FGF21在大脑神经元细胞和脑内皮细胞中的表达和与受体的相互作用

发布时间：2018-05-11 08:37

本文选题：T2D鼠 + 胰岛素敏感性　；参考：《湖南师范大学》2015年博士论文

【摘要】：第一部分成纤维生长因子21(FGF21)对糖尿病鼠的快速降糖作用目的：已有大量研究表明FGF21对糖代谢有调控作用,连续给药7到14天后可以降低2型糖尿病型高血糖,还能增强胰岛素的敏感性,降低胰岛素抵抗。但很少有研究FGF21的快速降糖的作用。快速降糖对于以高血糖为并发症或者为主要危险因素的疾病例如急性创伤、炎症以及心脑血管疾病等有重要的意义。方法：本实验先用温州医学院提供的人重组FGF21蛋白注射2型糖尿病小鼠(T2D小鼠)14天,用血糖试纸检测小鼠随机血糖。再用Elisa法检测小鼠胰岛素水平。之后,在注射FGF211小时后,用血糖试纸检测小鼠血糖。结果：小鼠随机血糖明显下降并且增强了胰岛素敏感性和降低了胰岛素抵抗。注射FGF211小时后,T2D小鼠血糖和模拟应激性高血糖小鼠血糖都明显下降。提示FGF 21可能也有快速降低血糖的作用。第二部分血清FGF21水平对脑内FGF21表达水平的影响目的：有研究表明FGF21在患有心血管疾病、肝脏疾病、高血压、肥胖、糖尿病等疾病时血清水平显著增加。可能的机制是机体是对FGF21的信号传导发生了障碍,对FGF21的敏感性减弱,才使FGF21过量表达。非常重要一点是有研究表明FGF21可以穿过血脑屏障(BBB)。但同时有研究发现FGF21需要通过细胞上的穿膜蛋白β-Klotho才能与成纤维因子受体(FGFR)结合而激活受体,这使FGF21的作用有组织选择性。而FGF21是否能进入大脑,大脑中是否有β-Klotho的表达和FGF21的受体激活机制还没有研究。方法：本实验用Elisa法检测非高血糖小鼠(ND小鼠)和2型糖尿病高血糖小鼠(T2D) FGF21血清水平。用western blot检测ND小鼠的T2D小鼠的全脑蛋白做中FGF21表达水平。用western blot检测静脉注射外源性FGF21至小鼠后,同的时间点小鼠大脑的不同部位的FGF21、pFGFRl和β-Klotho的表达水平。结果：血清中FGF21在T2D小鼠中明显高于ND小鼠。T2D小鼠大脑蛋白中的FGF21表达显著性高于ND小鼠。静脉注射外源性FGF21至小鼠后不同的时间点收集大脑蛋白,做western blot检测,发现大脑不同部分FGF21的表达的和成纤维成长因子受体1(FGFR1)的磷酸化都发生了变化,并且检测到了β-Klotho的表达。这表明了血清中FGF21的升高对大脑中FGF21的含量和FGFR1受体的激活有影响。但脑中FGF21的含量升高除了外源性FGF21直接进入,是否还存在被诱导升高尚不清楚；FGFR1的磷酸化是FGF21直接激活还是FGF21诱导了其他FGF家族的因子升高而激活的也尚不能定论,但由于检测到了β-Klotho的表达,也有可能可以直接激活受体。这一结果表明FGF21可以穿过血脑屏障进入大脑,也许可以直接作用于中枢神经系统产生作用。第三部分 FGF21对脑内皮细胞的影响目的：神经保护不局限于神经元细胞的保护,对血脑屏障的保护也非常关键。血清中升高的FGF21直接接触到血管内皮细胞,但FGF21对血管内皮细胞是否有保护作用目前以及保护机制没有研究。在血管内皮中FGF21的表达和损伤后的表达变化,传导通路和作用机制,以及受体复合物形式都有待研究。方法：本研究应用Western blot检测FGF21在血管内皮细胞中的表达,缺氧缺糖(OGD)4小时再复氧复糖20小时候后FGF21的表达变化,以及血管内皮细胞被重组FGF21蛋白处理后磷酸化FGFR1和β-Klotho的表达变化,是否对FGF21有时间依赖和剂量依赖。使用RNA干扰(SiRNA)技术,转染FGF21 SiRNA后用Western blot检测分析,以确认血管内皮中FGF21细胞中的表达。转染β-Klotho SiRNA后用Western blot和免疫荧光来观察在β-Klotho基因沉默后,FGFR1能否被FGF21激活以及FGF21／磷酸化FGFRl/p-Klotho在血管内皮细胞上是否共定位。用MTT检测外源性FGF21能否对脑血管内皮细胞在OGD 4小时复糖复氧20小时的损伤产生保护作用。用FITC检测高糖炎症损伤下FGF21对内皮细胞渗透性保护。用western blot检测FGF21通过诱导哪个蛋白升高来进行保护作用。结果：首次发现脑血管内皮细胞中FGF21有表达且能被细胞缺氧缺糖后再灌注损伤诱导表达增加。FGF21在脑血管内皮细胞中能激活FGFR1磷酸化并同时诱导β-Klotho表达增高,且FGF21／磷酸化FGFR1/β-Klotho这三者是在细胞是是共定位的。但在β-Klotho基因沉默后FGF21不能激活FGFR1。这一结果说明了在除了脂肪细胞,在脑血管内皮细胞中,FGF21的传导通路也是通过FGF21/FGFR1/βKlotho三聚体复合物的形成,激活FGFR1磷酸化而进行信号传导的。这是首次发现FGF21在非脂肪细胞组织,在脑内皮细胞中也是通过FGF21/FGFR1/β-Klotho三聚体复合物进行信号传导的,提示这也许是FGF21信号传导的共性。并且外源性FGF21对脑血管内皮细胞的缺氧缺糖后再灌注损伤和高糖合并炎症损伤都有保护作用。FGF21还对内皮细胞在高糖合并炎症损伤下减少其通透性的保护作用,其作用是通过诱导升高PPARγ实现的。FGF21还可以升高细胞膜连接蛋白ZO-1、VE-Cadherin连减少细胞通透性。第四部分： FGF21对神经元细胞的影响目的：之前的实验证实了FGF21能穿过血脑屏障,而神经元作为最重要的神经功能性细胞,FGF21对神经元细胞能否产生保护作用还没有研究。并且FGF21在神经元细胞中有无表达,或能否对FGF21的刺激产生反应,以及FGF21在神经元细胞的受体和信号传导通路是什么。都还有待研究。方法：本研究应用Western blot检测分析小鼠原代培养大脑皮层神经元细胞正常情况下和缺氧后的FGF21表达变化,和小鼠原代培养神经元细胞被重组FGF21蛋白处理后磷酸化纤维生长因子受体1(FGFR1)和β-Klotho的表达变化,是否对FGF21有时间依赖和剂量依赖。用免疫荧光染色检测磷酸化FGFR1和β-Klotho能否被FGF21诱导和是否与FGF21共定位。用C-IP和Western blot检测分析FGF21是否能与FGFR1和β-Klotho形成三聚体复合物。用LDH检测外源性FGF21蛋白能否对小鼠原代培养神经元细胞缺氧或高糖损伤时产生保护作用。结果：小鼠原代培养神经元细胞正常情况下FGF21的表达量极低,但在缺氧损伤后FGF21能被诱导表达增加。在小鼠原代培养神经元细胞中可以看到FGF21/磷酸化FGFR1/β-Klotho是共定位的,且FGFR1被FGF21激活磷酸化。在小鼠原代培养神经元细胞正常状态下,FGF21表达量太低,并不会形成FGF21/FGFR1/β-Klotho三聚体复合物,FGFR1/β-Klotho也不会形成二聚体复合物。但小鼠原代培养神经元细胞被重组FGF21蛋白处理后,可以观测到FGF21/FGFR1/β—Klotho三聚体复合物的形成。这一结果可以证明在小鼠原代培养神经元细胞中,FGF21的传导通路与脂肪细胞中和之前实验的脑内皮细胞一样,都是通过FGF21/FGFR1/β-Klotho三聚体复合物的形成,激活FGFR1磷酸化而进行信号传导的。并且外源性FGF21对小鼠原代培养神经元细胞在缺氧和高糖的损伤下都有保护作用。
