低氧誘導雪旺細胞AQP1表達機制的相關(guān)實驗研究
發(fā)布時間：2018-08-02 17:27
【摘要】：【研究背景】BELL’s面癱在面神經(jīng)損傷中最為常見,其初始病因目前尚未明確,但通過實驗室解剖以及臨床面神經(jīng)管減壓術(shù)中發(fā)現(xiàn)面神經(jīng)因缺血缺氧水腫致滋養(yǎng)血管受狹窄骨性管腔的卡壓而形成的“水腫-缺血缺氧-水腫”惡性循環(huán)是導致神經(jīng)變性壞死的直接原因。因此,終止“水腫-缺血缺氧-水腫”的惡性循環(huán)在面神經(jīng)損傷的治療中顯得更加重要。水通道蛋白是一類在細胞膜上對水分子具有快速轉(zhuǎn)運功能的膜通道蛋白家族,目前已于各種不同的組織器官中發(fā)現(xiàn)了13種水通道蛋白(AQP0-12),我們課題組前期工作已證實AQP1存在于面神經(jīng)的雪旺細胞上,并且與面神經(jīng)及雪旺細胞的水腫緊密相關(guān)。MAPK信號通路是細胞信息傳遞的交匯點,能夠?qū)⒓毎獾男畔鬟f至細胞核,可以直接或間接的調(diào)控轉(zhuǎn)錄因子活性及基因的表達,是細胞應(yīng)激和損傷反饋中的主要信號通路,廣泛存在于人體各類細胞中。有研究表明,AQP1表達可被低氧、滲透性應(yīng)激、炎癥介質(zhì)等調(diào)節(jié),同時研究還發(fā)現(xiàn)外界不同應(yīng)激條件誘導AQP1的表達與MAPK信號通路各亞族存在一定的相關(guān)性,但是對于MAPK信號通路是否參與低氧誘導雪旺細胞AQP1的表達,至今尚不清楚。因此,本課題通過三氣培養(yǎng)箱體外構(gòu)建雪旺細胞低氧模型,模擬面神經(jīng)受損后形成的低氧環(huán)境,研究低氧對雪旺細胞形態(tài)變化及AQP1、MAPK(P-p38、P-ERK、P-JNK)表達變化的影響;分別使用p38、ERK、JNK信號通路的抑制劑在低氧前1h對雪旺細胞預(yù)處理,研究其對低氧環(huán)境誘導雪旺細胞AQP1表達變化的影響,探討低氧誘導AQP1的表達與MAPK信號通路各個亞族之間的關(guān)系,揭示低氧損傷誘導雪旺細胞AQP1表達變化的相關(guān)分子機制,為終止面神經(jīng)受損傷后的“水腫-缺血缺氧-水腫”惡性循環(huán)提供理論基礎(chǔ)�！灸康摹�1、體外構(gòu)建雪旺細胞低氧模型,模擬面神經(jīng)受損后形成的低氧環(huán)境,為后續(xù)實驗提供可靠而穩(wěn)定的細胞模型;2、研究低氧條件下雪旺細胞AQP1在蛋白水平和mRNA水平的表達變化;3、研究低氧條件下雪旺細胞P-p38、P-ERK、P-JNK的表達變化;4、初步研究低氧誘導AQP1表達與MAPK信號通路各亞族的關(guān)系,揭示低氧條件下AQP1表達變化可能分子機制�！痉椒ā�1、雪旺細胞低氧模型的構(gòu)建(1)細胞培養(yǎng);(2)免疫熒光染色鑒定rsc96細胞aqp1表達;(3)cck8檢測低氧對細胞活力的影響。2、低氧對雪旺細胞aqp1及mapk信號通路的影響的研究(1)westernblot檢測低氧條件下aqp1表達變化;(2)realtime-pcr檢測低氧條件下aqp1mrna表達變化;(3)westernblot檢測低氧條件下p-p38、p38、p-erk、erk、p-jnk、jnk表達變化。3、低氧誘導雪旺細胞aqp1表達機制的研究(1)p38信號通路對低氧誘導雪旺細胞aqp1表達變化的研究westernblot檢測低氧條件下sb203580預(yù)處理的雪旺細胞p-p38表達變化westernblot檢測低氧條件下sb203580預(yù)處理的雪旺細胞aqp1表達變化(2)erk信號通路對低氧誘導雪旺細胞aqp1表達變化的研究westernblot檢測低氧條件下u0126預(yù)處理的雪旺細胞p-erk表達變化westernblot檢測低氧條件下u0126預(yù)處理的雪旺細胞aqp1表達變化(3)jnk信號通路對低氧誘導雪旺細胞aqp1表達變化的研究westernblot檢測低氧條件下sp600125預(yù)處理的雪旺細胞p-jnk表達變化westernblot檢測低氧條件下sp600125預(yù)處理的雪旺細胞aqp1表達變化【結(jié)果】1、免疫熒光顯示rsc96細胞表達aqp1;2、cck8細胞活力檢測顯示在低氧9小時內(nèi)各時間點細胞吸光度差異均無統(tǒng)計學意義(p0.05),低氧時間小于9小時對細胞活力無明顯影響,低氧12小時和24小時實驗組與對照組吸光度的差異有統(tǒng)計學意義(p0.05),說明在低氧12小時以后細胞的活力明顯受到抑制;3、rsc96細胞低氧后蛋白表達水平逐漸增加,在低氧1小時aqp1表達無明顯變化(p0.05),在低氧3小時、6小時aqp1表達呈逐漸上升趨勢,低氧6小時aqp1表達最多,差異有統(tǒng)計學意義(p0.05),隨后aqp1的表達逐漸降低;4、rsc96細胞低氧后mrna表達水平逐漸增加,低氧1h、3h、6h組aqp1mrna水平呈逐漸上升趨勢,且在低氧6小時aqp1mrna表達最多,差異有統(tǒng)計學意義(p0.05),隨后逐漸降低;5、rsc96細胞低氧后p-p38、p-erk、p-jnk均被激活,從低氧15min到低氧1小時呈逐漸上升趨勢,且在低氧1小時達到最大,差異有統(tǒng)計學意義(p0.05),隨后逐漸降低;6、低氧+抑制劑組與低氧組相比P-p38、P-ERK、P-JNK表達均被抑制(P0.05);7、在低氧前分別使用SB203580、U0126以及SP600125干預(yù),低氧+SB203580組以及低氧+U0126組與低氧組相比AQP1表達降低,差異有統(tǒng)計學意義(P0.05),低氧+SP600125組與低氧組相比AQP1表達未見明顯變化,差異無統(tǒng)計學意義(P0.05)�！窘Y(jié)論】1、AQP1表達于RSC96細胞;2、低氧可誘導雪旺細胞AQP1在基因及蛋白水平表達增加;3、低氧可誘導雪旺細胞p38、ERK、JNK磷酸化表達改變;4、低氧可能通過p38及ERK信號通路調(diào)節(jié)AQP1表達變化。
[Abstract]:[background] BELL 's facial paralysis is most common in facial nerve injury, and its initial cause is not yet clear. But through laboratory anatomy and clinical facial nerve canal decompression, the "edema ischemic anoxedematous circulation" cycle of the nourishing vessels of the facial nerve caused by ischemic anoxia edema caused by the stenosis of the osseous lumen is found. It is the direct cause of neurodegenerative necrosis. Therefore, the vicious cycle that terminates the "edema ischemic hypoxia edema" is more important in the treatment of facial nerve injury. The aquaporin is a class of membrane protein family with rapid transport function on the membrane of the cell membrane, which is now occurring in various tissues and organs. 