α-细辛醚开启斑马鱼血脑屏障作用机制及其生物安全性的研究

发布时间：2018-08-21 13:45

【摘要】：本文主要研究α-细辛醚对斑马鱼血脑屏障(Blood brain barrier,BBB)结构及紧密连接蛋白家族Claudin、occludin、ZO、JAM基因表达的影响,以阐明α-细辛醚开启血脑屏障的作用机制,并对α-细辛醚的生物安全性进行评价,主要对斑马鱼的生殖毒性和胚胎发育毒性进行研究,为人畜临床应用α-细辛醚奠定理论基础。采用的方法主要利用心脏注射方式将大分子荧光物质Dextran Texas Red注入斑马鱼幼鱼心脏,之后将其浸泡在α-细辛醚溶液中,利用共聚焦显微镜观察α-细辛醚是否能帮助大分子荧光物质通过血脑屏障进入脑内;成年斑马鱼通过腹腔注射方式给予α-细辛醚后,利用透射电镜观察α-细辛醚作用斑马鱼成鱼后,斑马鱼血脑屏障超微结构的变化;利用脑组织切片HE染色,显微镜观察斑马鱼脑组织结构的影响;利用实时荧光定量PCR(Real-time quantitative,RT-qPCR)来研究α-细辛醚对斑马鱼紧密连接蛋白家族Claudin、ZO、occludin、JAM基因表达及时效性的影响;α-细辛醚的生物安全性胚胎发育毒性,取受精后3h(Hours post-ferilization,hpf)的斑马鱼胚胎放入不同浓度梯度的α-细辛醚溶液中暴露,分别观察暴露在α-细辛醚在24hpf,48hpf,72hpf,96hpf不同时间点胚胎发育的形态现象,统计斑马鱼胚胎自主抽动的频率、孵化率、心率、畸形率及死亡率等指标的变化,包括心包的水肿、脊柱的弯曲、尾巴的弯曲、卵黄囊水肿等现象;利用RT-qPCR方法来检测斑马鱼浸泡α-细辛醚96hpf后对sepn1基因表达的影响。利用斑马鱼行为学系统Noldus来评价α-细辛醚对斑马鱼幼鱼运动方式及运动能力的影响。由试验得到的结果如下:斑马鱼心脏注射大分子荧光染料后,浸泡在养殖水和α-细辛醚中,发现浸泡α-细辛醚30min后斑马鱼的血管有些模糊,周围已经有少量的荧光物质渗出,浸泡α-细辛醚60min后可观察到斑马鱼血管更加的模糊,说明已经有大量的荧光物质从血管内渗出,而对照组却没有看到荧光物质渗出。通过电镜观察的结果显示,给予α-细辛醚后,内皮细胞出现轻度的皱缩,BBB的基膜出现膨大断裂,紧密连接出现疏松。而对照组的内皮细胞未出现皱缩,比较饱满,紧密连接致密,基膜结构完整连续。利用HE组织切片来观察斑马鱼脑组织结构的结果与BBB超微结构的结果相一致。腹腔注射α-细辛醚作用1h后,大部分的Claudin家族基因如Claudin-19、-k、-j、-5a、-7a、-7b、-a、-h、-11a下调。其中Claudin-5b、-11b上调。Claudin-2上下调没有太明显的变化。其他家族的基因如JAM,-2A、-2B、-3A、-3B、occludin-A、occludin-B均下调,下调的幅度却是不尽相同。腹腔注射α-细辛醚后,Claudin5a在1h下调最明显,作用2h又逐渐的恢复,到8h基本恢复正常水平。将斑马鱼胚胎暴露在不同浓度的α-细辛醚溶液中,24hpf斑马鱼自主抽动的次数降低;48hpf斑马鱼的心率减低;72hpf斑马鱼幼鱼有脊柱弯曲、心包水肿、尾巴弯曲、卵黄囊水肿等畸形的表征;96hpf斑马鱼幼鱼的孵化率、死亡率有很大的差异;通过斑马鱼幼鱼运动行为学表明随着α-细辛醚浓度增加,斑马鱼幼鱼运动受到抑制。本文的结论为芳香开窍药能使紧密连接蛋白claudin家族基因表达下调,致使紧密连接结构疏松,加之基膜结构破坏,内皮细胞皱缩,使得血脑屏障的屏障作用减弱,从而开启了血脑屏障,发挥了开窍的作用。基因可逆性的下调说明α-细辛醚对血脑屏障是生理性的,对脑部不会带来病理学损伤。从用药安全性来看,高浓度的α-细辛醚对斑马鱼胚胎发育有明显毒性作用。提示人畜临床慎用α-细辛醚。
[Abstract]:The effect of alpha-asarone on the structure of blood brain barrier (BBB) and the expression of tight junction protein family Claudin, occludin, ZO, JAM genes in zebrafish was studied in order to elucidate the mechanism of alpha-asarone opening blood brain barrier, and to evaluate the biological safety of alpha-asarone, mainly on the reproductive toxicity and the expression of JAM genes in zebrafish. The embryo development toxicity was studied to lay a theoretical foundation for the clinical application of alpha-asarone in human and livestock.Dextran Texas Red was injected into the heart of juvenile zebrafish by cardiac injection and then immersed in alpha-asarone solution. Macromolecular fluorescent substances entered the brain through the blood-brain barrier (BBB), and the ultrastructure of the BBB was observed by transmission electron microscopy (TEM) after the adult zebrafish were injected with alpha-asarone via abdominal cavity. Real-time quantitative PCR (RT-qPCR) was used to study the effects of alpha-asarone on the expression and timeliness of Claudin, ZO, occludin and JAM genes in zebrafish tightly junction proteins; the biological safety of alpha-asarone on embryonic development toxicity and