本文選題：脊髓水腫 + AQP4�。� 參考：《吉林大學(xué)》2015年博士論文

【摘要】：急性脊髓損傷是脊髓外科領(lǐng)域中一種常見(jiàn)的且破壞性極大的疾病。隨著急性脊髓損傷發(fā)生率的不斷增加，脊髓損傷的治療成為重要的醫(yī)療問(wèn)題。脊髓損傷病人的生理和心理傷害以及高成本的醫(yī)療，給家庭和社會(huì)帶來(lái)了沉重負(fù)擔(dān)。急性脊髓損傷具有復(fù)雜的病理機(jī)制，很難找到有效的治療方法。原發(fā)性脊髓損傷直接破壞組織并造成不可恢復(fù)的機(jī)體損傷，隨之引起脊髓組織中釋放大量自毀性介質(zhì)而導(dǎo)致脊髓組織缺血缺氧、脊髓水腫以及脊髓變性和壞死。而脊髓損傷后的脊髓水腫，是繼發(fā)性脊髓損傷的重要病理生理過(guò)程之一，對(duì)預(yù)后影響較大。除了應(yīng)用藥物治療外，目前人們對(duì)于脊髓水腫的發(fā)生機(jī)制尚不清楚。本文對(duì)脊髓水腫過(guò)程中重要相關(guān)分子的變化進(jìn)行研究，從細(xì)胞和分子水平探討急性脊髓損傷后，脊髓水腫的發(fā)生機(jī)制。期望能夠找到相應(yīng)的藥物治療靶點(diǎn)，為脊髓損傷治療提供更充分的理論依據(jù)。 目的：本研究通過(guò)制作大鼠急性脊髓損傷模型，觀察大鼠急性脊髓損傷后不同時(shí)間點(diǎn)的病理學(xué)改變與脊髓含水量變化，了解脊髓水腫的發(fā)展變化規(guī)律。研究大鼠急性脊髓損傷后，水通道蛋白AQP4與內(nèi)向整流型鉀通道Kir4.1在脊髓水腫期間的表達(dá)變化，探討兩者在脊髓水腫形成過(guò)程中的作用機(jī)制，以及兩者是否存在共表達(dá)關(guān)系。通過(guò)觀察在脊髓水腫期間AQP4、Kir4.1、P38、p-P38、IκB、NF-κB p65的表達(dá)變化，初步判定脊髓損傷后，AQP4和Kir4.1是否相互作用，探討各因子在脊髓水腫中的作用機(jī)制，為今后治療急性脊髓損傷提供理論依據(jù)及新的藥物治療靶點(diǎn)。 方法：本研究通過(guò)鉗夾法制作急性脊髓損傷模型，將體重250-300g的SD大鼠66只，隨機(jī)分為四組：正常組，假手術(shù)組（只手術(shù)無(wú)脊髓損傷），急性脊髓損傷組（ASCI），MP干預(yù)組（正常急性脊髓損傷造模，造模后1h內(nèi)，尾部靜脈注射30mg/kgMP）。假手術(shù)組與急性脊髓損傷組按時(shí)間點(diǎn)不同又分為8h、24h、3d、7d四個(gè)亞組，MP干預(yù)組分為3d、7d兩個(gè)亞組。術(shù)后分籠飼養(yǎng)，早期輔助排便排尿。術(shù)后通過(guò)Tarlov評(píng)分對(duì)大鼠的神經(jīng)癥狀和體征進(jìn)行鑒定。用干濕重法測(cè)定脊髓含水量。用常規(guī)HE染色觀察組織的病理變化。采用免疫熒光雙標(biāo)記方法檢測(cè)各組脊髓組織AQP4和Kir4.1的表達(dá)變化。利用Real-timePCR檢測(cè)各組脊髓組織中AQP4和Kir4.1的mRNA表達(dá)變化。用Western blot方法檢測(cè)各組脊髓組織中AQP4、Kir4.1、p-P38、P38、IκB、NF-κB p65的蛋白表達(dá)變化。結(jié)果統(tǒng)計(jì)采用SPSS18.0統(tǒng)計(jì)軟件處理，所有計(jì)量資料均采用方差分析或t檢驗(yàn)進(jìn)行統(tǒng)計(jì)，P0.05為差異有顯著性。 結(jié)果：大鼠急性脊髓損傷后，運(yùn)動(dòng)神經(jīng)元遭受損害，形成運(yùn)動(dòng)功能障礙，且隨著時(shí)間的推移損害加重，MP藥物可以減輕脊髓損傷后的運(yùn)動(dòng)功能障礙。 大鼠急性脊髓損傷后，脊髓組織的含水量隨著損傷時(shí)間的延長(zhǎng)而逐漸加重，用MP藥物可以明顯減輕脊髓損傷后的水腫現(xiàn)象。 