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  本文選題：Notch1 + 糖尿病視網(wǎng)膜病變　； 參考：《吉林大學(xué)》2011年博士論文

【摘要】：本實驗成功建立了糖尿病視網(wǎng)膜病變的小鼠模型,應(yīng)用免疫組織化學(xué)法和Western Blot方法檢測糖尿病視網(wǎng)膜病變小鼠視網(wǎng)膜中Notch1、Dll4、PARP、Akt、NF-κB和caspase-3表達(dá)量的變化,同時體外培養(yǎng)的人視網(wǎng)膜血管內(nèi)皮細(xì)胞HRVEC,建立高糖培養(yǎng)的視網(wǎng)膜內(nèi)皮細(xì)胞模型,應(yīng)用免疫印記方法檢測高糖培養(yǎng)的視網(wǎng)膜血管內(nèi)皮細(xì)胞由Notch1、Dll4、PARP、Akt、NF-κB和caspase-3表達(dá)量的變化,發(fā)現(xiàn)糖尿病小鼠模型的視網(wǎng)膜和高糖培養(yǎng)的HRVECs中的PARP和caspase-3表達(dá)量比正常對照組增高顯著,并與葡萄糖濃度正相關(guān)；而Notch1、Dll4及p-Akt表達(dá)量隨葡萄糖濃度的降低而顯著降低,說明高糖引起的細(xì)胞凋亡與PARP增加和Notch1、p-Akt下降有關(guān)。進(jìn)一步通過免疫共沉淀及激光共聚焦的方法證明了PARP和NF-κB是相互作用的蛋白質(zhì),在高糖情況下,與PARP結(jié)合的NF-κB蛋白明顯增多,說明PARP通過激活NF-κB誘導(dǎo)細(xì)胞凋亡。另外通過Western Blot檢測高糖下Notch1拮抗PARP-1和NF-κB介導(dǎo)的細(xì)胞凋亡,發(fā)現(xiàn)Notch1能夠抑制高糖高糖引起的細(xì)胞凋亡。另外本實驗還體外構(gòu)建了siNotch1表達(dá)載體,應(yīng)用免疫共沉淀及免疫印記的方法檢測siNotch1對D114的抑制作用,發(fā)現(xiàn)siNotch1能抑制D114的抗凋亡作用,即說明Notch信號對高糖導(dǎo)致的細(xì)胞凋亡有保護(hù)作用。另外,利用Akt特異抑制劑wortmannin研究Akt與Notch1的關(guān)系,發(fā)現(xiàn)wortmannin能抑制高糖情況下Notch1對細(xì)胞的保護(hù)作用。 本實驗的創(chuàng)新之處在于： 1、證明了Notch1、p-Akt及D114蛋白表達(dá)量在高糖培養(yǎng)的HRVEC中比正常HRVEC顯著降低。 2、本實驗發(fā)現(xiàn)并且證明了在高糖培養(yǎng)的視網(wǎng)膜血管內(nèi)皮細(xì)胞中,Notch1/Akt信號途徑能夠抑制PARP-1和NF-κB介導(dǎo)的HRVECs凋亡
[Abstract]:The mouse model of diabetic retinopathy was established successfully. The expression of Notch1Dll4pPARPER-Aktn- 魏 B and caspase-3 in the retina of diabetic retinopathy mice was detected by immunohistochemical method and Western Blot method. Human retinal vascular endothelial cells (HRVECs) were cultured in vitro, and high glucose cultured retinal endothelial cells (RECs) were established. The changes of the expression of NF- 魏 B and caspase-3 in cultured retinal vascular endothelial cells (RECs) cultured with high glucose by Notch1Dll4- PARPNF-kB and caspase-3 were detected by immunoblotting. It was found that the expression of PARP and caspase-3 in the retina of diabetic mice and HRVECs cultured with high glucose was significantly higher than that in the normal control group, and positively correlated with glucose concentration, while the expression of Notch1Dll4 and p-Akt decreased with the decrease of glucose concentration. The results showed that the apoptosis induced by high glucose was related to the increase of PARP and the decrease of Notch1 p-Akt. It was further proved that PARP and NF- 魏 B were interacting proteins by immunoprecipitation and laser confocal method, and that the NF- 魏 B protein binding to PARP increased significantly under high glucose condition, which indicated that PARP induced apoptosis by activating NF- 魏 B. In addition, Notch1 antagonized apoptosis induced by PARP-1 and NF- 魏 B under high glucose by Western Blot. It was found that Notch1 could inhibit apoptosis induced by high glucose and high glucose. In addition, the expression vector of siNotch1 was constructed in vitro. The inhibitory effect of siNotch1 on D114 was detected by immunoprecipitation and imprinting. It was found that siNotch1 could inhibit the anti-apoptosis of D114. That is to say, Notch signal has protective effect on apoptosis induced by high glucose. In addition, the relationship between Akt and Notch1 was studied by wortmannin, a specific inhibitor of Akt. It was found that wortmannin could inhibit the protective effect of Notch1 on cells under high glucose. The innovation of this experiment lies in: 1. The results showed that the expression of Notch1p-Akt and D114 protein in HRVEC cultured with high glucose was significantly lower than that in normal HRVEC. 2. We found and proved that the Notch1 / Akt signaling pathway can inhibit the apoptosis of HRVECs mediated by PARP-1 and NF- 魏 B in high glucose cultured retinal vascular endothelial cells.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R774.1

【共引文獻(xiàn)】

相關(guān)期刊論文 前8條

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7 王s，

本文編號：1784509