硫化氢合酶CBS调控电压门控性钠通道参与大鼠自体髓核移植致下肢痛觉过敏的分子机制研究

发布时间：2018-05-24 18:36

本文选题：腰椎间盘突出 + 自体髓核移植　；参考：《苏州大学》2015年硕士论文

【摘要】：目的1.初步阐明自体髓核(nucleus pulposus,NP)移植诱导的成年大鼠下肢痛觉过敏中背根神经节(DRG)神经元上电压门控性钠通道(VGSCs)的表达和功能的变化特征。2.探讨胱硫醚-?-合成酶(CBS)在下肢痛觉过敏中的作用及其对电压门控性钠通道表达和功能调控的影响。方法1.选择成年SD雄性大鼠随机分为LDH组和Sham组,LDH组在左侧L5、L6背根神经节处以自体尾椎髓核移植来建立大鼠腰椎间盘突出模型,Sham组仅暴露L5、L6神经根,其他处理与LDH组相同。测量两组大鼠术前及术后7天,14天,21天的左后肢机械刺激缩足反射阈值(PWT)和热刺激缩足反射阈值(PWL)的变化。鞘内注射CBS拮抗剂羧甲基羟胺半盐酸(AOAA),同样运用以上PWT、PWL测量方法评测大鼠的下肢疼痛反射阈值,从而检测CBS在LDH模型中的作用。2.运用逆行标记的荧光素(Di I)来标记支配后肢的L5-L6背根神经节(DRG)中、小神经元,运用全细胞膜片钳方法研究LDH大鼠L5-L6 DRG神经元兴奋性及电压门控性钠通道电流的变化特性。3.运用Western Blotting方法检测大鼠L5-L6 DRG神经元中Na V1.7,Na V1.8以及CBS蛋白表达情况。4.运用免疫组织化学检测LDH大鼠L5-L6 DRG神经元中CBS和Na V1.7,Na V1.8蛋白共表达情况。结果1.自体髓核移植明显增加大鼠下肢痛觉过敏,机械刺激缩足反射阈值(PWT)在术后1周至3周显著低于Sham组,1周时达到疼痛阈值的最低值,热刺激缩足反射阈值(PWL)在术后1周时显著降低,2周时基本恢复到原基线水平,并且PWT和PWL的显著降低与CBS的表达上调相关联;鞘内注射CBS的拮抗剂AOAA后,可以显著翻转LDH大鼠疼痛阈值,并呈现时间和剂量依赖性。2.全细胞膜片钳记录显示,与Sham组相比,LDH组L5-L6 DRG神经元兴奋性显著增加;进一步检测显示自体髓核移植后电压门控性钠通道的电流密度显著增加。3.数据分析后显示,与Sham组相比,LDH组的失活曲线显著左移,激活曲线没有显著变化。另外,自体髓核移植处理后,电压门控性钠通道的失活时间显著增长,激活时间没有显著变化。4.运用免疫组化的实验方法检测后发现CBS与Na V1.7,Na V1.8在L5-L6神经元中能够共表达。自体髓核移植大鼠L5-L6 DRG神经元中Na V1.7和Na V1.8受体蛋白表达显著增加,与移植无关的T10-T12 DRG神经元Na V1.7和Na V1.8的蛋白表达水平没有显著差异。5.连续鞘内注射AOAA可以显著翻转LDH组大鼠L5-L6相关DRG神经元的兴奋性,还可以显著降低电压门控性钠通道的电流密度,并使电压门控性钠通道的失活曲线右移。另外AOAA还可以显著降低LDH组大鼠L5-L6相关DRG神经元中Na V1.7和Na V1.8的高表达。结论以上实验结果表明硫化氢合酶CBS可能是通过电压门控性钠通道的激活和Na V1.7和Na V1.8蛋白的高表达,增加电压门控性钠通道电流密度,导致自体髓核移植处相关DRG神经元兴奋性的增加进而参与外周痛觉信号的调制,最终导致大鼠下肢痛觉过敏。本研究在一定程度上揭示了LDH病人疼痛产生的部分分子机制,可能会为LDH的治疗提供一些有效的治疗方案。-?-
[Abstract]:Objective 1. to preliminarily elucidate the expression and function of voltage gated sodium channel (VGSCs) on the dorsal root ganglion (DRG) neurons of the lower extremities induced by autologous nucleus pulposus (NP) transplantation in adult rats. The role of.2. to explore the role of cystthioether - synthetase (CBS) in the hyperalgesia of the lower extremities and its voltage gated sodium channel Method 1. the adult SD male rats were randomly divided into LDH group and Sham group. The LDH group was in the left L5 and L6 dorsal root ganglion with autologous tail marrow nucleus transplantation to establish the rat lumbar disc herniation model. The Sham group only exposed L5, L6 nerve root, and the other treatments were the same as LDH group. The two groups of rats were measured before and 7 days, 14 after the operation. The changes of the left hind limb mechanical stimulation of the contraction foot reflex threshold (PWT) and the thermal stimulation contraction reflex threshold (PWL). The intrathecal injection of CBS antagonist carboxymethyl hydroxylamine semi hydrochloric acid (AOAA) is also used to evaluate the pain reflex threshold of the lower extremities of the rats by the above PWT and PWL methods, and to detect the function of CBS in LDH model by.2. to use retrograde labeling. Di I to mark the L5-L6 dorsal root ganglion (DRG) in the hind limbs (DRG), small neurons, using whole cell patch clamp method to study the excitatory and voltage-gated sodium channel current change characteristics of L5-L6 DRG neurons in LDH rats.3. using Western Blotting method to detect the L5-L6 DRG neurons