13 kinds of aquaporins (AQP0-12) have been presented. Our previous work has confirmed that AQP1 exists on Schwann cells in facial nerve and is closely related to the edema of the facial nerve and Schwann cells. The.MAPK signal pathway is the intersection point of cell information transmission. It can transfer information from the cell to the nucleus, which can be directly or indirectly regulated. The transcriptional activity and gene expression are the main signaling pathways in cell stress and damage feedback, which exist widely in all kinds of human cells. Some studies have shown that AQP1 expression can be regulated by hypoxia, osmotic stress, and inflammatory mediators. At the same time, the expression of AQP1 and the subgroups of MAPK signaling pathway are also found to be induced by different stress conditions. In a certain correlation, however, it is not clear whether the MAPK signaling pathway is involved in hypoxia induced Schwann cell AQP1 expression, so it is not clear so far. Therefore, we constructed the hypoxic model of Schwann cells in the three gas incubator to simulate the hypoxia environment after the damage of the facial nerve, and study the morphologic changes of Schwann cells and the AQP1, MAPK (P-p38, P-) of hypoxia. The effect of ERK, P-JNK) on the expression changes; using the inhibitors of p38, ERK, and JNK signaling pathway to pre treat Schwann cells before hypoxia, respectively, to study the effect of 1H on the expression of Schwann cells induced by hypoxia environment, and to explore the relationship between the expression of hypoxia induced AQP1 and the subgroups of MAPK signaling pathway, and to reveal the induced hypoxia injury induced Schwann fine. The molecular mechanism of the change of AQP1 expression provides a theoretical basis for the termination of the "edema ischemic anoxedemanoedema" vicious cycle after the facial nerve injury. [Objective] 1, to construct a model of Schwann cell hypoxia in vitro, to simulate the hypoxic environment formed after the damage of the facial nerve, and to provide a reliable and stable cell model for subsequent test. 2, low research. Changes in protein level and mRNA level of Schwann cell AQP1 under oxygen condition; 3, study the expression changes of P-p38, P-ERK, P-JNK of Schwann cells under hypoxia; 4, preliminary study on the relationship between AQP1 expression and MAPK signaling subgroups in hypoxia, and reveal the possible molecular mechanism of AQP1 expression changes under hypoxia. [method] 1, Schwann cells are low Construction of oxygen model (1) cell culture (1) cell culture, (2) immunofluorescence staining identification of AQP1 expression in rsc96 cells; (3) the effect of hypoxia on cell viability,.2, the effect of hypoxia on the AQP1 and MAPK signaling pathway of Schwann cells (1) Westernblot detection of AQP1 table changes under the condition of hypoxia; (2) realtime-pcr detection of aqp1mrna expression changes under hypoxic conditions (3) (3) Westernblot detection of p-p38, p38, p-ERK, ERK, p-JNK, JNK expression,.3, and AQP1 expression mechanism of Schwann cells induced by hypoxia (1) p38 signaling pathway on the AQP1 expression of Schwann cells induced by hypoxia Changes in AQP1 expression of Schwann cells in SB203580 pretreated