【摘要】：目的：利用體外細胞氧化應激模型探索在缺血性腦損傷中,高爾基體囊泡轉(zhuǎn)運蛋白p115表達及裂解變化,及其與凋亡信號因子激活的關(guān)系,探討p115介導高爾基體應激的機制。 方法： 1.培養(yǎng)小鼠神經(jīng)瘤N2a細胞,通過不同濃度H2O2干預建立氧化應激模型,采用MTT法、流式細胞術(shù)等評價氧化應激模型。 2.實驗分為氧化應激組、保護性藥物干預后氧化應激組和正常對照組；其中氧化應激H2O2干預濃度分為40umol/l、80umol/l兩個濃度梯度；保護性藥物為抗氧化劑丙酮酸鈉,可以抑制caspase-3的激活,減少p115裂解。 3. Western blot法檢測各組中p115、p115C末端片段、p53、p-p53(ser15)的蛋白表達變化； 4.流式細胞術(shù)測定各組凋亡率的變化(Annexin V-FITC/PI); 5.MTT法測定并計算各組存活率的變化； 6.免疫熒光共聚焦方法觀察p115的亞細胞定位及在應激中碎裂情況。 實驗結(jié)果： 1.H2O2干預氧化應激后細胞形態(tài)的變化、細胞存活率及凋亡率檢測顯示：隨著H2O2濃度的增加,干預組小鼠N2a細胞的MTT值與正常對照組相比,MTT值顯著減少(P0.05),凋亡率明顯增加(P0.05),表明不同濃度H2O2造成的氧化應激引起了細胞損傷,導致了細胞凋亡的發(fā)生,H2O2的濃度可以造成不同程度的氧化應激損傷。 2.藥物干預后再予以H2O2處理,24小時后觀察細胞變化細胞存活率及凋亡率檢測顯示：N2a細胞形態(tài)變化較非藥物干預組變化不明顯,MTT值高于非藥物干預組(P0.05),凋亡率小于非藥物干預組(P0.05),表明抗氧化預處理可以減輕H2O2誘發(fā)的氧化應激損傷。 3.p115、p53表達的變化：H2O280umol/l干預后的2小時、4小時、8小時、12小時和24小時,隨著時間的延長,p115(115KD)全長表達逐漸減低,p115-CTF表達持續(xù)上升,p53表達較對照組無明顯變化,而p-p53(ser15)表達上升并在4小時達到峰值,此后下降。說明在H2O2的干預下,p115發(fā)生了持續(xù)性的裂解,p53發(fā)生了磷酸化；在H2O240umol/l,80umol/l及藥物處理4小時后,高濃度H2O2干預下,p115C末端片段、p-p53(ser15)的表達變化一致,均較低濃度組增加(P0.05),p115的表達較低濃度組減少(P0.05),而p53表達無顯著差異(P0.05),在藥物干預組p115、p115C末端片段、p53及p-p53(ser15)的表達均無顯著差異。結(jié)果提示p115C末端片段的生成與p53在ser15位點上的磷酸化有關(guān),藥物干預減少了p115的裂解,繼而減少了p53的磷酸化。 4.p115細胞內(nèi)定位及形態(tài)變化：免疫共聚焦顯微鏡下觀察到隨著H2O2濃度的增加,p115的紅色熒光由正常狀態(tài)下的環(huán)繞于核周的緊湊囊泡樣結(jié)構(gòu)逐漸向胞漿散開,部分呈點狀散布于整個細胞質(zhì)中。結(jié)果提示在氧化應激模型中,隨著應激程度的加重,p115C末端片段表達上調(diào),形態(tài)上發(fā)生裂解,與高爾基體碎裂發(fā)生相同的形態(tài)變化。 結(jié)論： 1.p115是高爾基體應激相關(guān)蛋白,在氧化應激中p115裂解,高爾基體發(fā)生碎裂。 2.p115介導的高爾基體應激性碎裂,參與了凋亡信號的擴大,可能與磷酸化p53蛋白有關(guān)。
[Abstract]:Objective: To explore the mechanism of p115-mediated Golgi stress in ischemic brain injury by using in vitro oxidative stress model to explore the expression and change of p115 and its relationship with the activation of apoptosis signal. Methods: 1. The N2a cells of mouse neuroma were cultured. The oxidative stress model was established by different concentration of H2O2, and the oxygen was evaluated by MTT and flow cytometry. 2. The experiment was divided into the oxidative stress group, the oxidative stress group after the protective drug intervention and the normal control group, wherein the concentration of the oxidative stress H2O2 intervention is divided into the concentration gradient of 40uml/ l and 80umol/ l; the protective medicine is an antioxidant pyruvate, and the activation of the caspase-3 can be inhibited, p115, p115C terminal fragment, p53, p-p53 (se) were detected by Western blot. r15) protein expression changes; 4. Flow cytometry to determine the change in apoptotic rates in each group (Ann exin V-FITC/ PI); 5. The change of the survival rate of each group was determined by the MTT method, and the method of fluorescence confocal microscopy was used. tp115 The results of the experiment were as follows: 1. The changes of cell morphology, cell survival rate and apoptosis rate after oxidative stress in the intervention group showed that, with the increase of the concentration of H2O2, the MTT value of the N2a cells in the intervention group was compared with that of the normal control group. The apoptosis rate was significantly increased (P0.05), and the apoptosis rate was significantly increased (P0.05). The results showed that the concentration of H2O2 could lead to different degree of oxidative stress injury. 2. After the intervention of the drug, the H2O2 treatment was carried out, and the cell survival rate and the apoptosis rate of the cells were observed after 24 hours. The change of the cell morphology of the N2a cells was less obvious than that of the non-drug intervention group, and the MTT value was higher than that of the non-drug. In the intervention group (P0.05), the apoptosis rate was lower than that of the non-drug intervention group (P0. The changes of the expression of p115 and p53: 2 hours, 4 hours, 8 hours, 12 hours and 24 hours after the intervention of H2O280umol/ l, the full-length expression of p115 (115KD) was gradually reduced, and the expression of p115-CTF continued to increase, and the expression of p53 was higher in the control group. No significant change The expression of p-p53 (ser15) and p-p53 (ser15) increased and reached the peak at 4 h, and then decreased. The results showed that under the intervention of H2O2, p115 had persistent lysis, and p53 was phosphorylated; after 4 hours of treatment with H2O240umol/ l, 80umol/ l and drug, the end fragment of p115C, p-p The expression of 53 (ser15) was consistent with that of low-concentration group (P0.05). The expression of p115 was lower than that of low-concentration group (P0.05), while the expression of p53 was not significant (P0.05). There was no significant difference in the expression of the terminal fragment, p53 and p-p53 (ser15) at the end of the 5C. The results suggested that the formation of the terminal fragment of p115C and the expression of p53 at the site of ser15 acidification-related, drug intervention reduced the lysis of p115, which in turn reduced the phosphorylation of p53. 4. p115 intracellular localization and morphological changes: the red fluorescence of p115 was observed under normal conditions as the concentration of h2o2 increased under an immunocofocus microscope. The compact vesicle-like structure around the core was gradually spread to the cytoplasm and partially dispersed throughout the cytoplasm. The results suggest that in the oxidative stress model, the p11 5C鏈，

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