内源性硫化氢合酶介导颞下颌关节炎症疼痛的机制研究

发布时间：2018-06-19 02:44

本文选题：颞下颌关节炎症 + 疼痛　；参考：《苏州大学》2014年硕士论文

【摘要】：研究背景硫化氢（H2S）是继NO、CO后的第三种新型气体信使，，在哺乳动物体内作为一种内源性气体分子发挥着非常重要的生理病理作用，但其是否在关节炎症疼痛中也起着调制作用还鲜有报道。本研究正是基于探究内源性硫化氢（H2S）在颞下颌关节炎症疼痛模型中的作用及其分子机制。实验方法 1.6~8周龄的SD雄性大鼠的颞下颌关节腔注射完全弗氏佐剂（completeFreund’s adjuvant，CFA）从而诱导SD大鼠的颞下颌关节炎症疼痛模型。运用VFF（Von Frey filaments）的方法来评测SD大鼠的行为学疼痛阈值反应。 2.运用逆行标记的荧光素（DiI）来标记支配颞下颌关节（temporomandibularjoint，TMJ）的特异性三叉神经节（trigeminal ganglion，TG）神经元，运用全细胞膜片钳方法研究炎症组SD大鼠TMJ特异性TG神经元兴奋性，电压依赖性钾通道特性以及ATP电流的变化。 3.运用Western Blotting方法检测颞下颌关节炎症疼痛模型SD大鼠TMJ特异性TG神经元中硫化氢合酶CBS和CSE的蛋白以及P2X3R表达情况。 4.运用Quantitative Real-time PCR检测颞下颌关节炎症疼痛模型SD大鼠颞下颌特异性TG神经元中硫化氢合酶CBS mRNA的表达情况。 5.运用免疫组织化学检测SD大鼠TG神经元中CBS和P2X3R蛋白共表达情况。结果 1. CFA能够显著降低SD大鼠颞下颌关节的机械性疼痛阈值，并且这一现象是与TG神经元内硫化氢合酶CBS蛋白及mRNA的表达上调相关联的。 2.对SD大鼠注射硫化氢合酶CBS的抑制剂AOAA后，发现AOAA能够显著的翻转炎症组SD大鼠的疼痛阈值，并呈现剂量和时间上的依赖性。而作为对照，AOAA并不能够对对照组SD大鼠的疼痛阈值产生作用。 3.采用全细胞电生理膜片钳技术对颞下颌关节特异性TG神经元细胞进行记录后发现，连续给予炎症组SD大鼠AOAA能够翻转CFA引起的颞下颌关节特异性TG神经元细胞的兴奋性增强，电压门控型钾通道的电流密度的减小以及ATP电流密度的增大。 4.为了进一步确认CBS-H2S信号通路在CFA诱导的颞下颌关节炎症性疼痛模型中发挥的作用，硫氢化钠被用来模拟CBS产生的硫化氢。结果发现NaHS能够显著的增加正常SD大鼠颞下颌关节特异性TG神经元的兴奋性。 5.运用免疫组化的实验方法检测后发现硫化氢合酶CBS与P2X3R在TG神经元中能够共表达。而且运用Western Blotting的方法进行检测后发现，连续给予炎症组SD大鼠AOAA能够显著翻转CFA引起的TG神经元中的P2X3R表达的上调。结论我们的研究结果表明，内源性硫化氢（H2S）合酶CBS表达的上调可能在SD大鼠颞下颌关节炎症疼痛过程中扮演着重要的角色。因此，本研究能够在一定程度上揭示颞下颌关节炎症疼痛产生的分子机制，从而为炎症疼痛的治疗提供更为有效的治疗方案。
[Abstract]:Background hydrogen sulfide (H _ 2S) is the third new gas messenger after NO-CO and plays a very important role in physiology and pathology as an endogenous gas molecule in mammals. But whether it also plays a modulating role in arthritis pain is rarely reported. The purpose of this study was to explore the role of endogenous hydrogen sulfide (H _ 2S) in temporomandibular arthritis (TMJ) pain model and its molecular mechanism. Methods the temporomandibular arthritis pain model of SD rats was induced by injecting complete Freundant adjuvant CFAA into the temporomandibular joint (TMJ) of male SD rats at the age of 1.6 weeks. The behavioral pain threshold response of SD rats was evaluated by VFFFV on Frey filaments. 2. 2. The specific trigeminal ganglion (TGG) neurons innervating temporomandibular joint (TMJ) were labeled with retrograde fluorescein (Dii). The excitability of TMJ specific TG neurons in inflammatory group was studied by whole-cell patch clamp method. Characteristics of voltage-dependent potassium channels and changes of ATP currents. 3. Western blotting was used to detect the expression of hydrogen sulfide synthase CBS and CSE and P2X3R in TMJ specific TG neurons of SD rat model of temporomandibular arthritis pain. 4. The expression of hydrogen sulfide synthase (CBS) mRNA in temporomandibular specific TG neurons was detected by quantitative Real-time PCR in SD rats with temporomandibular arthritis pain model. The co-expression of CBS and P2X 3R protein in TG neurons of SD rats was detected by immunohistochemistry. Result 1. CFA can significantly reduce the mechanical pain threshold of temporomandibular joint in SD rats, and this phenomenon is associated with the up-regulation of the expression of H2S CBS protein and mRNA in TG neurons. After injection of AOAA, an inhibitor of hydrogen sulfide synthase (CBS), it was found that AOAA could significantly reverse the pain threshold of SD rats in inflammatory group, and showed a dose-dependent and time-dependent manner. AOAA, as a control group, did not have an effect on the pain threshold of SD rats in the control group. Using whole-cell electrophysiological patch clamp technique to record the temporomandibular joint specific TG neurons, it was found that AOAA could reverse the excitability of temporomandibular joint specific TG neurons induced by CFA. The decrease of current density and the increase of ATP current density in voltage-gated potassium channels. 4. In order to further confirm the role of CBS-H2S signaling pathway in CFA induced temporomandibular arthritis (TMJ) pain model, sodium sulfide was used to simulate hydrogen sulfide produced by CBS. The results showed that NaHS could significantly increase the excitability of temporomandibular joint specific TG neurons in normal SD rats. The expression of hydrogen sulfide synthase CBS and P2X3R in TG neurons was detected by immunohistochemistry. Furthermore, Western blotting was used to detect the up-regulation of P2X3R expression in TG neurons induced by CFA in SD rats treated with continuous injection of AOAA. Conclusion our results suggest that the up-regulated expression of endogenous hydrogen sulfide (H2S) synthase CBS may play an important role in the process of temporomandibular arthritis (TMJ) pain in SD rats. Therefore, to some extent, this study can reveal the molecular mechanism of temporomandibular arthritis pain, so as to provide a more effective treatment for inflammatory pain.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R782.6

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