本文選題：異丙酚 + 內(nèi)質(zhì)網(wǎng)應(yīng)激　； 參考：《安徽醫(yī)科大學(xué)》2014年博士論文

【摘要】：內(nèi)質(zhì)網(wǎng)(endoplasmic reticulum ER)是真核細(xì)胞中的重要細(xì)胞器,其基本生理功能包括Ca2+調(diào)節(jié)、生物大分子的合成、加工等。內(nèi)質(zhì)網(wǎng)非常敏感,葡萄糖或營養(yǎng)素缺乏、蛋白質(zhì)糖基化抑制、二硫鍵形成障礙、蛋白質(zhì)轉(zhuǎn)運(yùn)異常、Ca2+耗竭等刺激均可導(dǎo)致ER功能失調(diào),導(dǎo)致ER應(yīng)激反應(yīng)(ER stress,ERS)。ERS持續(xù)存在或過強(qiáng)時(shí)將誘導(dǎo)ER相關(guān)性死亡(endoplasmic reticulum associated death,ERAD),造成細(xì)胞損傷。有研究表明,當(dāng)腦組織缺血時(shí),大量ATP耗竭、Ca2+超載及自由基生成等因素,均可以誘導(dǎo)過度ERS而導(dǎo)致神經(jīng)細(xì)胞損傷。異丙酚(2,6-diisopropylphenol)是一種非巴比妥類靜脈麻醉藥,具有快速、可靠的鎮(zhèn)靜催眠作用,臨床廣泛應(yīng)用于麻醉誘導(dǎo)、麻醉維持和ICU鎮(zhèn)靜。因其化學(xué)結(jié)構(gòu)類似于內(nèi)源性抗氧化劑維生素E和外源性抗氧化劑丁羥基甲苯(BHT),因而具有清除自由基作用及抗氧化作用。諸多研究表明異丙酚具有神經(jīng)細(xì)胞保護(hù)作用,但其神經(jīng)保護(hù)機(jī)制仍不清楚。本研究擬探討ERS在異丙酚發(fā)揮神經(jīng)保護(hù)中的作用及機(jī)制。目的:觀察異丙酚是否具有抑制ERS引起的細(xì)胞損傷作用,并探討ERS相關(guān)蛋白在其保護(hù)中的作用。 使用ERS的誘導(dǎo)劑依霉素(tunicamycin Tm)誘導(dǎo)SH-SY5Y和neuro-2a神經(jīng)細(xì)胞出現(xiàn)ERS,MTT法檢測(cè)SH-SY5Y細(xì)胞活力,流式細(xì)胞技術(shù)(Flow cytometry FCM)驗(yàn)證Tm引起的neuro-2a神經(jīng)細(xì)胞凋亡;逆轉(zhuǎn)錄聚合酶聯(lián)反應(yīng)(Reverse Transcription Polymerase Chain Reaction,RT-PCR)和免疫印跡(Western Blot,WB)檢測(cè)ERS引起B(yǎng)i P和CHOP的m RNA及蛋白水平的變化;免疫熒光(Immunofluorescence IF)觀察細(xì)胞形態(tài)和兩種蛋白的表達(dá)和胞內(nèi)定位;免疫印跡法檢測(cè)ERS三條通路相關(guān)蛋白的表達(dá);用si RNA沉默內(nèi)源性的Bi P的表達(dá),觀察neuro-2a神經(jīng)細(xì)胞活力變化,同時(shí)觀察凋亡蛋白的表達(dá)。結(jié)果:1.異丙酚能防止ERS導(dǎo)致的神經(jīng)細(xì)胞損傷我們首先利用MTT法檢測(cè)不同濃度的異丙酚對(duì)SH-SY5Y細(xì)胞增殖活性的影響,結(jié)果顯示,與對(duì)照組相比,10μM的異丙酚能提高SH-SY5Y細(xì)胞增殖活性。預(yù)先在SH-SY5Y細(xì)胞培養(yǎng)液中加入不同濃度的異丙酚6小時(shí)后,再用Tm誘導(dǎo)6小時(shí)。結(jié)果顯示當(dāng)異丙酚濃度在1、10、20μM和40μM時(shí),異丙酚能不同程度的抑制Tm引起的細(xì)胞活力下降,10μM濃度作用最為顯著,DAPI/PI雙染法也驗(yàn)證這一結(jié)果。流式細(xì)胞技術(shù)亦顯示異丙酚預(yù)先給藥可顯著降低Tm引起的neuro-2a細(xì)胞凋亡率。2.異丙酚提高Bi P的表達(dá)Bi P被認(rèn)為是ERS標(biāo)志性蛋白,為了解異丙酚對(duì)Tm引起的細(xì)胞損傷保護(hù)機(jī)制,我們檢測(cè)了該藥與Bi P表達(dá)的量效關(guān)系。將不同濃度異丙酚共孵育對(duì)數(shù)生長期的SH-SY5Y細(xì)胞后,提取總蛋白,WB結(jié)果顯示1、10、20、40、80μM異丙酚均可不同程度的上調(diào)Bi P的表達(dá),且濃度為10μM時(shí),Bi P的表達(dá)水平最高。同時(shí)我們還檢測(cè)了ERS凋亡途徑重要的信號(hào)分子CHOP,發(fā)現(xiàn)CHOP沒有明顯表達(dá)。異丙酚導(dǎo)致Bi P表達(dá)上調(diào)還具有時(shí)間依賴性,共孵育3小時(shí)后Bi P表達(dá)開始上調(diào),6小時(shí)達(dá)峰值,持續(xù)到24小時(shí)。IF實(shí)驗(yàn)同時(shí)證實(shí)10μM的異丙酚上調(diào)Bi P的表達(dá)。3.異丙酚抑制Tm誘導(dǎo)的CHOP蛋白的表達(dá)在了解單純異丙酚不影響SH-SY5Y細(xì)胞系中的CHOP表達(dá)之后,我們?cè)噲D知曉該藥對(duì)Tm引起的CHOP表達(dá)是否有影響。用不同濃度(0.01、0.1、1、10、20、40、80μM)異丙酚共孵育SH-SY5Y細(xì)胞6小時(shí)后,再用Tm(5μg/ml)刺激細(xì)胞6小時(shí)后提取總蛋白,WB顯示0.1、1、10、20μM異丙酚均能顯著抑制Tm誘導(dǎo)的CHOP蛋白的表達(dá),且以10μM效果最為顯著。選取10μM為異丙酚終濃度,同樣方法處理不同濃度的Tm(2.5、5、10μg/ml)刺激SH-SY5Y細(xì)胞,WB結(jié)果顯示CHOP蛋白均顯著下降。為進(jìn)一步了解異丙酚預(yù)處理對(duì)于兩種ERS相關(guān)蛋白表達(dá)的影響,我們還選用了小鼠神經(jīng)瘤neuro-2a細(xì)胞,用異丙酚(10μM)和Tm(5μg/ml)處理,分別提取總RNA和總蛋白,進(jìn)一步驗(yàn)證異丙酚對(duì)Bi P和CHOP的影響,結(jié)果與前類似。IF實(shí)驗(yàn)也證實(shí)異丙酚不僅能抑制Tm誘導(dǎo)的CHOP的表達(dá),而且抑制CHOP蛋白從胞漿向胞核轉(zhuǎn)移。4.異丙酚對(duì)UPR三種通路蛋白的影響鑒于異丙酚差異性地調(diào)節(jié)ERS標(biāo)志性蛋白Bi P和CHOP,我們繼續(xù)觀察了異丙酚對(duì)UPR通路中幾個(gè)關(guān)鍵蛋白表達(dá)水平的影響。用異丙酚(10μM)和Tm(5μg/ml)處理neuro-2a細(xì)胞,方法同前,提取總蛋白做WB分析。結(jié)果顯示,異丙酚能顯著上調(diào)s-XBP1和cleaved ATF6蛋白水平,對(duì)磷酸化的PERK和e IFα水平無明顯影響。而用Tm刺激后,異丙酚能抑制磷酸化PERK和e IFα的的升高,而對(duì)升高的s-XBP1和cleaved ATF6蛋白水平無明顯影響。5.