低氧诱导雪旺细胞AQP1表达机制的相关实验研究
发布时间：2018-08-02 17:27
【摘要】：【研究背景】BELL’s面瘫在面神经损伤中最为常见,其初始病因目前尚未明确,但通过实验室解剖以及临床面神经管减压术中发现面神经因缺血缺氧水肿致滋养血管受狭窄骨性管腔的卡压而形成的“水肿-缺血缺氧-水肿”恶性循环是导致神经变性坏死的直接原因。因此,终止“水肿-缺血缺氧-水肿”的恶性循环在面神经损伤的治疗中显得更加重要。水通道蛋白是一类在细胞膜上对水分子具有快速转运功能的膜通道蛋白家族,目前已于各种不同的组织器官中发现了13种水通道蛋白(AQP0-12),我们课题组前期工作已证实AQP1存在于面神经的雪旺细胞上,并且与面神经及雪旺细胞的水肿紧密相关。MAPK信号通路是细胞信息传递的交汇点,能够将细胞外的信息传递至细胞核,可以直接或间接的调控转录因子活性及基因的表达,是细胞应激和损伤反馈中的主要信号通路,广泛存在于人体各类细胞中。有研究表明,AQP1表达可被低氧、渗透性应激、炎症介质等调节,同时研究还发现外界不同应激条件诱导AQP1的表达与MAPK信号通路各亚族存在一定的相关性,但是对于MAPK信号通路是否参与低氧诱导雪旺细胞AQP1的表达,至今尚不清楚。因此,本课题通过三气培养箱体外构建雪旺细胞低氧模型,模拟面神经受损后形成的低氧环境,研究低氧对雪旺细胞形态变化及AQP1、MAPK(P-p38、P-ERK、P-JNK)表达变化的影响;分别使用p38、ERK、JNK信号通路的抑制剂在低氧前1h对雪旺细胞预处理,研究其对低氧环境诱导雪旺细胞AQP1表达变化的影响,探讨低氧诱导AQP1的表达与MAPK信号通路各个亚族之间的关系,揭示低氧损伤诱导雪旺细胞AQP1表达变化的相关分子机制,为终止面神经受损伤后的“水肿-缺血缺氧-水肿”恶性循环提供理论基础。【目的】1、体外构建雪旺细胞低氧模型,模拟面神经受损后形成的低氧环境,为后续实验提供可靠而稳定的细胞模型;2、研究低氧条件下雪旺细胞AQP1在蛋白水平和mRNA水平的表达变化;3、研究低氧条件下雪旺细胞P-p38、P-ERK、P-JNK的表达变化;4、初步研究低氧诱导AQP1表达与MAPK信号通路各亚族的关系,揭示低氧条件下AQP1表达变化可能分子机制。【方法】1、雪旺细胞低氧模型的构建(1)细胞培养;(2)免疫荧光染色鉴定rsc96细胞aqp1表达;(3)cck8检测低氧对细胞活力的影响。2、低氧对雪旺细胞aqp1及mapk信号通路的影响的研究(1)westernblot检测低氧条件下aqp1表达变化;(2)realtime-pcr检测低氧条件下aqp1mrna表达变化;(3)westernblot检测低氧条件下p-p38、p38、p-erk、erk、p-jnk、jnk表达变化。3、低氧诱导雪旺细胞aqp1表达机制的研究(1)p38信号通路对低氧诱导雪旺细胞aqp1表达变化的研究westernblot检测低氧条件下sb203580预处理的雪旺细胞p-p38表达变化westernblot检测低氧条件下sb203580预处理的雪旺细胞aqp1表达变化(2)erk信号通路对低氧诱导雪旺细胞aqp1表达变化的研究westernblot检测低氧条件下u0126预处理的雪旺细胞p-erk表达变化westernblot检测低氧条件下u0126预处理的雪旺细胞aqp1表达变化(3)jnk信号通路对低氧诱导雪旺细胞aqp1表达变化的研究westernblot检测低氧条件下sp600125预处理的雪旺细胞p-jnk表达变化westernblot检测低氧条件下sp600125预处理的雪旺细胞aqp1表达变化【结果】1、免疫荧光显示rsc96细胞表达aqp1;2、cck8细胞活力检测显示在低氧9小时内各时间点细胞吸光度差异均无统计学意义(p0.05),低氧时间小于9小时对细胞活力无明显影响,低氧12小时和24小时实验组与对照组吸光度的差异有统计学意义(p0.05),说明在低氧12小时以后细胞的活力明显受到抑制;3、rsc96细胞低氧后蛋白表达水平逐渐增加,在低氧1小时aqp1表达无明显变化(p0.05),在低氧3小时、6小时aqp1表达呈逐渐上升趋势,低氧6小时aqp1表达最多,差异有统计学意义(p0.05),随后aqp1的表达逐渐降低;4、rsc96细胞低氧后mrna表达水平逐渐增加,低氧1h、3h、6h组aqp1mrna水平呈逐渐上升趋势,且在低氧6小时aqp1mrna表达最多,差异有统计学意义(p0.05),随后逐渐降低;5、rsc96细胞低氧后p-p38、p-erk、p-jnk均被激活,从低氧15min到低氧1小时呈逐渐上升趋势,且在低氧1小时达到最大,差异有统计学意义(p0.05),随后逐渐降低;6、低氧+抑制剂组与低氧组相比P-p38、P-ERK、P-JNK表达均被抑制(P0.05);7、在低氧前分别使用SB203580、U0126以及SP600125干预,低氧+SB203580组以及低氧+U0126组与低氧组相比AQP1表达降低,差异有统计学意义(P0.05),低氧+SP600125组与低氧组相比AQP1表达未见明显变化,差异无统计学意义(P0.05)。【结论】1、AQP1表达于RSC96细胞;2、低氧可诱导雪旺细胞AQP1在基因及蛋白水平表达增加;3、低氧可诱导雪旺细胞p38、ERK、JNK磷酸化表达改变;4、低氧可能通过p38及ERK信号通路调节AQP1表达变化。
[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.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R745.12
本文编号：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.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R745.12
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