本文選題：細胞粘附激酶β + 血管內皮細胞生長因子��； 參考：《蘇州大學》2010年博士論文

【摘要】：【目的】視網膜新生血管形成(retinal neovascularization,RNV)是多種缺血缺氧性眼底病共有的病理改變。RNV會引起視網膜纖維組織增生,視網膜脫離,甚至視力喪失,已成為發(fā)達國家致盲性眼病的首要原因,也成為威脅我國人民視力健康的重要原因。明確RNV發(fā)生發(fā)展及其調控的分子機制,并進而尋找新的非侵入性的治療方法已成為眼底病研究的重要課題。細胞粘附激酶β(cellular adhesion kinaseβ, CAKβ)是一種能被細胞內鈣濃度增高及多種刺激信號激活的具有明顯的酪氨酸激酶和自身激酶活性的信號分子,參與細胞的生長、增殖、分化等,與細胞多種信號轉導途徑和多種生物學功能有密切關系。然而,CAKβ在RNV中的作用及其與VEGF的關系尚不清楚。本研究建立氧誘導視網膜新生血管形成模型,觀察VEGF、CAKβ基因及蛋白的表達,探討CAKβ通路在RNV中的作用以及氟伐他汀、厄貝沙坦干預對其的影響。 【方法】參照文獻建立氧誘導的小鼠視網膜新生血管形成(oxygen-induced retinopathy, OIR)模型。(1)選擇健康7日齡(P7)C57BL/6J小鼠,共80只,雌雄不拘。隨機分為高氧組(OIR組)和對照組(C組)。分別在P12、P14、P17、P19處死幼鼠。(2)選擇P7 C57BL/6J小鼠共180只,雌雄不拘。隨機分為C組、OIR組及模型+貝伐單抗組(BVZ組)。BVZ組在P12時腹腔麻醉后玻璃體內注射貝伐單抗5mg/Kg,分別在P12、P14、P17處死幼鼠。(3)選擇P7 C57BL/6J小鼠共140只,雌雄不拘。隨機分為5組:C組、模型+氟伐他汀組(FVS組)(P12時腹腔麻醉后雙眼玻璃體內注射氟伐他汀10mg/Kg/d)、模型+厄貝沙坦組(IBS組)(P12時腹腔麻醉后雙眼玻璃體內注射厄貝沙坦50mg/Kg/d)、模型+氟伐他汀組+厄貝沙坦組(FVS+IBS組)(P12時腹腔麻醉后雙眼玻璃體內注射同上劑量的氟伐他汀及厄貝沙坦)、OIR組。P17處死幼鼠。(4)視網膜組織切片及鋪片定性及定量觀察RNV;2’,7’-二氯雙氫熒光素雙乙酸酯(DCFH-DA)熒光探針檢測細胞內活性氧(ROS)水平,實時RT-PCR檢測VEGF mRNA、CAKβmRNA表達,Western blot技術檢測VEGF蛋白、CAKβ及其Tyr402磷酸化蛋白表達。 【結果】(1)將P7幼鼠置于75%高氧環(huán)境5天后移至正常氧環(huán)境,可成功誘導出RNV模型。P14即可產生視網膜新生血管,P17新生血管最明顯。新生血管內皮細胞內ROS水平改變與定量及定性觀察到的視網膜新生血管變化相一致。(2)正常新生小鼠P12時,視網膜VEGF mRNA及蛋白表達,CAKβmRNA及蛋白表達,CAKβTyr402/CAKβ比值均處于最高;OIR組自P12開始,VEGF mRNA及蛋白表達,CAKβmRNA及蛋白及磷酸化蛋白表達顯著升高,到P17時達高峰,CAKβTyr402/CAKβ比值在P14,P17也顯著高于對照組;貝伐單抗干預后第2天,VEGF mRNA及蛋白表達即顯著低于同時段OIR組,與同時段C組無顯著差異,其后一直處于較低水平。CAKβmRNA及蛋白表達也顯著低于同時段OIR組,但仍顯著高于同時段C組。表明貝伐單抗對于VEGF及CAKβ的表達均具有良好的抑制作用,但對CAKβ的抑制效率不及VEGF。(3)厄貝沙坦干預組及氟伐他汀干預組新生血管增生情況明顯延緩,突破內界膜的細胞核計數均顯著少于非干預組,細胞內ROS水平、VEGF mRNA及蛋白表達、CAKβmRNA以及CAKβ及其Tyr402磷酸化蛋白均較非干預組有顯著下降。而兩藥聯用在上述指標均優(yōu)于各自單獨用藥。 【結論】(1)氧化應激、VEGF及CAKβ在RNV中起重要作用。(2)貝伐單抗對VEGF及CAKβ均具有良好的抑制作用,但對CAKβ的抑制效率不及VEGF。(3)厄貝沙坦及氟伐他汀均能有效降低ROS水平、下調VEGF及CAKβ表達,改善血管新生。兩藥聯用更有利于改善視網膜新生血管形成。(4)CAKβ參與了RNV的多種信號通路, CAKβ可能是抗RNV治療的一個新的靶分子。
[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.
【學位授予單位】：蘇州大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R774.1

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