異丙酚對(duì)ERS相關(guān)凋亡蛋白表達(dá)的影響前面實(shí)驗(yàn)結(jié)果表明異丙酚能抑制Tm引起的神經(jīng)細(xì)胞凋亡,我們進(jìn)一步用WB法驗(yàn)證ERS途徑中的凋亡前蛋白ATF4表達(dá)和凋亡通路共有的終末效應(yīng)蛋白caspase3的活化水平。結(jié)果顯示Tm誘導(dǎo)的ATF4的高表達(dá)和活化的caspase 3水平均被異丙酚明顯抑制。6.內(nèi)源性Bi P敲低抑制異丙酚對(duì)CHOP和caspase 3的影響為了解異丙酚抑制ERS導(dǎo)致的細(xì)胞損傷的潛在機(jī)制,我們應(yīng)用RNAi技術(shù)對(duì)neuro-2a細(xì)胞中內(nèi)源性Bi P進(jìn)行敲低。轉(zhuǎn)染三條Bi P-si RNA 24小時(shí)后提取neuro-2a細(xì)胞總蛋白,WB法檢測(cè)Bi P-si RNA的敲低效果。結(jié)果顯示其中有一條si RNA對(duì)Bi P的沉默效果明顯,Bi P蛋白質(zhì)表達(dá)水平下降,遂選取此條si RNA進(jìn)行后續(xù)實(shí)驗(yàn)。將驗(yàn)證有效的Bi P-si RNA轉(zhuǎn)染neuro-2a細(xì)胞24小時(shí)后,加入10μM異丙酚,6小時(shí)后予5μg/ml Tm處共孵育,提取總蛋白后檢測(cè)CHOP和活化的caspase 3表達(dá)水平。結(jié)果顯示內(nèi)源性Bi P被敲除后,異丙酚不能有效抑制Tm引起的CHOP表達(dá)和caspase 3活化。7.Bi P基因敲低后異丙酚的神經(jīng)細(xì)胞保護(hù)作用消失為進(jìn)一步明確異丙酚引起的Bi P高表達(dá)是否具有抗ERS引起的凋亡作用,我們?cè)趎euro-2a細(xì)胞中瞬時(shí)轉(zhuǎn)染si RNA-Bi P后,給予異丙酚處理,MTT結(jié)果顯示在轉(zhuǎn)染si RNA-Bi P后,異丙酚所導(dǎo)致的細(xì)胞增殖活性增加的作用消失,流式細(xì)胞技術(shù)亦顯示無論是否預(yù)先予以異丙酚處理,兩組的細(xì)胞凋亡率基本一致。提示在瞬時(shí)轉(zhuǎn)染si RNA-Bi P后,異丙酚的神經(jīng)保護(hù)作用消失。結(jié)論:以上研究結(jié)果表明,臨床應(yīng)用濃度的異丙酚神經(jīng)保護(hù)作用可能與其上調(diào)Bi P表達(dá)、抑制PERK/e IFα/ATF4/CHOP通路引起的細(xì)胞凋亡有關(guān)。
[Abstract]:Endoplasmic reticulum ER is an important organelle in eukaryotic cells. Its basic physiological functions include Ca2+ regulation, biosynthesis of biological macromolecules, processing and so on. Endoplasmic reticulum is very sensitive, glucose or nutrient deficiency, protein glycosylation inhibition, two sulfur bond formation obstacle, protein transport abnormal, Ca2+ exhaustion and so on can lead to ER Dysfunction of the ER stress response (ER stress, ERS).ERS will induce ER related death (endoplasmic reticulum associated death, ERAD) and cause cell damage when the.ERS is persistent or too strong. Cell injury. Propofol (2,6-diisopropylphenol) is a non barbiturate intravenous anesthetic. It has a rapid and reliable sedative hypnotic effect. It is widely used in anesthesia induction, anesthesia maintenance and ICU sedative. Its chemical structure is similar to endogenous antioxidant vitamin E and exogenous antioxidant butyl hydroxy toluene (BHT). A number of studies have shown that propofol has neuroprotective effects, but its neuroprotective mechanism is still unclear. This study intends to explore the role and mechanism of ERS in the protection of propofol. Objective: To observe whether propofol can inhibit cell damage induced by ERS and to explore ERS phase. SH-SY5Y and Neuro-2a neurons were induced by ERS inducer (tunicamycin Tm), and SH-SY5Y cell viability was detected by MTT method. Flow cytometry (Flow cytometry FCM) was used to verify the apoptosis of neuronal cells caused by Tm, and reverse transcription polymerase chain reaction (RT PCR). Se Chain Reaction, RT-PCR) and immunoblotting (Western Blot, WB) detected the changes in ERS causing Bi P and CHOP m and protein levels; immunofluorescence observed cell morphology and the expression of two proteins and intracellular localization; the expression of three pathway related proteins was detected by immunoblotting; The expression of Neuro-2a nerve cell activity was observed and the expression of apoptotic protein was observed. Results: 1. propofol