YAP/TAZ 转录因子调控骨癌疼痛的分子机制研究

研究背景：骨癌痛（bone cancer pain, BCP）是癌症患者最典型、最严重的症状之一。治疗BCP 是临床上重大挑战，但是，目前引起BCP的机制尚不清楚。疼痛造成神经损伤和应急性刺激，随后引起的修复机制与神经组织的发育过程具有一定的相似性。因此，有关发育的信号通路在近几年在疼痛领域的研究中受到关注。Hippo 通路是一个与细胞发育密切相关的通路，在癌症发展和病理性疼痛中起重要作用。YAP/TAZ、MST、LATS 是Hippo 通路中的关键蛋白。本实验利用骨癌痛模型，研究Hippo通路中关键蛋白对癌性疼痛的影响。实验方法：通过将大鼠乳腺癌细胞Walker256 注射到大鼠的胫骨中；或小鼠肺癌细胞LLC 注射到小鼠胫骨中诱发BCP ，探究Hippo 通路中关键蛋白对BCP 的影响。

主要结果：
（1）为了阐明Hippo 通路在BCP 中的作用，采用Western blot 技术检测大鼠在产生BCP 后脊髓（ spinal cord ， SC ） 和背根神经节（ dorsalroot ganglia ， DRG ）中Hippo 通路关键蛋白LATS 、MST 、YAP/TAZ 的表达。结果发现， BCP 导致YAP/TAZ 上调、p-LATS/LATS 和p-MST/MST 下调，这意味着在BCP 后Hippo 通路由正常的激活状态转为关闭状态。且YAP/TAZ 蛋白与BCP 的变化在时间上更为一致，因此我们重点研究了YAP/TAZ 蛋白对BCP 的作用。
（2）为了进一步探讨Hippo 通路中YAP/TAZ 对BCP 的调控作用，将YAP/TAZ 的抑制剂在大鼠上进行鞘内给药，采用Von Frey 方法来测试YAP/TAZ 抑制剂对BCP 的影响。结果显示，YAP/TAZ 的抑制剂有明显镇痛作用， 说明YAP/TAZ 对BCP 有调控作用。为了进一步探讨YAP/TAZ调控BCP 的内在机制， 我们采用免疫荧光与RNA scope 方法检测了YAP/TAZ 在SC 和DRG 的分布。结果显示，YAP/TAZ 在SC 和DRG 的胶质细胞和神经元中均有分布。
（3）为了探讨YAP/TAZ 对于神经元的调控作用，在BCP 大鼠鞘内注射YAP/TAZ 抑制剂后，采用Western blot 技术检测神经元兴奋相关蛋白和离子通道的表达。采用Von Frey 方法评估BCP 的发展。研究结果表明，YAP/TAZ 抑制剂明显抑制了神经元兴奋相关蛋白p-NR2B 、p-CaMKII 、p-CREB 的表达。此外， YAP/TAZ 抑制剂可以调控P2X3R 受体的表达与功能。注射YAP/TAZ 抑制剂后BCP 的发展得到明显缓解。进一步的实验表明，在兴奋性神经元中消减YAP/TAZ 也可以抑制BCP 的发展。这些结果说明YAP/TAZ 可能通过调控P2X3R 从而调节神经元兴奋性来调控BCP的发展。
（4）为了探讨YAP/TAZ 对胶质细胞的调控作用，在BCP 大鼠鞘内注射YAP/TAZ 抑制剂后， 采用Western blot 、RT-qPCR 和免疫荧光方法检查胶质细胞的激活情况和炎症介质的表达。YAP/TAZ 抑制剂抑制了小胶质细胞和星型胶质细胞的激活及IL-6、TNF-α、IL-1β等炎症因子的表达。进一步的实验结果表明，在星型胶质细胞上消减YAP/TAZ 后， 炎症因子表达下调。这些实验结果说明YAP/TAZ 可以调节胶质细胞的激活，进而调控炎症因子的表达来调节BCP。
实验结论： 我们的结果表明，YAP/TAZ 对于BCP 的调控起到重要作用。YAP/TAZ 可以通过调节P2X3 受体的表达与功能，从而影响神经元的兴奋性来调控BCP。此外，YAP/TAZ 可以通过调节胶质细胞的激活和IL-6、TNF-α、IL-1β等炎症因子的表达来调节BCP。我们的研究表明，YAP和TAZ 可能是BCP 发生发展的重要因素，以YAP/TAZ 为靶向的药物可能成为BCP 潜在的有效镇痛剂。本项目研究揭示了癌性疼痛的新机制，这将为阐明癌性疼痛机制、探讨癌痛精准治疗的分子靶点提供研究思路和策略。

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