced action potential number.4. indicated that both P2X3R and p65 were involved in the development of hyperalgesia induced by cancer cells, and the function of P2X3R was regulated by p65. The second part: the study of the molecular and epigenetic mechanism of NF- kappa B involved in the regulation of P2X3R expression and the mechanism of apparent relic study (1) analysis of NF- kappa B p65 and exports The correlation of the expression of X3R and whether it is located in the rat tibial specific DRG neurons; (2) whether the p65 specific inhibitor or the silent p65 expression of LV-p65 sh RNA can inhibit the expression of P2X3R; (3) biological software predicts the possible presence of the p65 binding site on Cp G island of the p2x3r promoter region, and further uses chromatin free The pestilence coprecipitation method (Ch IP) was used to verify, (4) to detect the DNA methylation of Cp G island in the promoter region of the p2x3r gene, and to explore the influence of the methylation status on the p65 and its binding; (5) the mechanism of the alteration of the methylation status of Cp G island in the promoter region of p2x3r gene was preliminarily discussed, that is to detect the active demethylation phase of the tibia related DRG in the bone cancer rat Protein MBD2, MBD4, Gadd45a, TDG, and DNA methylation transferase DNMT3a and DNMT3b expression changes in passive demethylation.2. experimental method (1) statistical analysis of the correlation between the expression of both p65 and P2X3R. The co localization of p65 and P2X3R in the tibial specific neuron was detected by immunofluorescence staining; (2) detection of the expression of p65 and P2X3R on the tibial specific neuron. Whether the p65 inhibitor PDTC or p65 sh RNA lentivirus can reverse the high expression of P2X3R, to further verify whether p65 regulates P2X3R expression; (3) the application of the biological software Gene regulation (alibaba2) to predict the binding site on the p2x3r gene promoter on line, further verified by the chromatin immunoprecipitation method; (4) application of methyl methylene (methyl) The methylation of the p2x3r gene promoter region in the tibial associated DRG of rats was detected by chemical specific PCR) and BSP (heavy hydrogen sulfite sequencing PCR). (5) the Real-time PCR method was used to detect DNMT3a, DNMT3b, Gadd45a, MBD2, Gadd45a, and expression in the tibial associated DRG of rats (1) in the tibial associated rat model of bone cancer pain model The expression of both p65 and P2X3R was correlated; p65 and P2X3R were co expressed in the specific DRG neurons of the tibia of rats; (2) p65 inhibitor PDTC or p65 sh RNA lentivirus could significantly inhibit the expression of P2X3R in the DRG of the tibia associated with bone cancer rats; (3) the online software predicted that there were 5 binding sites on the island of the promoter region of the gene, and the chromatin immunity was immunized. Ch IP detection showed that p65 was combined with 1,4 and 5 on Cp G Island, and the combination of bone cancer pain group was significantly enhanced at the first site. (4) the Cp G island of the p2x3r gene promoter region of the tibial associated DRG of the bone cancer rats was obviously demethated; (5) the level of DNMT3a and expression in the tibia associated DRG of bone cancer rats decreased significantly. Low, but the expression of Gadd45a, MBD2, MBD4 and TDG did not change significantly. The high expression of P2X3R in the tibial associated DRG induced by.4. nodule injection may be due to the enhancement of the Cp G Island demethylation in the promoter region and the increase of p65 binding with p65. The results showed that the expression of NMT3b was reduced. These results suggest that DNA methylation participates in the regulation of the development of bone cancer pain. Conclusion (1) the enhancement of DNA demethylation in the promoter region of the p2x3r gene and the increase of the binding with p65 lead to the increase of P2X3R expression and the increase of the ATP current, resulting in the hyperalgesia of the bone cancer rats, and (2) the decrease of DNMT3A and DNMT3B expression. It may be one of the causes of p2x3r gene promoter DNA demethylation, suggesting that apparent regulation participates in the development of bone cancer pain; (3) P2X3R and p65 are involved in the formation of bone cancer pain, suggesting that P2X3R and p65 can be used as potential targets for the treatment of bone cancer pain.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R730

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