本文選題：大黃素乙酰化產(chǎn)物B + 放射增敏�。� 參考：《廣西醫(yī)科大學(xué)》2012年碩士論文

【摘要】：目的：研究大黃素乙�；a(chǎn)物B放射增敏過程中對鼻咽癌CNE-1細(xì)胞的線粒體功能的影響,應(yīng)用基于二維液相色譜/質(zhì)譜聯(lián)用的穩(wěn)定同位素標(biāo)記(iTRAQ-2D-LC/MALDI-TOF/TOF/MS)的方法分析放射增敏前后線粒體的蛋白質(zhì)差異,初步確定大黃素乙�；a(chǎn)物B放射增敏作用的線粒體蛋白靶點。 方法：(1)透射電鏡觀察大黃素乙�；a(chǎn)物B對鼻咽癌CNE-1細(xì)胞放射增敏過程中線粒體超微結(jié)構(gòu)的改變；(2)羅丹明123染色結(jié)合激光共聚焦顯微鏡測定大黃素乙酰化產(chǎn)物B對CNE-1細(xì)胞放射增敏過程中線粒體跨膜電位的變化；(3)Fluo-3AM染色結(jié)合激光共聚焦顯微鏡測定大黃素乙酰化產(chǎn)物B對CNE-1細(xì)胞放射增敏過程中細(xì)胞內(nèi)鈣離子濃度的變化；(4)采用線粒體蛋白提取試劑盒提取鼻咽癌CNE-1細(xì)胞線粒體蛋白；(5)采用iTRAQ-2D-LC/MALDI-TOF/TOF/MS方法定量分析大黃素乙�；a(chǎn)物B對鼻咽癌CNE-1細(xì)胞放射增敏前后細(xì)胞的線粒體蛋白質(zhì)組,ProteinPilot2.0對NCBInr數(shù)據(jù)庫進(jìn)行檢索鑒定蛋白,報告置信度在95%以上的蛋白質(zhì)；(6)通過gene ontology、David、NCBI等數(shù)據(jù)庫分析差異蛋白的細(xì)胞定位、分子功能和生物過程,并應(yīng)用string數(shù)據(jù)庫對差異表達(dá)蛋白質(zhì)進(jìn)行相互作用網(wǎng)絡(luò)分析,初步確定大黃素乙�；a(chǎn)物B放射增敏作用的線粒體節(jié)點蛋白。 結(jié)果： (1)透射電鏡結(jié)果顯示,空白對照組細(xì)胞形態(tài)正常,細(xì)胞器結(jié)構(gòu)完整豐富,線粒體內(nèi)膜清晰呈現(xiàn)；2Gy照射組和10μ g/ml大黃素乙�；a(chǎn)物B作用組,細(xì)胞的超微結(jié)構(gòu)變化不明顯；而10μg/ml大黃素乙�；a(chǎn)物B聯(lián)合放射增敏組可以觀察到細(xì)胞膨脹、體積增大、細(xì)胞內(nèi)線粒體腫脹、脊斷裂等改變。 (2)空白對照組細(xì)胞羅丹明123染色后熒光強(qiáng)度均值為40.63±2.36,2Gy照射組和10μg/ml大黃素乙�；a(chǎn)物B作用組熒光強(qiáng)度均下降,熒光強(qiáng)度值分別為36.73±2.62和26.56±1.29,與空白對照組比較差異有統(tǒng)計學(xué)意義(P0.05),10μ/ml大黃素乙�；a(chǎn)物B聯(lián)合放射增敏組熒光強(qiáng)度值最低,為19.14±3.84,與2Gy照射組和10μ g/ml大黃素乙�；a(chǎn)物B作用組比較差異有統(tǒng)計學(xué)意義(P0.05),與空白對照組比較差異有明顯的統(tǒng)計學(xué)意義(P0.01)。(3)空白對照組細(xì)胞Fluo-3AM染色后熒光強(qiáng)度均值為1164.17+68.69,2Gy照射組和10μg/ml大黃素乙�；a(chǎn)物B作用組熒光強(qiáng)度均上升,熒光強(qiáng)度值分別為1391.83±33.35和1406.0±48.02,與空白對照組比較差異有統(tǒng)計學(xué)意義(P0.05),10μ g/ml大黃素乙酰化產(chǎn)物B聯(lián)合放射增敏組熒光強(qiáng)度值最高,為1940.08±55.74,與2Gy照射組和10μ g/ml大黃素乙�；a(chǎn)物B作用組比較差異有統(tǒng)計學(xué)意義(P0.05),與空白對照組比較差異有明顯的統(tǒng)計學(xué)意義(P0.01)。(4)提取所得線粒體,經(jīng)詹納斯綠B染色呈現(xiàn)藍(lán)綠色的顆粒狀或線狀結(jié)構(gòu)；BCA法測定線粒體蛋白濃度,標(biāo)準(zhǔn)曲線相關(guān)系數(shù)r=0.99,空白組,2Gy照射組、10μ g/ml大黃素乙酰化產(chǎn)物B作用組和10μ g/ml大黃素乙�；a(chǎn)物B聯(lián)合放射增敏組4組細(xì)胞線粒體蛋白濃度分別為：2.07,2.10,1.93和2.07μg/μL (5)采用iTRAQ-2D-LC/MALDI-TOF/TOF/MS方法分析增敏過程中各組線粒體蛋白質(zhì)組,共鑒定出線粒體相關(guān)蛋白998個(置信度95%),分子量范圍在555.62KD～6.92KD之間。 (6)對所鑒定蛋白進(jìn)行差異分類,與空白對照組比較,各組線粒體蛋白表達(dá)量均有顯著性差異(≥1.5fold)的蛋白有101種,其中與創(chuàng)傷、應(yīng)激反應(yīng)相關(guān)的蛋白最多占19.8%。差異表達(dá)蛋白質(zhì)相互作用網(wǎng)絡(luò)分析結(jié)果表明差異蛋白質(zhì)HspA8、Cox7C、ATP5A1等是大黃素乙�；a(chǎn)物B放射增敏作用的節(jié)點蛋白。