[Abstract]:The first part is the rapid hypoglycemic effect of fibroblast growth factor 21 (FGF21) on diabetic rats: a large number of studies have shown that FGF21 has a regulatory effect on glucose metabolism. Continuous administration of drugs for 7 to 14 days can reduce type 2 diabetes type hyperglycemia, enhance insulin sensitivity and lower insulin resistance. But few studies have studied the rapid decline of FGF21. The effect of sugar is of great significance for high blood sugar as a complication or a major risk factor such as acute trauma, inflammation, and cardiovascular and cerebrovascular diseases. Methods: this experiment first injected the recombinant FGF21 protein from the Wenzhou Medical College (T2D mice) to test the mice with blood glucose test paper for 14 days. Elisa method was used to detect the insulin level in mice. After the injection of FGF211 hours, the blood glucose of mice was detected with blood glucose test paper. Results: the blood glucose of mice was obviously decreased and the insulin sensitivity was increased and the insulin resistance was reduced. After FGF211 hours, the blood glucose of T2D rats and the simulated stress hyperglycemia mice blood sugar The effect of FGF 21 may also have a rapid decrease in blood sugar. Second the effect of serum level of FGF21 on the level of FGF21 expression in the brain: a study shows that FGF21 has a significant increase in blood level in diseases such as cardiovascular disease, liver disease, hypertension, obesity, diabetes and other diseases. The possible mechanism is that the body is a FGF21 The signal transduction was impaired and the sensitivity of FGF21 weakened to overexpression of FGF21. It was very important that research showed that FGF21 could pass through the blood brain barrier (BBB). But at the same time, it was found that FGF21 needed to bind to the fibroblast receptor (FGFR) through the membrane protein beta -Klotho on the cell to activate the receptor, which made FGF21 Whether or not FGF21 can enter the brain, whether there is the expression of beta -Klotho in the brain and the mechanism of FGF21 receptor activation have not yet been studied. Methods: the Elisa assay was used to detect the level of FGF21 blood in non hyperglycemic mice (ND mice) and type 2 diabetic hyperglycemia mice (T2D). Western blot was used to detect T2D mice in ND mice. The expression level of FGF21 in the whole brain protein. The expression level of FGF21, pFGFRl and beta -Klotho in different parts of the brain of mice was detected at the same time point after intravenous injection of exogenous FGF21 to mice by Western blot. Results: the significance of FGF21 in serum in T2D mice was significantly higher than that in.T2D mice of ND mice. The brain protein was collected at different time points after intravenous injection of exogenous FGF21 to mice. Western blot detection was performed. The phosphorylation of FGF21 in different parts of the brain and phosphorylation of fibroblast growth factor receptor 1 (FGFR1) were changed, and the expression of beta -Klotho was detected. This indicates that the elevation of