TAT-HaFGF经鼻入脑的吸收特点和分子机制研究
发布时间：2019-02-26 13:14
【摘要】：目的： 研究TAT-HaFGF经鼻给药后的安全性、局部药动学和TAT-HaFGF穿过血脑屏障的分子机制。 方法： 1、采用蟾蜍上腭离体法考察TAT-HaFGF对鼻纤毛运动和形态影响,评价其鼻腔应用的安全性。TAT-HaFGF连续给药五周后，通过嗅神经标记蛋白（olfactorymarker protein, OMP)和组织生理病理学变化的情况评价其长期给药的安全性。 2、用放射性核素标记和共聚焦技术研究TAT-HaFGF经单次滴鼻给药后15min、30min、45min在鼻腔、脑部、胸腺和胰腺的分布。 3、用小鼠脑微血管内皮细胞系（bEnd.3）建立单层血脑屏障（blood brainbarrier，BBB）模型，通过Elisa、扫描电镜、western blotting （WB）技术研究TAT-HaFGF穿过BBB的机制。 结果： 1、鼻粘膜纤毛毒性试验表明TAT-HaFGF不影响鼻纤毛的摆动，也不会使鼻纤毛脱落。鼻腔连续给予SD大鼠100/300/600μg/kg TAT-HaFGF五周，与对照组相比， OMP蛋白表达量无显著性差异（P0.05)，组织形态无病理性改变。 2、SD大鼠经滴鼻给予125I-TAT-HaFGF15min（包含滴鼻给药所需时长12~13min）后在嗅球、小脑、脑干、垂体和血清中检测到125I-TAT-HaFGF，，给药30min后在脑干、大脑、颈脊髓达到了峰值。给药后30min的免疫荧光结果显示较多的TAT-HaFGF包绕于嗅神经周围。 3、BBB模型的transwell上室加入TAT-HaFGF、HaFGF、TAT， Elisa方法检测下室中HaFGF含量，结果发现：0~4h后TAT-HaFGF与HaFGF穿过BBB的量没有差异，但给药后8~16h，TAT-HaFGF穿过BBB的量明显多于HaFGF。扫描电镜结果显示TAT与aFGF的物理混合组可明显使得脑微血管内皮细胞间隙变大。WB结果显示，TAT-HaFGF可上调MLC、CREB等Rho信号通路标志蛋白的磷酸化；HaFGF仅促进了p-CREB的表达，对p-MLC的表达没有影响。 结论： 1、TAT-HaFGF对鼻粘膜纤毛无毒性；连续滴鼻五周对SD大鼠鼻腔整体结构无损伤，不改变鼻粘膜结构和嗅球上特异性嗅感觉神经元数量。TAT-HaFGF滴鼻给药相对安全。 2、TAT-HaFGF经滴鼻给药后可进入中枢神经系统，分布于大脑、小脑、脑干和垂体等部位。 3、体外BBB单层细胞模型表明TAT-HaFGF和HaFGF可通过降低BBB间ZO-1表达来加大细胞间隙，从细胞间隙穿过BBB进入中枢神经系统。TAT-HaFGF调控ZO-1表达与Rho信号通路有关，通过上调p-CREB的表达来抑制ZO-1的表达。
[Abstract]:Aim: to study the safety, local pharmacokinetics and molecular mechanism of TAT-HaFGF crossing the blood-brain barrier (BBB) of TAT-HaFGF after nasal administration. Methods: 1. The effects of TAT-HaFGF on nasal cilia movement and morphology of toad were investigated by the method of suprapalatal isolation. The safety of nasal application of TAT-HaFGF was evaluated. After five weeks of continuous administration of TAT-HaFGF, the olfactory nerve marker protein (olfactorymarker protein, was used to evaluate the safety of nasal application. OMP) and histopathological changes were used to evaluate the safety of long-term administration. 2. Radionuclide labeling and confocal technique were used to study the distribution of TAT-HaFGF in nasal cavity, brain, thymus and pancreas 15 min, 30 min, 45 min after single nasal infusion. 3. The monolayer blood-brain barrier (blood brainbarrier,BBB) model was established with mouse cerebral microvascular endothelial cell line (bEnd.3). The mechanism of TAT-HaFGF passing through BBB was studied by Elisa, scanning electron microscopy (, western blotting (WB). Results: 1. The nasal mucociliary toxicity test showed that TAT-HaFGF did not affect the swinging of nasal cilia, nor could it make nasal cilia fall off. SD rats were given 100 渭 g / 600 渭 g / kg TAT-HaFGF in nasal cavity for five weeks. Compared with the control group, there was no significant difference in the expression of OMP protein (P0.05), and there was no pathological change in tissue morphology. (2) after administration of 125I-TAT-HaFGF15min (including the length of 12~13min), 125i TATA HaFGF was detected in olfactory bulb, cerebellum, brainstem, pituitary and serum of SD rats. After administration of 30min, the peak value was found in brain stem, brain, neck and spinal cord. The immunofluorescence results of 30min showed that more TAT-HaFGF surrounded the olfactory nerve after administration. 3. The content of HaFGF in the transwell upper chamber of BBB model was detected by TAT-HaFGF,TAT, Elisa method. The results showed that there was no difference in the amount of TAT-HaFGF and HaFGF passing through BBB after 0 h, but the amount of TAT-HaFGF passing through BBB was significantly higher than that of HaFGF. at 8 h 16 h after administration. Scanning electron microscopy (SEM) showed that the physical mixture of TAT and aFGF could significantly enlarge the gap of cerebral microvascular endothelial cells. WB results showed that TAT-HaFGF could up-regulate the phosphorylation of Rho signaling marker proteins such as MLC,CREB. HaFGF only promoted the expression of p-CREB, but had no effect on the expression of p-MLC. Conclusion: 1. TATA-HaFGF has no toxicity to nasal cilia, and has no damage to the whole nasal structure of SD rats for five weeks, does not change the structure of nasal mucosa and the number of olfactory-specific olfactory sensory neurons in olfactory bulb. The administration of TAT-HaFGF is relatively safe. 2. TATA-HaFGF can enter the central nervous system and distribute in the brain, cerebellum, brainstem and pituitary after nasal administration. 3. The BBB monolayer cell model in vitro showed that TAT-HaFGF and HaFGF could increase the intercellular space by decreasing the expression of ZO-1 between BBB and entering the central nervous system from the cell gap through BBB. TAT-HaFGF regulated ZO-1 expression related to Rho signaling pathway. The expression of ZO-1 was inhibited by up-regulating the expression of p-CREB.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965