結(jié)論： (1)經(jīng)10μg/ml大黃素乙�；a(chǎn)物B聯(lián)合2Gy照射對鼻咽癌CNE-1細(xì)胞的放射增敏作用,與細(xì)胞內(nèi)線粒體損傷,線粒體跨膜電位下降,細(xì)胞內(nèi)鈣超載等密切相關(guān)。 (2)采用iTRAQ-2D-LC/MALDI-TOF/TOF/MS的方法成功的鑒定大黃素乙�；a(chǎn)物B對鼻咽癌CNE-1細(xì)胞放射增敏線粒體相關(guān)差異蛋白101種,這些蛋白主要涉及應(yīng)激反應(yīng)和氧化還原反應(yīng)過程。 (3)對差異蛋白進(jìn)行分類及生物信息學(xué)分析,節(jié)點蛋白HspA8、Cox7C、ATP5A1等有望成為大黃素乙�；a(chǎn)物B放射增敏作用的蛋白靶點。
[Abstract]:Objective: To study the effect of emodin acetylation product (B) on mitochondrial function of nasopharyngeal carcinoma CNE-1 cells during radiosensitization of nasopharyngeal carcinoma (nasopharyngeal carcinoma), and to analyze the protein difference of the grain body before and after radiosensitization by two-dimensional liquid chromatography / mass spectrometry (iTRAQ-2D-LC/MALDI-TOF/TOF/MS), and to determine the acetyl of emodin The mitochondrial protein target of radiosensitization of the product B.
Methods: (1) the ultrastructural changes of mitochondria were observed by emodin acetylation product B during radiosensitization of nasopharyngeal carcinoma CNE-1 cells. (2) Luo Danming 123 staining and laser confocal microscopy were used to determine the changes of mitochondrial transmembrane potential during radiosensitization of CNE-1 cells by the acetylation of emodin product B; (3) Fluo-3AM staining The changes in intracellular calcium concentration of emodin acetylation product B during radiosensitization of CNE-1 cells were measured by laser confocal microscopy. (4) mitochondrial protein extraction kit was used to extract mitochondrial protein from CNE-1 cells of nasopharyngeal carcinoma; (5) iTRAQ-2D-LC/MALDI-TOF/ TOF/MS method was used to quantitatively analyze the B pairs of emodin acetylation products. The mitochondrial protein group of CNE-1 cells before and after radiosensitization of nasopharyngeal carcinoma cells, ProteinPilot2.0 to retrieve and identify the protein of NCBInr database, report the protein with confidence of more than 95%; (6) analyze the cell location, molecular function and biological process of differential protein by gene ontology, David, NCBI and so on, and apply string data. Based on the interaction network analysis of differentially expressed proteins, the mitochondrial node protein of radiosensitization of emodin acetylated product B was preliminarily determined.
Result錛，

本文編號：1986427