under hypoxia (2) a study on the changes of AQP1 expression in Schwann cells induced by hypoxia in ERK signal pathway Westernblot detection of p-ERK expression change of U0126 pretreated Schwann cells under hypoxia condition Westernblot detection of AQP1 expression of Schwann cells in U0126 pretreated under hypoxia condition (3) JNK letter Study on changes of expression of AQP1 in hypoxia induced Schwann cells Westernblot detection of p-JNK expression changes in sp600125 pretreated Schwann cells under hypoxic conditions Westernblot detection of AQP1 expression in sp600125 pretreated Schwann cells under hypoxia [results] 1, immunofluorescent rsc96 cells expressed AQP1; 2, CCK8 cell viability test The results showed that there was no significant difference in the cell absorbency at all time points in 9 hours (P0.05), and the hypoxia time was less than 9 hours, but the difference in the absorbance between the experimental group and the control group was statistically significant (P0.05) for 12 hours and 24 hours of hypoxia (P0.05), and the cell viability was obviously inhibited after 12 hours of hypoxia. 3, the protein expression level of rsc96 cells increased gradually after hypoxia (P0.05) in 1 hours of hypoxia (P0.05). The expression of AQP1 increased gradually in 3 hours and 6 hours in hypoxia. The expression of AQP1 was most significant in 6 hours of hypoxia. The difference was statistically significant (P0.05), and the expression of AQP1 decreased gradually. 4, the mRNA expression level of rsc96 cells increased gradually after hypoxia. In addition, the aqp1mrna level of the hypoxia 1H, 3h, 6h group increased gradually, and the expression of aqp1mrna was the most in the 6 hour hypoxia. The difference was statistically significant (P0.05), and then decreased gradually. 5, p-p38, p-ERK, p-JNK were activated after the hypoxia of rsc96 cells, and increased gradually from low oxygen 15min to low oxygen 1, and reached the maximum in 1 hours of hypoxia, and the difference was statistically significant. Learning significance (P0.05), and then gradually decreased; 6, P-p38, P-ERK, P-JNK expression in hypoxia + inhibitor group were inhibited compared with hypoxia group (P0.05); 7, SB203580, U0126 and SP600125 intervention were used before hypoxia, and low oxygen +SB203580 group and hypoxia +U0126 group were lower than hypoxia group, and the difference was statistically significant (P0.05) and hypoxia. There was no significant change in the expression of AQP1 in the 5 groups compared with the hypoxia group (P0.05). [Conclusion] 1, AQP1 was expressed in RSC96 cells; 2, hypoxia could induce the expression of AQP1 in the gene and protein level of Schwann cells; 3, hypoxia could induce p38, ERK, JNK, phosphorylated expression of Schwann cells; 4, hypoxia may be modulated by p38 and ERK signaling pathways. The expression of AQP1 was changed.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R745.12
本文編號：2160152
