细胞粘附激酶β信号通路在氧诱导小鼠视网膜新生血管中的作用及厄贝沙坦、氟伐他汀的干预研究

发布时间：2018-07-01 19:28

本文选题：细胞粘附激酶β + 血管内皮细胞生长因子　；参考：《苏州大学》2010年博士论文

【摘要】：【目的】视网膜新生血管形成(retinal neovascularization,RNV)是多种缺血缺氧性眼底病共有的病理改变。RNV会引起视网膜纤维组织增生,视网膜脱离,甚至视力丧失,已成为发达国家致盲性眼病的首要原因,也成为威胁我国人民视力健康的重要原因。明确RNV发生发展及其调控的分子机制,并进而寻找新的非侵入性的治疗方法已成为眼底病研究的重要课题。细胞粘附激酶β(cellular adhesion kinaseβ, CAKβ)是一种能被细胞内钙浓度增高及多种刺激信号激活的具有明显的酪氨酸激酶和自身激酶活性的信号分子,参与细胞的生长、增殖、分化等,与细胞多种信号转导途径和多种生物学功能有密切关系。然而,CAKβ在RNV中的作用及其与VEGF的关系尚不清楚。本研究建立氧诱导视网膜新生血管形成模型,观察VEGF、CAKβ基因及蛋白的表达,探讨CAKβ通路在RNV中的作用以及氟伐他汀、厄贝沙坦干预对其的影响。【方法】参照文献建立氧诱导的小鼠视网膜新生血管形成(oxygen-induced retinopathy, OIR)模型。(1)选择健康7日龄(P7)C57BL/6J小鼠,共80只,雌雄不拘。随机分为高氧组(OIR组)和对照组(C组)。分别在P12、P14、P17、P19处死幼鼠。(2)选择P7 C57BL/6J小鼠共180只,雌雄不拘。随机分为C组、OIR组及模型+贝伐单抗组(BVZ组)。BVZ组在P12时腹腔麻醉后玻璃体内注射贝伐单抗5mg/Kg,分别在P12、P14、P17处死幼鼠。(3)选择P7 C57BL/6J小鼠共140只,雌雄不拘。随机分为5组:C组、模型+氟伐他汀组(FVS组)(P12时腹腔麻醉后双眼玻璃体内注射氟伐他汀10mg/Kg/d)、模型+厄贝沙坦组(IBS组)(P12时腹腔麻醉后双眼玻璃体内注射厄贝沙坦50mg/Kg/d)、模型+氟伐他汀组+厄贝沙坦组(FVS+IBS组)(P12时腹腔麻醉后双眼玻璃体内注射同上剂量的氟伐他汀及厄贝沙坦)、OIR组。P17处死幼鼠。(4)视网膜组织切片及铺片定性及定量观察RNV;2’,7’-二氯双氢荧光素双乙酸酯(DCFH-DA)荧光探针检测细胞内活性氧(ROS)水平,实时RT-PCR检测VEGF mRNA、CAKβmRNA表达,Western blot技术检测VEGF蛋白、CAKβ及其Tyr402磷酸化蛋白表达。【结果】(1)将P7幼鼠置于75%高氧环境5天后移至正常氧环境,可成功诱导出RNV模型。P14即可产生视网膜新生血管,P17新生血管最明显。新生血管内皮细胞内ROS水平改变与定量及定性观察到的视网膜新生血管变化相一致。(2)正常新生小鼠P12时,视网膜VEGF mRNA及蛋白表达,CAKβmRNA及蛋白表达,CAKβTyr402/CAKβ比值均处于最高;OIR组自P12开始,VEGF mRNA及蛋白表达,CAKβmRNA及蛋白及磷酸化蛋白表达显著升高,到P17时达高峰,CAKβTyr402/CAKβ比值在P14,P17也显著高于对照组;贝伐单抗干预后第2天,VEGF mRNA及蛋白表达即显著低于同时段OIR组,与同时段C组无显著差异,其后一直处于较低水平。CAKβmRNA及蛋白表达也显著低于同时段OIR组,但仍显著高于同时段C组。表明贝伐单抗对于VEGF及CAKβ的表达均具有良好的抑制作用,但对CAKβ的抑制效率不及VEGF。(3)厄贝沙坦干预组及氟伐他汀干预组新生血管增生情况明显延缓,突破内界膜的细胞核计数均显著少于非干预组,细胞内ROS水平、VEGF mRNA及蛋白表达、CAKβmRNA以及CAKβ及其Tyr402磷酸化蛋白均较非干预组有显著下降。而两药联用在上述指标均优于各自单独用药。【结论】(1)氧化应激、VEGF及CAKβ在RNV中起重要作用。(2)贝伐单抗对VEGF及CAKβ均具有良好的抑制作用,但对CAKβ的抑制效率不及VEGF。(3)厄贝沙坦及氟伐他汀均能有效降低ROS水平、下调VEGF及CAKβ表达,改善血管新生。两药联用更有利于改善视网膜新生血管形成。(4)CAKβ参与了RNV的多种信号通路, CAKβ可能是抗RNV治疗的一个新的靶分子。
[Abstract]:[Objective] retinal neovascularization (retinal neovascularization, RNV) is a common pathological change of various hypoxic-ischemic fundus diseases..RNV can cause retinal fibrous tissue hyperplasia, retinal detachment and even loss of vision. It has become the first cause of blindness in developed countries. It has also become a serious threat to the vision and health of the people of our country. The molecular mechanism of RNV development and its regulation and the search for new non invasive treatment have become an important subject in the study of fundus disease. Cell adhesion kinase beta (cellular adhesion kinase beta, CAK beta) is a kind of tyrosine excitation with increased intracellular calcium concentration and activation of multiple stimuli. The signal molecules of enzyme and self kinase activity are involved in cell growth, proliferation and differentiation, which are closely related to a variety of signal transduction pathways and biological functions. However, the role of CAK beta in RNV and its relationship with VEGF is not clear. This study established an oxygen induced retinal neovascularization model and the observation of VEGF, CAK beta gene And protein expression, to explore the role of CAK beta pathway in RNV and the effect of fluvastatin on irbesartan intervention.