FGF21 in the serum to FGF2 in the brain. The content of 1 and the activation of FGFR1 receptors are affected. But the increase in the content of FGF21 in the brain is not clear except for the direct entry of exogenous FGF21. The activation of FGFR1 phosphorylation is directly activated by FGF21 or by FGF21 induced factors in the FGF family, but it is still inconclusive, but as a result of the detection of beta -Klotho The expression may also activate the receptor directly. This results suggest that FGF21 can penetrate the blood brain barrier into the brain, perhaps directly acting on the role of the central nervous system. Third part of the effect of FGF21 on the brain endothelial cells: neuroprotection is not limited to the protection of neuronal cells and the protection of the blood brain barrier is also not It is often crucial that the elevated FGF21 in the serum is directly exposed to vascular endothelial cells, but the protective mechanism of FGF21 on vascular endothelial cells and the protective mechanism are not studied. The expression of FGF21 in the vascular endothelium and the change of expression after injury, the transduction pathway and mechanism of action, and the form of receptor complex are still to be studied. Methods: Western blot was used to detect the expression of FGF21 in vascular endothelial cells, the changes of FGF21 expression after 4 hours of anoxic glucose deficiency (OGD) and 20 reoxygenated reoxygenation, and the changes in the expression of phosphorylated FGFR1 and beta -Klotho after the recombinant FGF21 protein was treated by recombinant FGF21 protein. SiRNA) technology, after transfection of FGF21 SiRNA, Western blot was used to detect the expression in FGF21 cells in vascular endothelial cells. After transfection of beta -Klotho SiRNA, Western blot and immunofluorescence were used to observe whether FGFR1 could be activated after beta -Klotho gene silencing and whether or not it was co determined on vascular endothelial cells. The protective effect of exogenous FGF21 on the damage of cerebral vascular endothelial cells at OGD 4 hours complex of reoxygenation for 20 hours was detected by MTT. FITC was used to detect the permeability of FGF21 to endothelial cells under high glucose inflammation. Western blot was used to detect the protective effect of FGF21 by inducing which protein to increase. Results: the first discovery of brain blood FGF21 is expressed in endothelial cells and can be induced by reperfusion injury induced by anoxia and glucose deficiency..FGF21 can activate FGFR1 phosphorylation in cerebral vascular endothelial cells and induce higher expression of beta -Klotho, and FGF21 / phosphorylation of FGFR1/ beta -Klotho, these three are Co located in cells, but after the silence of the beta -Klotho gene, FGF21 The failure to activate FGFR1. shows that in the brain vascular endothelial cells, the conduction pathway of FGF21 is also transmitted through the formation of the FGF21/FGFR1/ beta Klotho tripolymer complex in the brain vascular endothelial cells, activating FGFR1 phosphorylation and signaling. This is the first time that FGF21 is found in non adipocyte