[Abstract]:[background] BELL 's facial paralysis is most common in facial nerve injury, and its initial cause is not yet clear. But through laboratory anatomy and clinical facial nerve canal decompression, the "edema ischemic anoxedematous circulation" cycle of the nourishing vessels of the facial nerve caused by ischemic anoxia edema caused by the stenosis of the osseous lumen is found. It is the direct cause of neurodegenerative necrosis. Therefore, the vicious cycle that terminates the "edema ischemic hypoxia edema" is more important in the treatment of facial nerve injury. The aquaporin is a class of membrane protein family with rapid transport function on the membrane of the cell membrane, which is now occurring in various tissues and organs. 13 kinds of aquaporins (AQP0-12) have been presented. Our previous work has confirmed that AQP1 exists on Schwann cells in facial nerve and is closely related to the edema of the facial nerve and Schwann cells. The.MAPK signal pathway is the intersection point of cell information transmission. It can transfer information from the cell to the nucleus, which can be directly or indirectly regulated. The transcriptional activity and gene expression are the main signaling pathways in cell stress and damage feedback, which exist widely in all kinds of human cells. Some studies have shown that AQP1 expression can be regulated by hypoxia, osmotic stress, and inflammatory mediators. At the same time, the expression of AQP1 and the subgroups of MAPK signaling pathway are also found to be induced by different stress conditions. In a certain correlation, however, it is not clear whether the MAPK signaling pathway is involved in hypoxia induced Schwann cell AQP1 expression, so it is not clear so far. Therefore, we constructed the hypoxic model of Schwann cells in the three gas incubator to simulate the hypoxia environment after the damage of the facial nerve, and study the morphologic changes of Schwann cells and the AQP1, MAPK (P-p38, P-) of hypoxia. The effect of ERK, P-JNK) on the expression changes; using the inhibitors of p38, ERK, and JNK signaling pathway to pre treat Schwann cells before hypoxia, respectively, to study the effect of 1H on the expression of Schwann cells induced by hypoxia environment, and to explore the relationship between the expression of hypoxia induced AQP1 and the subgroups of MAPK signaling pathway, and to reveal the induced hypoxia injury induced Schwann fine. The molecular mechanism of the change of AQP1 expression provides a theoretical basis for the termination of the "edema ischemic anoxedemanoedema" vicious cycle after the facial nerve injury. [Objective] 1, to construct a model of Schwann cell hypoxia in vitro, to simulate the hypoxic environment formed after the damage of the facial nerve, and to provide a reliable and stable cell model for subsequent test. 2, low research. Changes in protein level and mRNA level of Schwann cell AQP1 under oxygen condition; 3, study the expression changes of P-p38, P-ERK, P-JNK of Schwann cells under hypoxia; 4, preliminary study on the relationship between AQP1 expression and MAPK signaling subgroups in hypoxia, and reveal the possible molecular mechanism of AQP1 expression changes under hypoxia. [method] 1, Schwann cells are low Construction of oxygen model (1) cell culture (1) cell culture, (2) immunofluorescence staining identification of AQP1 expression in rsc96 cells; (3) the effect of hypoxia on cell viability,.2, the effect of hypoxia on the AQP1 and MAPK signaling pathway of Schwann cells (1) Westernblot detection of AQP1 table changes under the condition of hypoxia; (2) realtime-pcr detection of aqp1mrna expression changes under hypoxic conditions (3) (3) Westernblot