水通道蛋白4基因对慢性高眼压小鼠视网膜中胶质纤维酸性蛋白表达的调控

背景 本课题组先前的研究发现,神经胶质纤维酸性蛋白（GFAP）在慢性高眼压模型小鼠视网膜星形胶质细胞和Müller细胞中表达增加,这一改变与视网膜神经节细胞（RGCs）的改变及疾病的发生及发展密切相关.水通道蛋白4（AQP4）在视网膜主要存在于神经胶质细胞中,青光眼发病过程中AQP4与GFAP表达的关系尚不清楚. 目的 研究慢性高眼压状态下AQP4基因在慢性高眼压情况下是否能够调控视网膜神经胶质细胞中GFAP的表达,探讨AQP4基因在青光眼RGCs损害中的作用.方法 利用小鼠巩膜外静脉烧烙法（烧烙静脉3～4支）建立雄性小鼠AQP4基因敲除（AQP4-/-）小鼠及其同背景雄性野生型（WT）小鼠左眼慢性高眼压模型,均取右眼作为对照眼.选择造模成功的两种小鼠各30只,分别于术后1、3、7、14和28 d各取6只小鼠用Icare回弹式眼压计测量小鼠眼压,分别于相应时间点分离取小鼠视网膜,通过Western blot 法检测AQP4-/-小鼠及WT小鼠视网膜神经胶质细胞中GFAP水平的变化,用t检验、三因素方差分析及SNK-q检验分析实验数据.结果 造模后1、3、7、14和28 d,AQP4-/-小鼠实验眼眼压均明显高于对照眼,差异均有统计学意义（t=15.29、16.02、13.77、14.34、12.40,均P＜0.05）；上述各时间点WT小鼠实验眼眼压均明显高于对照眼,差异均有统计学意义（t=17.65、14.91、15.97、13.41、12.53,均P＜0.05）.正常情况下AQP4-/-小鼠与WT小鼠视网膜组织中均有GFAP表达,WT小鼠对照眼、实验眼造模后1、3、7、14和28 d,视网膜中GFAP的表达量（GFAP/β-actin）分别为1.00±0.00、1.99 ±0.29、4.05±0.69、4.47±0.48、3.21±0.35、3.25±0.53;AQP4-/-小鼠对照眼、实验眼术后1、3、7、14和28 d视网膜中GFAP相对表达量（GFAP/β-actin）分别为1.00±0.00、1.69±0.31、2.27±0.55、2.79±0.39、1.93±0.31和1.54±0.40；造模后不同时间点小鼠视网膜中GFAP表达量差异有统计学意义（F=9.54,P＜0.05）,WT小鼠随着造模后时间的延长,视网膜总GFAP的表达量逐渐升高,而AQP4-/-小鼠视网膜中总GFAP的相对表达量逐渐下降,差异均有统计学意义（P＜0.05）.正常情况下WT小鼠与AQP4-/-小鼠视网膜组织中GFAP/β-actin差异无统计学意义（P＞0.05）,WT小鼠术后3、7、14和28 d视网膜中GFAP的表达值均显著高于AQP4-/-小鼠,差异均有统计学意义（t=4.51、7.95、6.12、5.76,P＜0.01）. 结论 慢性高眼压状态下AQP4基因可以上调小鼠视网膜神经胶质细胞中GFAP的表达,从而引起视网膜神经胶质细胞的活化,导致RGCs的损害.AQP4可能成为治疗青光眼的新靶点.

Regulating effect of aquaporin 4 gene on the expression of glial fibrillary acid protein in retina in chronic high intraocular pressure mice

Background Our previous study showed that the expression level of glial fibrillary acid protein （GFAP） increases in astrocytes and Müller cells of retina in chronic hypertensive eye,and this change was clarified to be associated with the damage process of the retinal ganglion cells （RGCs）.Aquaporin 4 （AQP4） exists in neural glial cells,so we conjecture AQP4 plays a role in the regulating GFAP expression in glaucomatous eye.Objectives This study was to investigate whether AQP4 gene can regulate the expression of GFAP in retina and explore the effect of AQP4 on RGCs damage of glaucoma.Methods Chronic ocular hypertensive eye models were established by cauterizing the scleral venous in the left eyes of 30 male AQP4-/-mice and 30 male wild type （WT） mice with the same background,and the right eyes served as control eyes.The intraocular pressure （IOP） was measured with Icare rebound tonometer at 1 day,3,7,14 and 28 days respectively and the retinas were isolated from 6 of each types of mice at the corresponding time points.The expression of GFAP in the retina was detected by Western blot.The use and care of the experimental animals followed ARVO Statement.Results The IOP was significantly higher in the model eyes than that of the control eyes 1 day,3,7,14 and 28 days in both AQP-/-mice （t =15.29,16.02,13.77,14.34,12.40,all at P＜0.05） and WT mice （t =17.65,14.91,15.97,13.41,12.53,all at P ＜0.05）.GFAP was expressed in the control eyes both of the AQP4-/-mice and the WT mice.The expressing level of GFAP （GFAP/β-actin） in retinas was 1.00±0.00,1.99±0.29,4.05±0.69,4.47±0.48,3.21±0.35 and 3.25±0.53 in the control eyes and 1-,3-,7-,14-,28-day model eyes of WT mice; and those in the AQP4-/-mice were 1.00±0.00,1.69±0.31,2.27 ±0.55,2.79 ± 0.39,1.93 ± 0.31 and 1.54 ± 0.40,with a significant difference in the expressing level of GFAP in various time points （F =9.54,P＜0.05）.In addition,significant gradually elevation of GFAP expression were seen in the WT mice and gradually decline of GFAP expression was found in the AQP4-/-mice with the lapse of time （all at P＜0.05）.No significant difference was seen in the expression of GFAP in the control eyes between the WT mice and AQP4-/-mice （P＞0.05）.However,the expression level of GFAP in retina was significantly higher in the WT mice than that of A QP4-/-mice 3,7,14 and 28 days after operation （t =4.51,7.95,6.12,5.76,all at P＜0.01）.tonelusions In chronic high IOP mice,AQP4 gene plays an important role in retinal damage by upregulating the expression of GFAP in retina and promoting the activation of RGCs.AQP4 probably is a new target of treatment of glaucoma.