【摘要】：目的： 研究TAT-HaFGF經(jīng)鼻給藥后的安全性、局部藥動學和TAT-HaFGF穿過血腦屏障的分子機制。 方法： 1、采用蟾蜍上腭離體法考察TAT-HaFGF對鼻纖毛運動和形態(tài)影響,評價其鼻腔應(yīng)用的安全性。TAT-HaFGF連續(xù)給藥五周后，通過嗅神經(jīng)標記蛋白（olfactorymarker protein, OMP)和組織生理病理學變化的情況評價其長期給藥的安全性。 2、用放射性核素標記和共聚焦技術(shù)研究TAT-HaFGF經(jīng)單次滴鼻給藥后15min、30min、45min在鼻腔、腦部、胸腺和胰腺的分布。 3、用小鼠腦微血管內(nèi)皮細胞系（bEnd.3）建立單層血腦屏障（blood brainbarrier，BBB）模型，通過Elisa、掃描電鏡、western blotting （WB）技術(shù)研究TAT-HaFGF穿過BBB的機制。 結(jié)果： 1、鼻粘膜纖毛毒性試驗表明TAT-HaFGF不影響鼻纖毛的擺動，也不會使鼻纖毛脫落。鼻腔連續(xù)給予SD大鼠100/300/600μg/kg TAT-HaFGF五周，與對照組相比， OMP蛋白表達量無顯著性差異（P0.05)，組織形態(tài)無病理性改變。 2、SD大鼠經(jīng)滴鼻給予125I-TAT-HaFGF15min（包含滴鼻給藥所需時長12~13min）后在嗅球、小腦、腦干、垂體和血清中檢測到125I-TAT-HaFGF，，給藥30min后在腦干、大腦、頸脊髓達到了峰值。給藥后30min的免疫熒光結(jié)果顯示較多的TAT-HaFGF包繞于嗅神經(jīng)周圍。 3、BBB模型的transwell上室加入TAT-HaFGF、HaFGF、TAT， Elisa方法檢測下室中HaFGF含量，結(jié)果發(fā)現(xiàn)：0~4h后TAT-HaFGF與HaFGF穿過BBB的量沒有差異，但給藥后8~16h，TAT-HaFGF穿過BBB的量明顯多于HaFGF。掃描電鏡結(jié)果顯示TAT與aFGF的物理混合組可明顯使得腦微血管內(nèi)皮細胞間隙變大。WB結(jié)果顯示，TAT-HaFGF可上調(diào)MLC、CREB等Rho信號通路標志蛋白的磷酸化；HaFGF僅促進了p-CREB的表達，對p-MLC的表達沒有影響。 結(jié)論： 1、TAT-HaFGF對鼻粘膜纖毛無毒性；連續(xù)滴鼻五周對SD大鼠鼻腔整體結(jié)構(gòu)無損傷，不改變鼻粘膜結(jié)構(gòu)和嗅球上特異性嗅感覺神經(jīng)元數(shù)量。TAT-HaFGF滴鼻給藥相對安全。 2、TAT-HaFGF經(jīng)滴鼻給藥后可進入中樞神經(jīng)系統(tǒng)，分布于大腦、小腦、腦干和垂體等部位。 3、體外BBB單層細胞模型表明TAT-HaFGF和HaFGF可通過降低BBB間ZO-1表達來加大細胞間隙，從細胞間隙穿過BBB進入中樞神經(jīng)系統(tǒng)。TAT-HaFGF調(diào)控ZO-1表達與Rho信號通路有關(guān)，通過上調(diào)p-CREB的表達來抑制ZO-1的表達。
[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.
【學位授予單位】：暨南大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R965

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相關(guān)期刊論文 前1條

1 蔣新國，崔景斌，方曉玲，韋陽，奚念朱;藥物的鼻粘膜纖毛毒性及評價方法[J];藥學學報;1995年11期

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