核黄素缺乏对HepG2细胞蛋白质表达谱影响的研究
发布时间：2018-07-24 11:39
【摘要】：目的建立核黄素缺乏的细胞模型,用含不同浓度核黄素的培养基培养细胞,观测可使细胞维持正常状态的适宜核黄素水平,用蛋白质组学技术检测核黄素缺乏对HepG2蛋白质表达谱的影响,进一步分析验证蛋白质组学结果,为今后深入研究核黄素缺乏对人体影响的机制提供科学依据。方法1.胎牛血清(FBS)中核黄素的去除:无菌条件下用紫外线照射方法去除FBS中核黄素,采用高效液相色谱(HPLC)法测定经不同照射时间后FBS中核黄素的含量,并用不同照射时间后的血清培养细胞,观测细胞活力。选择使FBS中核黄素显著减少且可维持细胞正常状态的紫外线照射时间,作为进一步模型建立的前处理方法。2.核黄素缺乏的模型建立与适宜干预浓度探讨:通过定制无核黄素培养基、紫外线清除FBS中核黄素建立核黄素缺乏培养条件,在此基础上,以不同浓度核黄素(0.76、3.76、6.76、12.76、24.76、48.76nmol/L)培养HepG2细胞96小时,期间于不同时间点取样测定细胞活力、细胞凋亡率,于72小时测定培养上清液的丙二醛(MDA)含量、丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)活性,以及细胞中谷胱甘肽还原酶(GR)、谷胱甘肽过氧化物酶(GSH-Px)与超氧化物歧化酶(SOD)活性,细胞内还原型谷胱甘肽(GSH)与氧化型谷胱甘肽(GSSG)含量。3.蛋白质组学技术检测核黄素缺乏对HepG2细胞蛋白表达谱的影响:运用非标记(labelfree)定量蛋白质组学技术分析比较在核黄素缺乏(0.76nmol/l)和核黄素适宜(12.76nmol/l)培养基中培养4天后的hepg2细胞中蛋白质差异表达情况,并进行了生物信息学分析(包括go富集分析、kegg通路分析,差异蛋白质互相作用网络分析),然后对筛选出的5个关键差异蛋白(ndufs1,ndufv2,sdha,sqstm1,ero1a)进行蛋白免疫印迹(westernbolt)验证。4.核黄素缺乏对hepg2细胞载脂蛋白b100折叠及其表达通路的影响:因蛋白质组学结果显示核黄素缺乏导致了可能影响载脂蛋白(apo)b100二硫键折叠的关键蛋白(ero1a)显著下调,故采用酶联免疫(elisa)法定量检测细胞内和细胞外分泌apob100的含量,用westernbolt和逆转录实时荧光定量(rt-qpcr)技术分别检测核黄素缺乏对apob100表达通路基因和蛋白的影响,随后用ellman法测定细胞中蛋白结合巯基以间接反映apob100的二硫键形成情况。结果1.fbs中的核黄素含量随紫外线照射时间的延长而逐渐减少,fbs经照射30min后核黄素含量较未照射组极显著下降(p0.01),继续延长照射时间核黄素含量下降趋势逐渐减弱;用照射30min血清培养细胞,我们发现其活力与未照射组无显著差异,而照射时间大于30min的血清组细胞活力显著下降(p0.05)。2.培养72至96小时,随着核黄素浓度的降低,hepg2细胞活力显著下降、细胞凋亡率显著升高(p0.05),培养液中ast、alt活性显著升高、mda含量显著升高(p0.05),细胞内gr活性显著下降、而gsh-px的活性显著上升、gssg含量显著上升、gsh含量显著降低、gsh/gssg显著下降(p0.05)。上述大部分指标的核黄素剂量效应拐点在12.76nmol/l左右。证明核黄素缺乏细胞模型建立成功,维持细胞正常状态的核黄素浓度应高于12.76nmol/l。3.本研究共鉴定到3730个蛋白质,其中在核黄素缺乏组鉴定到2830个蛋白质,在对照组鉴定到3020个蛋白质,即85个蛋白是核黄素缺乏组特有、275个蛋白是对照组特有,在两组共有的2745个蛋白质中经差异性比较后获得37个差异表达倍数大于2的蛋白,其中,与核黄素适宜组相比,核黄素缺乏组有13个蛋白表达显著上调,24个蛋白显著下调。采用GO富集分析发现37个差异蛋白在在线粒体氧化呼吸链有较高富集,分子功能主要为氧化还原活性,主要参与了电子转移,氧化还原,能量代谢等生物学过程,随后我们采用KEGG信号通路富集分析发现这些差异蛋白主要参与了18个信号通路,其中富集度较高的通路为帕金森病、脂肪酸代谢、内质网应激等信号通路。Western bolt结果显示:与核黄素适宜组相比,核黄素缺乏组的NDUFS1、NDUFV2、SDHA、ERO1A表达显著上调,SQSTM1表达显著下调。验证结果与蛋白质组学结果基本一致。4.与核黄素适宜组相比,核黄素缺乏组ApoB100细胞内含量及细胞外分泌量均显著下降(P0.05),二硫键异构酶(PDI)蛋白表达量显著降低、内质网应激标志性蛋白(GRP78,GADD153)表达量显著升高(P0.05),细胞内总蛋白结合巯基含量显著下降(P0.05)。结论核黄素缺乏显著影响人肝癌HepG2细胞的正常状态,维持该细胞状态的培养液中核黄素浓度应高于12.76nmol/L。核黄素缺乏显著影响了HepG2细胞蛋白质表达谱,受影响的相关蛋白可使能量代谢和脂质代谢下调,且可促进内质网应激和凋亡的发生等,进一步针对ApoB100的通路研究发现核黄素缺乏可导致ApoB100二硫键形成障碍从而影响脂质转运。此研究为深入探索核黄素乏对人体影响的分子机制提供线索和实验基础。
[Abstract]:Objective to establish the cell model of riboflavin deficiency and to observe the riboflavin level in the normal state by culture medium containing different concentrations of riboflavin. The effect of riboflavin deficiency on the expression of HepG2 protein was detected by proteomics technology, and the results of proteomics were further analyzed and verified for future research. To investigate the mechanism of riboflavin deficiency on human body, the removal of riboflavin in 1. fetal bovine serum (FBS): removal of riboflavin in FBS by UV irradiation under aseptic conditions, and the determination of the content of riboflavin in FBS after different exposure time by high performance liquid chromatography (HPLC), and the serum culture after different irradiation time. Cells, observe cell vitality. Select the ultraviolet radiation time that significantly reduces riboflavin in FBS and can maintain cell normal state. As a preconditioning method established for further models, the model of.2. riboflavin deficiency is established and the appropriate intervention concentration is discussed: by customizing the riboflavin medium and eliminating riboflavin in FBS to establish nuclear yellow On this basis, HepG2 cells were cultured with different concentrations of riboflavin (0.76,3.76,6.76,12.76,24.76,48.76nmol/L) for 