本文選題：生物肽 + 神經(jīng)保護(hù)　； 參考：《上海交通大學(xué)》2014年博士論文

【摘要】：目的：篩選一種具有神經(jīng)保護(hù)作用的復(fù)合型神經(jīng)保護(hù)生物肽CBN-1，探討其神經(jīng)保護(hù)的作用機(jī)制。 方法：利用生物信息學(xué)技術(shù)，篩選一種具有神經(jīng)保護(hù)作用的復(fù)合型生物肽CBN-1，利用細(xì)胞和動物實(shí)驗(yàn)驗(yàn)證CBN-1的生物學(xué)活性，然后進(jìn)一步研究可能的作用機(jī)制。細(xì)胞實(shí)驗(yàn)使用大鼠視網(wǎng)膜神經(jīng)節(jié)細(xì)胞5（retinal ganglial cells，RGC-5cells），常規(guī)培養(yǎng)后加入生物肽CBN-1干預(yù)，觀察CBN-1對細(xì)胞Trk受體磷酸化水平、PI3K/Akt通路表達(dá)、ERK通路表達(dá)、BCL-2蛋白家族含量的影響；使用PI3K/Akt通路LY294002和Wortmannin以及ERK通路抑制劑PD098059，觀察上述信號通路抑制劑對細(xì)胞生存率的影響；建立RGC-5細(xì)胞的缺氧或NMDA損傷模型，觀察CBN-1對細(xì)胞死亡率、細(xì)胞微管蛋白β-III-tubulin含量、Caspase3含量的影響。動物實(shí)驗(yàn)選擇成年雄性Wistar大鼠，建立高眼壓模型或NMDA損傷模型，玻璃體腔注射生物肽CBN-1，7天后觀察視網(wǎng)膜神經(jīng)節(jié)細(xì)胞層細(xì)胞凋亡、視網(wǎng)膜厚度變化、組織中BCL-2蛋白家族含量和Caspase3含量。 結(jié)果：利用生物信息學(xué)技術(shù)，，篩選出一種含有17個氨基酸的新型生物肽CBN-1。細(xì)胞實(shí)驗(yàn)部分，CBN-1與RGC-5細(xì)胞作用24小時后，能夠顯著提高細(xì)胞Trk受體的磷酸化水平，提高細(xì)胞中Bcl-2含量，降低Bad和Bax的含量；CBN-1能夠提高Akt和Erk1/2的磷酸化水平，使用抑制劑LY294002、Wortmannin以及PD098059后，細(xì)胞死亡率顯著升高；生物肽CBN-1能夠降低NMDA損傷后細(xì)胞的凋亡，降低缺氧或NMDA損傷模型下細(xì)胞的死亡率，提高NMDA損傷后細(xì)胞微管蛋白β-III-tubulin的表達(dá)；生物肽CBN-1能夠降低NMDA損傷后細(xì)胞中Caspase3的含量，使用Caspase3抑制劑Z-DEVD-FMK后，細(xì)胞死亡率顯著降低。動物實(shí)驗(yàn)部分，玻璃體腔注射生物肽CBN-1七天后，實(shí)驗(yàn)組神經(jīng)節(jié)細(xì)胞層的細(xì)胞凋亡數(shù)量顯著低于對照組，視網(wǎng)膜組織中Bcl-2含量顯著高于對照組，而Bad和Bax的含量無顯著性差異；實(shí)驗(yàn)組視網(wǎng)膜組織中Caspase3含量顯著低于對照組，視網(wǎng)膜厚度無顯著變化。 結(jié)論：生物肽CBN-1具有一定的神經(jīng)保護(hù)活性，CBN-1的神經(jīng)保護(hù)活性可能主要通過結(jié)合Trk受體，激活PI3K/Akt和ERK信號通路來實(shí)現(xiàn)的；生物肽CBN-1的神經(jīng)保護(hù)活性在眼科具有一定的臨床應(yīng)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.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R77

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本文編號：2000053