[Methods] to establish oxygen induced retinal neovascularization (oxygen-induced retinopathy, OIR) model in mice. (1) a total of 80 healthy 7 days old (P7) C57BL/6J mice were selected and randomly divided into the hyperoxia group (group OIR) and the control group (group C). The mice were killed in P12, P14, P17, and P19. (2) 180 mice were selected for P7. Male and female were randomly divided into group C, group OIR and model + bevacizumab group (group BVZ), group.BVZ was injected with bevacizumab 5mg/Kg after P12 intraperitoneal anesthesia, in P12, P14, P17 to die young rats respectively. (3) a total of 140 P7 C57BL/6J mice were selected and randomly divided into 5 groups: C group, model + fluvastatin group after abdominal anesthesia. Intravitreal injection of fluvastatin 10mg/Kg/d), model + irbesartan group (group IBS) (group P12 after P12 intraperitoneal anaesthesia injection of erbesartan 50mg/Kg/d), model + fluvastatin group + erbesartan group (FVS+IBS group) (P12 at the same dose of fluvastatin and erbesartan in the vitreous body after P12), and OIR group.P17 to death (4) (4) qualitative and quantitative observation of retinal tissue sections and slices; 2 ', 7' - two chlorodihydrofluorescein diacetate (DCFH-DA) fluorescence probe for detecting intracellular reactive oxygen species (ROS) level; real-time RT-PCR detection of VEGF mRNA, CAK beta mRNA expression, Western blot assay VEGF protein, CAK beta and phosphorylated protein expression.
[results] (1) P7 young rats were placed in the 75% hyperoxic environment and moved to the normal oxygen environment for 5 days. The RNV model.P14 could be successfully induced to produce retinal neovascularization, and the P17 neovascularization was the most obvious. The changes in the level of ROS in the neovascular endothelial cells were consistent with the quantitative and qualitative changes in the retinal neovascularization. (2) P12 of normal newborn mice. The expression of VEGF mRNA and protein in the retina, the expression of CAK beta mRNA and protein, and the ratio of CAK beta Tyr402/CAK beta were the highest. The OIR group started from P12, VEGF mRNA and protein expression, and the expression of CAK beta mRNA and protein and phosphorylated protein was significantly higher than that of the control group. On the second day, the expression of VEGF mRNA and protein was significantly lower than that of the simultaneous segment OIR group. There was no significant difference between the C group and the simultaneous segment C group. After that, the expression of.CAK beta mRNA and protein was significantly lower than that of the simultaneous segment OIR group, but it was still significantly higher than that of the simultaneous segment C group, which showed that bevacizumab had a good inhibitory effect on the expression of VEGF and CAK beta, but to CAK. The inhibition efficiency of beta was less than VEGF. (3) the proliferation of neovascularization in the intervention group and the intervention group of fluvastatin was significantly delayed. The number of cells breaking through the inner boundary membrane was significantly less than that in the non intervention group, the intracellular ROS level, the VEGF mRNA and the protein expression, and the CAK beta mRNA, CAK beta and its Tyr402 phosphorylation protein were significantly lower than those in the non intervention group. The combined use of the two drugs was superior to the respective drugs alone.
[Conclusion] (1) (1) oxidative stress, VEGF and CAK beta play an important role in RNV. (2) bevacizumab has a good inhibitory effect on VEGF and CAK beta, but the inhibition efficiency of CAK beta is less than that of VEGF. (3) erbesartan and fluvastatin can effectively reduce the level of ROS, reduce the expression of VEGF and CAK beta, and improve angiogenesis. Two drug combination is more beneficial to improve the vision. Retinal neovascularization. (4) CAK beta is involved in many signaling pathways of RNV. CAK beta may be a new target molecule for RNV treatment.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R774.1

【共引文献】