本文编号：2430803
[Abstract]:Aim: to study the safety, local pharmacokinetics and molecular mechanism of TAT-HaFGF crossing the blood-brain barrier (BBB) of TAT-HaFGF after nasal administration. Methods: 1. The effects of TAT-HaFGF on nasal cilia movement and morphology of toad were investigated by the method of suprapalatal isolation. The safety of nasal application of TAT-HaFGF was evaluated. After five weeks of continuous administration of TAT-HaFGF, the olfactory nerve marker protein (olfactorymarker protein, was used to evaluate the safety of nasal application. OMP) and histopathological changes were used to evaluate the safety of long-term administration. 2. Radionuclide labeling and confocal technique were used to study the distribution of TAT-HaFGF in nasal cavity, brain, thymus and pancreas 15 min, 30 min, 45 min after single nasal infusion. 3. The monolayer blood-brain barrier (blood brainbarrier,BBB) model was established with mouse cerebral microvascular endothelial cell line (bEnd.3). The mechanism of TAT-HaFGF passing through BBB was studied by Elisa, scanning electron microscopy (, western blotting (WB). Results: 1. The nasal mucociliary toxicity test showed that TAT-HaFGF did not affect the swinging of nasal cilia, nor could it make nasal cilia fall off. SD rats were given 100 渭 g / 600 渭 g / kg TAT-HaFGF in nasal cavity for five weeks. Compared with the control group, there was no significant difference in the expression of OMP protein (P0.05), and there was no pathological change in tissue morphology. (2) after administration of 125I-TAT-HaFGF15min (including the length of 12~13min), 125i TATA HaFGF was detected in olfactory bulb, cerebellum, brainstem, pituitary and serum of SD rats. After administration of 30min, the peak value was found in brain stem, brain, neck and spinal cord. The immunofluorescence results of 30min showed that more TAT-HaFGF surrounded the olfactory nerve after administration. 3. The content of HaFGF in the transwell upper chamber of BBB model was detected by TAT-HaFGF,TAT, Elisa method. The results showed that there was no difference in the amount of TAT-HaFGF and HaFGF passing through BBB after 0 h, but the amount of TAT-HaFGF passing through BBB was significantly higher than that of HaFGF. at 8 h 16 h after administration. Scanning electron microscopy (SEM) showed that the physical mixture of TAT and aFGF could significantly enlarge the gap of cerebral microvascular endothelial cells. WB results showed that TAT-HaFGF could up-regulate the phosphorylation of Rho signaling marker proteins such as MLC,CREB. HaFGF only promoted the expression of p-CREB, but had no effect on the expression of p-MLC. Conclusion: 1. TATA-HaFGF has no toxicity to nasal cilia, and has no damage to the whole nasal structure of SD rats for five weeks, does not change the structure of nasal mucosa and the number of olfactory-specific olfactory sensory neurons in olfactory bulb. The administration of TAT-HaFGF is relatively safe. 2. TATA-HaFGF can enter the central nervous system and distribute in the brain, cerebellum, brainstem and pituitary after nasal administration. 3. The BBB monolayer cell model in vitro showed that TAT-HaFGF and HaFGF could increase the intercellular space by decreasing the expression of ZO-1 between BBB and entering the central nervous system from the cell gap through BBB. TAT-HaFGF regulated ZO-1 expression related to Rho signaling pathway. The expression of ZO-1 was inhibited by up-regulating the expression of p-CREB.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965
【参考文献】
相关期刊论文 前1条
1 蒋新国，崔景斌，方晓玲，韦阳，奚念朱;药物的鼻粘膜纤毛毒性及评价方法[J];药学学报;1995年11期
本文编号：2430803
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