AQP4和Kir4.1在急性脊髓损伤后脊髓水肿中的作用机制研究

发布时间：2018-06-27 06:17

本文选题：脊髓水肿 + AQP4　；参考：《吉林大学》2015年博士论文

【摘要】：急性脊髓损伤是脊髓外科领域中一种常见的且破坏性极大的疾病。随着急性脊髓损伤发生率的不断增加，脊髓损伤的治疗成为重要的医疗问题。脊髓损伤病人的生理和心理伤害以及高成本的医疗，给家庭和社会带来了沉重负担。急性脊髓损伤具有复杂的病理机制，很难找到有效的治疗方法。原发性脊髓损伤直接破坏组织并造成不可恢复的机体损伤，随之引起脊髓组织中释放大量自毁性介质而导致脊髓组织缺血缺氧、脊髓水肿以及脊髓变性和坏死。而脊髓损伤后的脊髓水肿，是继发性脊髓损伤的重要病理生理过程之一，对预后影响较大。除了应用药物治疗外，目前人们对于脊髓水肿的发生机制尚不清楚。本文对脊髓水肿过程中重要相关分子的变化进行研究，从细胞和分子水平探讨急性脊髓损伤后，脊髓水肿的发生机制。期望能够找到相应的药物治疗靶点，为脊髓损伤治疗提供更充分的理论依据。目的：本研究通过制作大鼠急性脊髓损伤模型，观察大鼠急性脊髓损伤后不同时间点的病理学改变与脊髓含水量变化，了解脊髓水肿的发展变化规律。研究大鼠急性脊髓损伤后，水通道蛋白AQP4与内向整流型钾通道Kir4.1在脊髓水肿期间的表达变化，探讨两者在脊髓水肿形成过程中的作用机制，以及两者是否存在共表达关系。通过观察在脊髓水肿期间AQP4、Kir4.1、P38、p-P38、IκB、NF-κB p65的表达变化，初步判定脊髓损伤后，AQP4和Kir4.1是否相互作用，探讨各因子在脊髓水肿中的作用机制，为今后治疗急性脊髓损伤提供理论依据及新的药物治疗靶点。方法：本研究通过钳夹法制作急性脊髓损伤模型，将体重250-300g的SD大鼠66只，随机分为四组：正常组，假手术组（只手术无脊髓损伤），急性脊髓损伤组（ASCI），MP干预组（正常急性脊髓损伤造模，造模后1h内，尾部静脉注射30mg/kgMP）。假手术组与急性脊髓损伤组按时间点不同又分为8h、24h、3d、7d四个亚组，MP干预组分为3d、7d两个亚组。术后分笼饲养，早期辅助排便排尿。术后通过Tarlov评分对大鼠的神经症状和体征进行鉴定。用干湿重法测定脊髓含水量。用常规HE染色观察组织的病理变化。采用免疫荧光双标记方法检测各组脊髓组织AQP4和Kir4.1的表达变化。利用Real-timePCR检测各组脊髓组织中AQP4和Kir4.1的mRNA表达变化。用Western blot方法检测各组脊髓组织中AQP4、Kir4.1、p-P38、P38、IκB、NF-κB p65的蛋白表达变化。结果统计采用SPSS18.0统计软件处理，所有计量资料均采用方差分析或t检验进行统计，P0.05为差异有显著性。结果：大鼠急性脊髓损伤后，运动神经元遭受损害，形成运动功能障碍，且随着时间的推移损害加重，MP药物可以减轻脊髓损伤后的运动功能障碍。大鼠急性脊髓损伤后，脊髓组织的含水量随着损伤时间的延长而逐渐加重，用MP药物可以明显减轻脊髓损伤后的水肿现象。常规HE染色观察发现大鼠急性脊髓损伤8h后，脊髓组织的中央管和中央灰质发生出血，血管的周围组织有轻度的水肿现象。24h后，脊髓灰质出血严重，部分坏死、部分形成囊腔，且神经元的肿胀严重。3d后，上述的病理变化全部加重，血管周围组织的水肿现象加重，细胞间隙明显增宽，神经元肿胀加重且细胞核发生固缩。7d后，脊髓组织大部分坏死，脊髓白质大部分发生退变，有囊腔和空泡形成。用MP干预处理3d、7d后，可发现脊髓灰白质的边界清晰，出血点的范围减小，细胞核固缩减少，血管周围组织及神经元细胞的水肿现象减轻，水肿体积变小。急性脊髓损伤后，AQP4和Kir4.1的mRNA和蛋白表达均随着时间的推移逐渐升高，且两者的表达变化与脊髓水肿的变化规律呈正相关。急性脊髓损伤后，AQP4和Kir4.1共表达于脊髓的软脊膜、中央管和血管周围胶质细胞的胞膜上，两者的表达随着脊髓损伤时间的延长逐渐增加，用MP药物进行干预治疗后，AQP4和Kir4.1的表达量均发生明显下调。大鼠急性脊髓损伤后，AQP4和Kir4.1在脊髓水肿组织中的表达量均增多。经过MP治疗后，脊髓水肿组织中AQP4和Kir4.1的表达量均减少。大鼠急性脊髓损伤后，脊髓水肿组织中产生大量p-P38，经过MP治疗后，脊髓水肿组织中p-P38的表达量减少。大鼠急性脊髓损伤后，脊髓水肿组织中IκB表达量减少，NF-κB表达量增加，经过MP治疗后，脊髓水肿组织中IκB表达量增多，，NF-κB表达量减少。结论：急性脊髓损伤会对大鼠的运动神经元造成损害，从而产生运动功能障碍，MP药物虽然可以减轻脊髓损伤后的运动功能障碍，但不能完全逆转。对SD大鼠造成急性脊髓损伤后，在早期就会出现脊髓水肿等一系列的病理变化，用MP药物进行干预后，病理变化得到改善并逐渐恢复。 AQP4和Kir4.1基因和蛋白表达情况与水肿具有相关性，提示，在大鼠急性脊髓损伤后，AQP4和Kir4.1的表达变化，直接影响脊髓的水肿程度。 AQP4和Kir4.1存在共表达现象，大部分存在于脊髓灰质，在脊髓白质中出现的较少，且两者的表达与脊髓水肿的变化规律呈正相关。 AQP4和Kir4.1共同参与了急性脊髓损伤后脊髓水肿的产生，可能在脊髓水肿形成机制中，AQP4和Kir4.1之间存在协同作用，共同促进了脊髓水肿的形成。P38MAPK和NF-κB信号通路都参与了急性脊髓损伤后AQP4和Kir4.1的表达调节及脊髓水肿的形成。