could prevent ERS induced nerve cell damage. First, we detected the effect of propofol on the proliferation of SH-SY5Y cells with different concentrations of propofol by MTT method. The results showed that, compared with the control group, 10 M of propofol could improve SH-SY5Y. Cell proliferation activity. After adding different concentrations of propofol in SH-SY5Y cell culture for 6 hours, Tm was induced for 6 hours. The results showed that when the concentration of propofol at 1,10,20 mu M and 40 mu M, the propofol could decrease the cell vitality of Tm in varying degrees, and the concentration of 10 mu M was the most significant. The DAPI/PI double staining method also verified this knot. Flow cytometry also showed that propofol could significantly reduce the apoptosis rate of Neuro-2a cells induced by Tm,.2. propofol increased Bi P expression, Bi P was considered as a ERS marker protein, to understand the protective mechanism of cell damage caused by propofol on Tm. We detected the dose effect relationship between the drug and Bi P expression. After incubation of the SH-SY5Y cells in the logarithmic growth period, the total protein was extracted, and the results of WB showed that 1,10,20,40,80 uh M could increase the expression of Bi P in varying degrees. When the concentration was 10 mu M, the expression level of Bi P was the highest. At the same time, we also detected the important signal molecule CHOP of ERS apoptosis pathway, and found that CHOP was not obviously expressed. The up-regulated expression of P was also time dependent. After 3 hours of incubation, the expression of Bi P began to rise, the peak value reached 6 hours, and the.IF test lasted to 24 hours. The expression of 10 mu M was confirmed by the expression of.3. propofol and the expression of CHOP protein inhibited by Tm induced CHOP protein after understanding the CHOP expression in the pure propofol SH-SY5Y cell line. We tried to know whether the drug had an effect on the expression of CHOP induced by Tm. After incubating SH-SY5Y cells with different concentration (0.01,0.1,1,10,20,40,80 M) propofol for 6 hours, the total protein was extracted after 6 hours by Tm (5 g/ml), and WB showed that 0.1,1,10,20 micron M propofol could significantly inhibit the Tm induced CHOP protein expression, and the maximum effect was 10 micron. 10 M was selected as the final concentration of propofol, and the same method was used to treat SH-SY5Y cells with different concentrations of Tm (2.5,5,10 mu g/ml). WB results showed that CHOP protein decreased significantly. In order to further understand the effect of propofol pretreatment on the expression of two ERS related proteins, we also selected the mice neuroma Neuro-2a cells, using propofol (10 micron M). And Tm (5 mu g/ml) treatment, the total RNA and total protein were extracted, and the effect of propofol on Bi P and CHOP was further verified. The results similar to the previous.IF test also proved that the propofol could not only inhibit the expression of CHOP induced by Tm, but also inhibit the effect of.4. Isoprophenol on the three pathway proteins from the cytoplasm to the nucleus in view of the difference of propofol. The effect