detection of p-p38, p38, p-ERK, ERK, p-JNK, JNK expression,.3, and AQP1 expression mechanism of Schwann cells induced by hypoxia (1) p38 signaling pathway on the AQP1 expression of Schwann cells induced by hypoxia Changes in AQP1 expression of Schwann cells in SB203580 pretreated under hypoxia (2) a study on the changes of AQP1 expression in Schwann cells induced by hypoxia in ERK signal pathway Westernblot detection of p-ERK expression change of U0126 pretreated Schwann cells under hypoxia condition Westernblot detection of AQP1 expression of Schwann cells in U0126 pretreated under hypoxia condition (3) JNK letter Study on changes of expression of AQP1 in hypoxia induced Schwann cells Westernblot detection of p-JNK expression changes in sp600125 pretreated Schwann cells under hypoxic conditions Westernblot detection of AQP1 expression in sp600125 pretreated Schwann cells under hypoxia [results] 1, immunofluorescent rsc96 cells expressed AQP1; 2, CCK8 cell viability test The results showed that there was no significant difference in the cell absorbency at all time points in 9 hours (P0.05), and the hypoxia time was less than 9 hours, but the difference in the absorbance between the experimental group and the control group was statistically significant (P0.05) for 12 hours and 24 hours of hypoxia (P0.05), and the cell viability was obviously inhibited after 12 hours of hypoxia. 3, the protein expression level of rsc96 cells increased gradually after hypoxia (P0.05) in 1 hours of hypoxia (P0.05). The expression of AQP1 increased gradually in 3 hours and 6 hours in hypoxia. The expression of AQP1 was most significant in 6 hours of hypoxia. The difference was statistically significant (P0.05), and the expression of AQP1 decreased gradually. 4, the mRNA expression level of rsc96 cells increased gradually after hypoxia. In addition, the aqp1mrna level of the hypoxia 1H, 3h, 6h group increased gradually, and the expression of aqp1mrna was the most in the 6 hour hypoxia. The difference was statistically significant (P0.05), and then decreased gradually. 5, p-p38, p-ERK, p-JNK were activated after the hypoxia of rsc96 cells, and increased gradually from low oxygen 15min to low oxygen 1, and reached the maximum in 1 hours of hypoxia, and the difference was statistically significant. Learning significance (P0.05), and then gradually decreased; 6, P-p38, P-ERK, P-JNK expression in hypoxia + inhibitor group were inhibited compared with hypoxia group (P0.05); 7, SB203580, U0126 and SP600125 intervention were used before hypoxia, and low oxygen +SB203580 group and hypoxia +U0126 group were lower than hypoxia group, and the difference was statistically significant (P0.05) and hypoxia. There was no significant change in the expression of AQP1 in the 5 groups compared with the hypoxia group (P0.05). [Conclusion] 1, AQP1 was expressed in RSC96 cells; 2, hypoxia could induce the expression of AQP1 in the gene and protein level of Schwann cells; 3, hypoxia could induce p38, ERK, JNK, phosphorylated expression of Schwann cells; 4, hypoxia may be modulated by p38 and ERK signaling pathways. The expression of AQP1 was changed.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R745.12
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，本文編號：2160152
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