[Abstract]:Acute spinal cord injury is a common and devastating disease in the field of spinal surgery. With the increasing incidence of acute spinal cord injury, the treatment of spinal cord injury has become an important medical problem. The physiological and psychological injuries of the patients with spinal cord injury and high cost medical care bring a heavy burden to the family and society. The intramedullary injury has a complicated pathological mechanism, and it is difficult to find an effective treatment. Primary spinal cord injury directly destroys the tissue and causes the non recoverable body damage, resulting in the release of a large number of self destruct media in the spinal cord, resulting in ischemic anoxia, spinal edema, degeneration and necrosis of the spinal cord, and spinal cord injury. Medullary edema, one of the important pathophysiological processes of secondary spinal cord injury, has a great influence on the prognosis. In addition to the application of drug therapy, the mechanism of the occurrence of spinal edema is still unclear. This paper studies the changes of important related molecules in the process of spinal edema, and studies the acute spinal cord injury from the cell and molecular level. The mechanism of spinal edema is expected to be able to find appropriate targets for drug therapy and provide a more sufficient theoretical basis for the treatment of spinal cord injury.
Objective: To observe the pathological changes of the spinal cord and the change of the spinal cord water content at different time points after acute spinal cord injury in rats, and to understand the development and changes of the spinal edema after acute spinal cord injury in rats. After the acute spinal cord injury in rats, the water channel protein AQP4 and the introverted rectifier potassium channel Kir4.1 were studied during the spinal edema. The changes in the expression of AQP4, Kir4.1, P38, p-P38, I kappa B, NF- kappa B p65 during the spinal edema were observed, and the interaction between AQP4 and Kir4.1 after spinal cord injury was preliminarily determined, and the role of each factor in the spinal edema was discussed. It provides a theoretical basis for the treatment of acute spinal cord injury and a new target for drug therapy.