of propofol on the expression level of several key proteins in the ERS pathway was observed. We continued to observe the effect of propofol on the expression level of several key proteins in the UPR pathway. Using propofol (10 mu M) and Tm (5 mu g/ml) to treat Neuro-2a cells, the method was used before and extracted the total protein for WB analysis. The results showed that isopropofol could significantly increase s-XBP1 and cleaved protein levels, and phosphorus. The levels of acidified PERK and E IF alpha were not significantly affected, and propofol could inhibit the increase of phosphorylated PERK and E IF alpha after Tm stimulation, but there was no significant effect on the elevated s-XBP1 and cleaved ATF6 protein levels. The previous experimental results of.5. propofol on the expression of ERS related apoptotic proteins showed that propofol could inhibit the neuronal decay caused by the inhibitory effect of propofol. WB method was further used to verify the activation level of terminal effector protein Caspase3 in the expression of pre apoptotic protein ATF4 and apoptosis pathway in ERS pathway. The results showed that the high expression of Tm induced ATF4 and the level of activated caspase 3 were all obviously inhibited by propofol and the effect of propofol on CHOP and caspase 3 In order to understand the potential mechanism of propofol inhibiting cell damage caused by ERS, we used RNAi technology to knock down endogenous Bi P in Neuro-2a cells. The total protein of Neuro-2a cells was extracted after transfection of three Bi P-si RNA, and WB method was used to detect the low effect of Bi P-si. The expression level of P protein was decreased, then the Si RNA was selected for the follow-up experiment. The effective Bi P-si RNA transfected to Neuro-2a cells for 24 hours, 10 u M propofol was added, and 5 mu g/ml Tm was reincubated after 6 hours, and the CHOP and activated caspase 3 was detected after extracting the total protein. No effective inhibition of Tm induced CHOP expression and caspase 3 activation of.7.Bi P gene knockout, the neuroprotective effect of propofol disappeared to further clarify whether the Bi P high expression induced by propofol has the effect of anti ERS induced apoptosis. We transiently transfect Si RNA-Bi P in Neuro-2a cells and give propofol treatment. After transfection of Si RNA-Bi P, the increase of cell proliferation activity caused by propofol disappeared. Flow cytometry also showed that the rate of apoptosis in the two groups was basically the same. It suggested that the neuroprotective effect of propofol disappeared after transient transfection of Si RNA-Bi P. Conclusion: the above results showed that The neuroprotective effect of propofol in clinical application may be related to its up regulation of Bi P expression and inhibition of apoptosis induced by PERK/e IF alpha /ATF4/CHOP pathway.

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R614

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 葉治;郭曲練;王鍔;;七氟醚遲發(fā)性預(yù)處理對(duì)大鼠局灶性腦缺血損傷后NF-κB表達(dá)的影響[J];中南大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2010年03期

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本文編號(hào)：1788402