本文關(guān)鍵詞： 顱面部神經(jīng)嵴 高糖環(huán)境 自噬 細(xì)胞凋亡 雞胚　出處：《暨南大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：目的高糖環(huán)境會導(dǎo)致胚胎中顱面部神經(jīng)嵴參與形成的組織和器官的畸形,然而其機(jī)制仍未得以闡明。本課題利用早期雞胚為模型,探索高糖影響顱面部神經(jīng)嵴發(fā)育的細(xì)胞及其分子機(jī)制。方法雞胚經(jīng)高糖處理后,首先通過阿利新藍(lán)/茜素紅染色觀察由顱面部神經(jīng)嵴細(xì)胞衍生而來的顱面部骨骼。其后,利用免疫熒光,原位雜交,q RT-PCR,Western blot、透射電鏡等方法對高糖環(huán)境下顱面部神經(jīng)嵴細(xì)胞中關(guān)鍵基因表達(dá),增殖,凋亡和自噬進(jìn)行觀察和檢測,探索自噬對顱面部神經(jīng)嵴產(chǎn)生的影響機(jī)制。并結(jié)合HEK293T細(xì)胞探索高糖誘導(dǎo)的自噬與凋亡之間的關(guān)系。結(jié)果在此研究中,我們首先觀察到了高糖環(huán)境導(dǎo)致的由顱面部神經(jīng)嵴衍生而來的雞胚顱頂骨骼的發(fā)育缺陷,尤其是頂骨的發(fā)育缺陷。在早期雞胚中,我們發(fā)現(xiàn)高糖環(huán)境抑制顱面部神經(jīng)嵴的產(chǎn)生,而神經(jīng)嵴細(xì)胞的增殖并未受到顯著影響。然而高糖環(huán)境促進(jìn)了顱面部神經(jīng)嵴細(xì)胞的死亡。并且高糖環(huán)境能夠促進(jìn)HEK293T細(xì)胞中凋亡和自噬相關(guān)基因的表達(dá),包括P53,cleaved-Caspase3、cleaved-Caspase7,LC3B,truncated-Atg5以及Beclin-1。通過微珠實驗結(jié)合自噬抑制劑(氯喹)和激動劑(衣霉素)處理雞胚,從功能上證實了自噬涉及高糖環(huán)境對神經(jīng)嵴細(xì)胞產(chǎn)生的抑制作用。而高糖環(huán)境激活自噬主要是通過ERK信號通路而非m TOR通路實現(xiàn)的。結(jié)論我們的結(jié)果表明胚胎暴露于高糖環(huán)境中會抑制顱面部神經(jīng)嵴的產(chǎn)生,這種抑制作用可能是由于高糖引起的過度自噬導(dǎo)致了神經(jīng)嵴細(xì)胞的死亡增加。
[Abstract]:Objective High glucose environment may lead to the deformity of tissues and organs involved in the formation of craniofacial nerve crest in embryos, but its mechanism has not been elucidated. In this study, early chicken embryos were used as model. Methods after high glucose treatment, the craniofacial bone derived from cranio-facial nerve crest cells was observed by Alisin blue / alizarin red staining. The expression, proliferation, apoptosis and autophagy of key genes in cranio-facial nerve crest cells were observed and detected by immunofluorescence, in situ hybridization and Western blot, transmission electron microscopy (TEM). To explore the mechanism of autophagy on craniofacial neural crest and to explore the relationship between high glucose induced autophagy and apoptosis in combination with HEK293T cells. We first observed the developmental defects of the craniocephalic bone, especially the parietal bone, induced by high glucose environment. In the early chicken embryos, we found that high glucose environment inhibited the formation of cranio-facial neural crest. But the proliferation of neural crest cells was not significantly affected. However, high glucose environment promoted the death of craniofacial neural crest cells. Furthermore, high glucose environment promoted apoptosis and autophagy related gene expression in HEK293T cells. Including P53 cleaved-Caspase3, cleaved-cleaved-caspase7, LC3Bnctruncated-Atg5 and Beclin-1. Chicken embryos were treated with microbead test combined with autophagy inhibitor (chloroquine) and agonist (chlortetracycline). It is proved that autophagy is related to the inhibitory effect of high glucose environment on neural crest cells, while high glucose environment activates autophagy mainly through ERK signaling pathway rather than m TOR pathway. Conclusion our results indicate that embryonic burst. Exposure to high glucose conditions inhibits the production of craniofacial nerve crest. This inhibition may be due to hyperglycemic autophagy resulting in increased death of neural crest cells.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R321.5

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