Methods: a model of acute spinal cord injury was made by clamp method. 66 SD rats with weight 250-300g were randomly divided into four groups: normal group, sham operation group (only operation without spinal cord injury), acute spinal cord injury group (ASCI), MP intervention group (normal acute spinal cord injury model, 1H, Obe Shizu injection 30mg/kgMP). The acute spinal cord injury group was divided into four subgroups of 8h, 24h, 3D and 7d at different time points. The MP intervention group was divided into 3D and 7d two subgroups. The nerve symptoms and signs were identified by Tarlov score after operation, and the water content of the spinal cord was measured by the dry wet weight method. The pathological pathology of the tissue was observed by routine HE staining. The changes in the expression of AQP4 and Kir4.1 in spinal cord tissues were detected by double labeling immunofluorescence. The changes in mRNA expression of AQP4 and Kir4.1 in spinal cord tissues were detected by Real-timePCR. Western blot method was used to detect AQP4, Kir4.1, p-P38, P38, kappa, and protein expression in all groups of spinal cord tissues. S18.0 statistical software processing, all the measurement data were analyzed by variance analysis or t test, P0.05 was significant difference.
Results: after the acute spinal cord injury, the motor neurons were damaged, and the motor dysfunction was formed, and the damage was aggravated with time. The MP drug could reduce the motor dysfunction after the spinal cord injury.
After acute spinal cord injury in rats, the water content of spinal cord gradually increased with the prolongation of injury time. The edema after spinal cord injury could be obviously reduced by MP drug.
After the routine HE staining, the central canal and central gray matter of the spinal cord tissue bleed after the acute spinal cord injury of 8h. After the mild edema phenomenon of.24h in the surrounding tissue of the blood vessel, the hemorrhage of the spinal cord was severe and partial necrosis was formed, and the swelling of the neurons was severely.3d, all of the above pathological changes were aggravated and around the vessels around the vessels. The edema of the tissue was aggravated, the intercellular space was widened obviously, the swelling of the neurons was aggravated and the nucleus occurred.7d, most of the spinal cord tissue was necrotic, most of the white matter in the spinal cord was degenerated and the cavity and vacuoles formed. After the treatment of 3D and 7d, the boundary of the gray matter of the spinal cord was clear, the range of the hemorrhagic point was reduced, and the nucleus retraction was found. The edema of perivascular tissues and neurons decreased and the volume of edema decreased.
After acute spinal cord injury, the expression of mRNA and protein in AQP4 and Kir4.1 increased with time, and the expression of both of them was positively correlated with the change of spinal edema.
After acute spinal cord injury, AQP4 and Kir4.1 were co expressed in the spinal cord membrane, the central canal and the membrane of the peripheral glial cells. The expression of both of them increased gradually with the prolongation of the time of spinal cord injury. The expression of AQP4 and Kir4.1 decreased significantly after the intervention of MP drugs.
After acute spinal cord injury, the expression of AQP4 and Kir4.1 increased in the edema tissue of the spinal cord. After MP treatment, the expression of AQP4 and Kir4.1 in the edema tissue of the spinal cord decreased. After the acute spinal cord injury, a large number of p-P38 was produced in the edema tissue of the spinal cord. After MP treatment, the expression of p-P38 in the edema tissue of the spinal cord decreased. After acute spinal cord injury, the expression of I kappa B in the edema tissue of spinal cord decreased and the expression of NF- kappa B increased. After MP treatment, the expression of I kappa B in the tissue of spinal cord edema increased, and the expression of NF- kappa B decreased.
Conclusion: acute spinal cord injury can cause damage to motor neurons in rats and thus produce motor dysfunction. Although MP drugs can reduce motor dysfunction after spinal cord injury, it can not be completely reversed.
After acute spinal cord injury in SD rats, a series of pathological changes such as edema, such as spinal cord edema, were found in the early stage. After the treatment of MP, the pathological changes were improved and gradually recovered.
The expression of AQP4 and Kir4.1 genes and proteins is associated with edema, suggesting that the changes in the expression of AQP4 and Kir4.1 directly affect the degree of edema in the spinal cord after acute spinal cord injury in rats.
The co expression of AQP4 and Kir4.1 exists in the gray matter of the spinal cord, and there is less in the white matter of the spinal cord, and the expression of the two is positively correlated with the changes in the spinal edema.
AQP4 and Kir4.1 participate in the formation of spinal edema after acute spinal cord injury. There is a synergistic effect between AQP4 and Kir4.1 in the formation mechanism of spinal edema, which jointly promote the formation of.P38MAPK and NF- kappa B signaling pathways in the formation of spinal edema and the regulation of the expression of AQP4 and Kir4.1 and the formation of spinal edema after acute spinal cord injury.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R651.2

【参考文献】