tissue, and in the brain endothelial cells is also through FGF2. The signal transduction of 1/FGFR1/ beta -Klotho trimer complexes suggests that this may be the commonality of FGF21 signal transduction. And exogenous FGF21 has protective effects on reperfusion injury after hypoxia and glucose deficiency and high glucose combined with inflammatory injury in cerebral vascular endothelial cells, and.FGF21 also reduces the permeability of endothelial cells under high glucose and inflammatory injury. The protective effect is that.FGF21 can increase the cell membrane connexin ZO-1 by inducing the increase of PPAR gamma, and VE-Cadherin even reduces cell permeability. The fourth part: the effect of FGF21 on neuron cells: previous experiments confirmed that FGF21 could pass through the blood brain barrier, and the neuron was the most important neural functional fine. There is no study on the protective effect of FGF21 on neuron cells. And whether FGF21 is expressed in neuron cells, or whether it can react to the stimulation of FGF21, and what the receptor and signal transduction pathway of FGF21 in the neuron cells are all still to be studied. Methods: This study used Western blot to detect and analyze the original mice. The changes in FGF21 expression in normal and anoxic neurons of cerebral cortex, and the changes in the expression of phosphorylated fibroblast growth factor receptor 1 (FGFR1) and beta -Klotho after the recombinant FGF21 protein were treated by recombinant FGF21 protein in the primary cultured neurons of the mice, were dependent on the time dependence and dose dependence on FGF21. The phosphorylated FGF was detected by immunofluorescence staining. Whether R1 and beta -Klotho can be induced by FGF21 and co location with FGF21. Use C-IP and Western blot to detect whether FGF21 can form a trimer complex with FGFR1 and beta -Klotho. The expression of FGF21 is very low in normal neuron cells, but the expression of FGF21 can be induced to increase after the hypoxia injury. In the primary cultured neuron cells of the mice, the FGF21/ phosphorylation FGFR1/ beta -Klotho is Co located, and FGFR1 is activated by FGF21 phosphorylation. The expression of FGF21 in the normal state of the primary cultured neuron cells in mice. The amount is too low to form a FGF21/FGFR1/ beta -Klotho trimer complex and FGFR1/ beta -Klotho does not form a two polymer complex. But the primary cultured neuron cells of mice can be treated with the recombinant FGF21 protein, and the formation of FGF21/FGFR1/ beta Klotho trimer complex can be observed. This result can prove that the primary cultured nerve in mice can be cultured. In the cell, the conduction pathway of FGF21 is the same as that of the adipocytes and the previous experimental endothelium cells, all through the formation of the FGF21/FGFR1/ beta -Klotho trimer complex, activating FGFR1 phosphorylation and conducting signal transduction. And exogenous FGF21 protects the primary cultured neurons of mice under the damage of hypoxia and high glucose. Use.

【学位授予单位】：湖南师范大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R587.1

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