【摘要】：目的：研究受體相互作用蛋白3(Receptor Interacting Protein3,RIP3)在氧-糖剝奪后(Oxygen glucose deprivation, OGD)視網(wǎng)膜節(jié)細(xì)胞(Retinal gonglion cell-5,RGC-5)程序性壞死過程中的表達(dá)變化,探討RIP3在視網(wǎng)膜節(jié)細(xì)胞程序性壞死過程中的可能作用及其下游機(jī)制。 方法：(1)從液氮罐中取出RGC-5,進(jìn)行細(xì)胞復(fù)蘇、傳代。待細(xì)胞長(zhǎng)到鋪滿約80%后,選取第3-8代細(xì)胞為實(shí)驗(yàn)細(xì)胞；(2)將傳代好的細(xì)胞進(jìn)行實(shí)驗(yàn)分組并標(biāo)記,將各組細(xì)胞放入37℃,5%CO2培養(yǎng)箱中培養(yǎng)至細(xì)胞鋪滿約80%,然后正常組不處理,其他各組用DMEM無(wú)糖培養(yǎng)基替換DMEM高糖培養(yǎng)基后將兩組細(xì)胞放入密閉容器中并用95%N2和5%CO2混合氣充滿密閉容器進(jìn)行OGD處理,8小時(shí)后取出細(xì)胞并用DMEM高糖培養(yǎng)基替換DMEM無(wú)糖培養(yǎng)基,將細(xì)胞放入37℃,5%CO2培養(yǎng)箱中恢復(fù)至相應(yīng)時(shí)間；(3)通過PI染色和Annexin V/PI雙染后流式細(xì)胞儀檢測(cè)探討OGD處理8小時(shí)的RGC-5的壞死中是否有程序性壞死；(4)OGD處理8小時(shí)后恢復(fù)至相應(yīng)的時(shí)間點(diǎn)后采用免疫蛋白印跡法分析RIP3的表達(dá)量；(5)加入嗎啉寡核苷酸制備RIP3基因敲低的RGC-5(RIP3i-RGC-5),通過AnnexinV/PI雙染后采用流式細(xì)胞儀檢測(cè)以及丙二醛(Maleic dialdehyde,MDA)含量檢測(cè)探索RIP3在OGD誘導(dǎo)的RGC-5細(xì)胞程序性壞死過程中的可能作用及其下游機(jī)制。 結(jié)果：(1)OGD處理后,PI染色發(fā)現(xiàn)壞死細(xì)胞增多,Annexin V/PI雙染色后采用流式細(xì)胞儀檢測(cè)出氧-糖剝奪8小時(shí)恢復(fù)6小時(shí)后部分細(xì)胞出現(xiàn)壞死,而預(yù)先用10μmol/L Nec-1(程序性壞死的特異性抑制劑)孵育24小時(shí)后壞死明顯減少；(2)OGD處理8小時(shí)恢復(fù)至相應(yīng)時(shí)間,Western blot顯示RIP3表達(dá)較正常對(duì)照組為強(qiáng)；(3)Annexin V/PI雙染色-流式細(xì)胞儀檢測(cè)顯示：RIP3i-RGC-5經(jīng)OGD處理后壞死細(xì)胞數(shù)量較正常模型組明顯減少；(4)MDA含量檢測(cè)結(jié)果顯示：OGD處理后正常模型組及RIP3i-RGC-5組的MDA含量均較正常對(duì)照組高,但RIP3i-RGC-5組的MDA含量較正常模型組明顯降低。 結(jié)論:OGD處理后RGC-5存在程序性壞死,且RIP3通過氧化應(yīng)激參與介導(dǎo)了RGC-5的程序性壞死。圖8幅,參考文獻(xiàn)49篇。
[Abstract]:Aim: to investigate the expression of receptor interaction protein 3 (Receptor Interacting Protein3,RIP3) in (Oxygen glucose deprivation, OGD) retinal ganglion cells (Oxygen glucose deprivation, OGD) with programmed necrosis after oxygen-glucose deprivation. To investigate the possible role of RIP3 in the process of programmed retinal ganglion cell necrosis and its downstream mechanism. Methods: (1) RGC-5, was removed from liquid nitrogen tank for cell resuscitation and subculture. When the cells grew to about 80%, the 3-8 passage cells were selected as experimental cells. (2) the cultured cells were divided into experimental groups and labeled. The cells in each group were placed at 37 鈩，

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