一种新型多肽CBN-1的神经保护作用及其机制研究

发布时间：2018-06-09 14:31

本文选题：生物肽 + 神经保护　；参考：《上海交通大学》2014年博士论文

【摘要】：目的：筛选一种具有神经保护作用的复合型神经保护生物肽CBN-1，探讨其神经保护的作用机制。方法：利用生物信息学技术，筛选一种具有神经保护作用的复合型生物肽CBN-1，利用细胞和动物实验验证CBN-1的生物学活性，然后进一步研究可能的作用机制。细胞实验使用大鼠视网膜神经节细胞5（retinal ganglial cells，RGC-5cells），常规培养后加入生物肽CBN-1干预，观察CBN-1对细胞Trk受体磷酸化水平、PI3K/Akt通路表达、ERK通路表达、BCL-2蛋白家族含量的影响；使用PI3K/Akt通路LY294002和Wortmannin以及ERK通路抑制剂PD098059，观察上述信号通路抑制剂对细胞生存率的影响；建立RGC-5细胞的缺氧或NMDA损伤模型，观察CBN-1对细胞死亡率、细胞微管蛋白β-III-tubulin含量、Caspase3含量的影响。动物实验选择成年雄性Wistar大鼠，建立高眼压模型或NMDA损伤模型，玻璃体腔注射生物肽CBN-1，7天后观察视网膜神经节细胞层细胞凋亡、视网膜厚度变化、组织中BCL-2蛋白家族含量和Caspase3含量。结果：利用生物信息学技术，，筛选出一种含有17个氨基酸的新型生物肽CBN-1。细胞实验部分，CBN-1与RGC-5细胞作用24小时后，能够显著提高细胞Trk受体的磷酸化水平，提高细胞中Bcl-2含量，降低Bad和Bax的含量；CBN-1能够提高Akt和Erk1/2的磷酸化水平，使用抑制剂LY294002、Wortmannin以及PD098059后，细胞死亡率显著升高；生物肽CBN-1能够降低NMDA损伤后细胞的凋亡，降低缺氧或NMDA损伤模型下细胞的死亡率，提高NMDA损伤后细胞微管蛋白β-III-tubulin的表达；生物肽CBN-1能够降低NMDA损伤后细胞中Caspase3的含量，使用Caspase3抑制剂Z-DEVD-FMK后，细胞死亡率显著降低。动物实验部分，玻璃体腔注射生物肽CBN-1七天后，实验组神经节细胞层的细胞凋亡数量显著低于对照组，视网膜组织中Bcl-2含量显著高于对照组，而Bad和Bax的含量无显著性差异；实验组视网膜组织中Caspase3含量显著低于对照组，视网膜厚度无显著变化。结论：生物肽CBN-1具有一定的神经保护活性，CBN-1的神经保护活性可能主要通过结合Trk受体，激活PI3K/Akt和ERK信号通路来实现的；生物肽CBN-1的神经保护活性在眼科具有一定的临床应用前景。
[Abstract]:Objective: to screen a compound neuroprotective biological peptide CBN-1 with neuroprotective effect, and to explore the mechanism of its neuroprotective effect. The biological activity of CBN-1 was verified by cell and animal experiments, and the possible mechanism was studied. Rat retinal ganglion cells (5(retinal ganglial cells) were treated with RGC-5 cells. The effects of CBN-1 on the expression of PI3K / Akt pathway and the expression of BCL-2 protein family were observed in normal cultured rat retinal ganglion cells (RGC / 5 cells), and the effect of CBN-1 on the expression of PI3K / Akt pathway was observed. PI3K / Akt pathway LY294002 and Wortmannin and ERK pathway inhibitor PD098059were used to observe the effect of these signaling pathway inhibitors on cell survival, and to establish a model of hypoxia or NMDA damage in RGC-5 cells. Effect of tubulin 尾-III-tubulin content and Caspase3 content. Adult male Wistar rats were selected to establish intraocular pressure model or NMDA injury model. The retinal ganglion cell layer apoptosis and retinal thickness were observed 7 days after vitreous injection of biological peptide CBN-1. Results: a new biological peptide CBN-1 containing 17 amino acids was selected by bioinformatics. After treated with RGC-5 cells for 24 hours, CBN-1 could significantly increase the phosphorylation level of Trk receptor, increase the Bcl-2 content and decrease the content of Bad and Bax. CBN-1 could increase the phosphorylation level of Akt and Erk 1 / 2. After treatment with LY294002Wortmannin and PD098059, the cell death rate increased significantly, and the biological peptide CBN-1 decreased the cell apoptosis after NMDA injury, decreased the cell death rate under hypoxia or NMDA injury model, and increased the expression of tubulin 尾 -III-tubulin after NMDA injury. Biopeptide CBN-1 could reduce the content of Caspase3 after NMDA injury, and the cell death rate was significantly decreased by Caspase3 inhibitor Z-DEVD-FMK. In the animal experiment, the number of apoptosis in ganglion cell layer of experimental group was significantly lower than that in control group, and the content of Bcl-2 in retinal tissue was significantly higher than that in control group, but the contents of Bad and Bax had no significant difference after 7 days of vitreous injection of CBN-1. The content of Caspase3 in retinal tissue of experimental group was significantly lower than that of control group, and the retinal thickness had no significant change. Conclusion: CBN-1 has some neuroprotective activity and the neuroprotective activity of CBN-1 may be mainly by binding to Trk receptor. Activation of the PI3K / Akt and ERK signaling pathways, the neuroprotective activity of the biological peptide CBN-1 has a certain clinical application prospect in ophthalmology.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R77

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