the insemination of 3 h (Hours post-ferilization, hpf) zebrafish embryos into different concentrations. The morphological changes of embryos at 24 hpf, 48 hpf, 72 HPF and 96 HPF were observed. The frequency of spontaneous twitching, hatching rate, heart rate, deformity rate and mortality of zebrafish embryos were measured, including pericardial edema, spinal curvature, tail curvature, yolk, etc. The effect of alpha-asarone on SEPN1 gene expression in zebrafish was detected by RT-qPCR. The effect of alpha-asarone on movement pattern and ability of juvenile zebrafish was evaluated by Noldus, a zebrafish behavioral system. After soaking in culture water and alpha-asarone ether, it was found that the blood vessels of zebrafish were blurred after soaking in alpha-asarone ether for 30 minutes, and a small amount of fluorescent substances were exuded. After soaking in alpha-asarone for 60 minutes, the blood vessels of zebrafish became more blurred, indicating that a large amount of fluorescent substances had exuded from the blood vessels, but not seen in the control group. The results of electron microscopy showed that after administration of alpha-asarone, the endothelial cells contracted slightly, the basement membrane of BBB expanded and ruptured, and the tight junction loosened. After intraperitoneal injection of alpha-asarone for one hour, most of the Claudin family genes, such as Claudin-19, -k, -j, -5a, -7a, -7b, -a, -h, -11a, were down-regulated. Claudin-5b, -11b was up-regulated. Claudin-2 was not up-regulated. Other family genes such as JAM, -2A, -2B, -3A, o, 3B, etc. After intraperitoneal injection of alpha-asarone, Claudin 5A was down-regulated most obviously at 1 h, and recovered gradually at 2 h, and returned to normal level at 8 h. After exposing zebrafish embryos to different concentrations of alpha-asarone, the number of spontaneous twitches of 24hpf zebrafish decreased. The results showed that the hatching rate and mortality of juvenile zebrafish at 96 HPF were significantly different, and the movement of juvenile zebrafish was inhibited with the increase of concentration of alpha-asarone. Aromatic rehabilitative drugs can down-regulate the gene expression of claudin family, resulting in loose tight junction structure, destruction of basement membrane structure and shrinkage of endothelial cells, weakening the barrier function of blood brain barrier, thus opening the blood brain barrier and playing an enlightening role. Physiologically, it does not cause pathological damage to the brain. In terms of drug safety, high concentration of alpha-asarone has obvious toxic effect on zebrafish embryo development. It suggests that human and animal clinical use of alpha-asarone cautiously.
【学位授予单位】：内蒙古民族大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S853.7

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