常規(guī)HE染色觀察發(fā)現(xiàn)大鼠急性脊髓損傷8h后，脊髓組織的中央管和中央灰質(zhì)發(fā)生出血，血管的周?chē)M織有輕度的水腫現(xiàn)象。24h后，脊髓灰質(zhì)出血嚴(yán)重，部分壞死、部分形成囊腔，且神經(jīng)元的腫脹嚴(yán)重。3d后，上述的病理變化全部加重，血管周?chē)M織的水腫現(xiàn)象加重，細(xì)胞間隙明顯增寬，神經(jīng)元腫脹加重且細(xì)胞核發(fā)生固縮。7d后，脊髓組織大部分壞死，脊髓白質(zhì)大部分發(fā)生退變，有囊腔和空泡形成。用MP干預(yù)處理3d、7d后，可發(fā)現(xiàn)脊髓灰白質(zhì)的邊界清晰，出血點(diǎn)的范圍減小，細(xì)胞核固縮減少，血管周?chē)M織及神經(jīng)元細(xì)胞的水腫現(xiàn)象減輕，水腫體積變小。 急性脊髓損傷后，AQP4和Kir4.1的mRNA和蛋白表達(dá)均隨著時(shí)間的推移逐漸升高，且兩者的表達(dá)變化與脊髓水腫的變化規(guī)律呈正相關(guān)。 急性脊髓損傷后，AQP4和Kir4.1共表達(dá)于脊髓的軟脊膜、中央管和血管周?chē)z質(zhì)細(xì)胞的胞膜上，兩者的表達(dá)隨著脊髓損傷時(shí)間的延長(zhǎng)逐漸增加，用MP藥物進(jìn)行干預(yù)治療后，AQP4和Kir4.1的表達(dá)量均發(fā)生明顯下調(diào)。 大鼠急性脊髓損傷后，AQP4和Kir4.1在脊髓水腫組織中的表達(dá)量均增多。經(jīng)過(guò)MP治療后，脊髓水腫組織中AQP4和Kir4.1的表達(dá)量均減少。大鼠急性脊髓損傷后，脊髓水腫組織中產(chǎn)生大量p-P38，經(jīng)過(guò)MP治療后，脊髓水腫組織中p-P38的表達(dá)量減少。大鼠急性脊髓損傷后，脊髓水腫組織中IκB表達(dá)量減少，NF-κB表達(dá)量增加，經(jīng)過(guò)MP治療后，脊髓水腫組織中IκB表達(dá)量增多，，NF-κB表達(dá)量減少。 結(jié)論：急性脊髓損傷會(huì)對(duì)大鼠的運(yùn)動(dòng)神經(jīng)元造成損害，從而產(chǎn)生運(yùn)動(dòng)功能障礙，MP藥物雖然可以減輕脊髓損傷后的運(yùn)動(dòng)功能障礙，但不能完全逆轉(zhuǎn)。 對(duì)SD大鼠造成急性脊髓損傷后，在早期就會(huì)出現(xiàn)脊髓水腫等一系列的病理變化，用MP藥物進(jìn)行干預(yù)后，病理變化得到改善并逐漸恢復(fù)。 AQP4和Kir4.1基因和蛋白表達(dá)情況與水腫具有相關(guān)性，提示，在大鼠急性脊髓損傷后，AQP4和Kir4.1的表達(dá)變化，直接影響脊髓的水腫程度。 AQP4和Kir4.1存在共表達(dá)現(xiàn)象，大部分存在于脊髓灰質(zhì)，在脊髓白質(zhì)中出現(xiàn)的較少，且兩者的表達(dá)與脊髓水腫的變化規(guī)律呈正相關(guān)。 AQP4和Kir4.1共同參與了急性脊髓損傷后脊髓水腫的產(chǎn)生，可能在脊髓水腫形成機(jī)制中，AQP4和Kir4.1之間存在協(xié)同作用，共同促進(jìn)了脊髓水腫的形成。P38MAPK和NF-κB信號(hào)通路都參與了急性脊髓損傷后AQP4和Kir4.1的表達(dá)調(diào)節(jié)及脊髓水腫的形成。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R651.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 楊迎暴,樸英杰;白藜蘆醇對(duì)大鼠脊髓急性損傷后繼發(fā)性損傷的影響(英文)[J];Acta Pharmacologica Sinica;2003年07期

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