96 hours. The cell viability and apoptosis rate were measured at different time points, and the content of malondialdehyde (MDA), alanine aminotransferase (ALT) and aspartate aminotransferase (AS) were measured at 72 hours. T) activity, as well as cell Nakatani Ka reductase (GR), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activity, intracellular reduced glutathione (GSH) and oxidized glutathione (GSSG) content.3. proteomics techniques to detect the effect of riboflavin deficiency on the protein expression profiles of HepG2 cells: using unlabeled (labelfr) EE) quantitative proteomics technology analysis and comparison of protein differential expression in HepG2 cells of 4 days after 4 days of riboflavin deficiency (0.76nmol/l) and riboflavin suitable (12.76nmol/l) culture medium, and bioinformatics analysis (including go enrichment analysis, KEGG pathway analysis, differential protein interaction network analysis), and then sieved. 5 key differential proteins (NDUFS1, ndufv2, sdhA, sqstm1, ero1a) were selected to carry out protein immunoblotting (westernbolt) to verify the effect of.4. riboflavin deficiency on the B100 folding and expression of apolipoprotein in HepG2 cells. The results of proteomics showed that the deficiency of riboflavin was responsible for the possible influence of the apolipoprotein (apo) B100 two sulfur bond folding. The bond protein (ero1a) was significantly downregulated, so the content of intracellular and extracellular secretory apoB100 was detected by the enzyme linked immunosorbent assay (ELISA). The effect of riboflavin deficiency on the gene and protein of apoB100 expression pathway was detected by westernbolt and reverse transcriptase real-time quantitative (RT-qPCR) technique. Then the protein binding sulfhydryl in cells was determined by Ellman method. The formation of the two sulfur bond of apoB100 was indirectly reflected. Results the riboflavin content in 1.fbs decreased gradually with the prolongation of ultraviolet irradiation time. After 30min irradiation, the riboflavin content of FBS decreased significantly (P0.01), and continued to decrease under the riboflavin content of the irradiation time. We found that there was no significant difference between the activity and the unirradiated group, but the cell vitality of the serum group of the serum group was significantly lower than 30min (P0.05) for 72 to 96 hours. With the decrease of the riboflavin concentration, the activity of HepG2 cells decreased significantly, the apoptosis rate increased significantly (P0.05), the activity of AST, ALT in the culture medium increased significantly, and the MDA content was significant. The activity of GR in cells decreased significantly (P0.05), while the activity of GSH-Px increased significantly, the content of GSSG increased significantly, the content of GSH decreased significantly, and gsh/gssg decreased significantly (P0.05). The riboflavin dose effect inflection point was around 12.76nmol/l. It proved that the riboflavin deficiency cell model was established successfully and maintained the normal state of the nuclear yellow. The element concentration should be higher than the 12.76nmol/l.3. study to identify 3730 proteins, of which 2830 proteins were identified in the riboflavin deficiency group and 3020 proteins were identified in the control group, that is, 85 proteins were endemic to the riboflavin deficiency group and 275 proteins were specific in the control group, and 3 of the 2745 proteins in the two groups were compared to 3. 