in rats. .4. immunohistochemical staining was used to detect the co expression of CBS and Na V1.7, Na V1.8 protein in the L5-L6 DRG neurons of LDH rats. Results 1. autologous nucleus pulposus transplantation significantly increased the hyperalgesia of the lower limbs of the rats. The threshold of mechanical stimulation contraction reflex (PWT) was significantly lower than that in the Sham group from 1 to 3 weeks after the operation, and the minimum value of the pain threshold was reached at 1 weeks, and the heat stimulation was achieved. The threshold of the contraction reflex (PWL) decreased significantly at 1 weeks after the operation and was basically restored to the original baseline level at 2 weeks, and the significant reduction of PWT and PWL was associated with the up regulation of CBS expression. After the intrathecal CBS antagonist AOAA, the pain threshold of LDH rats could be reversed significantly, and the time and dose dependent.2. whole cell patch clamp recording showed, and Sha. Compared with group M, the excitability of L5-L6 DRG neurons in group LDH increased significantly. Further detection showed that the current density of voltage gated sodium channel after autotransplantation of autologous nucleus pulposus was significantly increased after.3. data analysis. Compared with the Sham group, the inactivation curve in the LDH group was significantly left, and the activation curve was not significantly changed. In addition, after autologous nucleus pulposus transplantation, the voltage was changed. The inactivation time of the gated sodium channel increased significantly and the activation time did not change significantly..4. was detected by CBS and Na V1.7, and Na V1.8 was co expressed in L5-L6 neurons. The Na V1.7 and Na receptor receptor protein expression in L5-L6 DRG neurons in the autologous nucleus pulposus transplanted rats increased significantly. There is no significant difference in the protein expression level of Na V1.7 and Na V1.8 in the T12 DRG neurons..5. continuous intrathecal AOAA can significantly overturn the excitatory of L5-L6 related DRG neurons in the LDH group, and can significantly reduce the current density of the voltage gated sodium channel and move the inactivation curve of the voltgated sodium channel to the right. To reduce the high expression of Na V1.7 and Na V1.8 in the L5-L6 related DRG neurons in the LDH group. Conclusion the above experimental results suggest that the hydrogen sulfide synthase CBS may be activated by voltage gated sodium channels and the high expression of Na V1.7 and Na V1.8 protein, increasing the current density of voltage gated sodium channels, leading to the associated nerve related to the transplantation of autologous nucleus pulposus. The increase of element excitability then participates in the modulation of peripheral pain signals and eventually leads to the hyperalgesia of the lower extremities. This study reveals some of the molecular mechanisms of pain in LDH patients, and may provide some effective treatment for the treatment of LDH. - -
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R681.5

【参考文献】