7 proteins were expressed in more than 2, of which 13 protein expressions were significantly up-regulated in riboflavin deficiency group and 24 proteins were significantly downregulated in riboflavin deficiency group. GO enrichment analysis found that 37 differential proteins were enriched in mitochondrial oxidative respiration chain, and the molecular function was mainly redox activity, mainly involved in the electron. Biological processes such as metastasis, redox, energy metabolism, and so on, and then we use KEGG signal pathway enrichment analysis to find that these differential proteins are mainly involved in 18 signal pathways, of which the high concentration pathway is Parkinson's disease, fatty acid metabolism, endoplasmic reticulum stress and other signal passway.Western bolt results: compared with the riboflavin suitable group The expression of NDUFS1, NDUFV2, SDHA, ERO1A in the riboflavin deficiency group was significantly up-regulated, and the expression of SQSTM1 was down significantly. The results were in agreement with the results of the proteomics. The content of ApoB100 cells in the riboflavin deficiency group and the exocrine volume of the riboflavin deficiency group decreased significantly (P0.05), and the expression of two sulfur bond isomerase (PDI) protein was significant. The expression of endoplasmic reticulum stress marker protein (GRP78, GADD153) increased significantly (P0.05), and the total protein binding sulfhydryl content in the cells decreased significantly (P0.05). Conclusion riboflavin deficiency significantly affected the normal state of human hepatocellular carcinoma HepG2 cells. The concentration of riboflavin in the cultured cultured fluid of this cell should be higher than that of 12.76nmol/L. riboflavin deficiency. The protein expression profiles of HepG2 cells are affected. The related proteins can reduce the energy metabolism and lipid metabolism, and promote the occurrence of endoplasmic reticulum stress and apoptosis. Further research on ApoB100 pathway has found that riboflavin deficiency can lead to the formation of ApoB100 two sulfur bonds and thus affect lipid transport. Huang Sufa provides clues and experimental basis for the molecular mechanism of human impact.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R151
本文编号：2141258
[Abstract]:Objective to establish the cell model of riboflavin deficiency and to observe the riboflavin level in the normal state by culture medium containing different concentrations of riboflavin. The effect of riboflavin deficiency on the expression of HepG2 protein was detected by proteomics technology, and the results of proteomics were further analyzed and verified for future research. To investigate the mechanism of riboflavin deficiency on human body, the removal of riboflavin in 1. fetal bovine serum (FBS): removal of riboflavin in FBS by UV irradiation under aseptic conditions, and the determination of the content of riboflavin in FBS after different exposure time by high performance liquid chromatography (HPLC), and the serum culture after different irradiation time. Cells, observe cell vitality. Select the ultraviolet radiation time that significantly reduces riboflavin in FBS and can maintain cell normal state. As a preconditioning method established for further models, the model of.2. riboflavin deficiency is established and the appropriate intervention concentration is discussed: by customizing the riboflavin medium and eliminating riboflavin in FBS to establish nuclear yellow On this basis, HepG2 cells were cultured with different concentrations of riboflavin (0.76,3.76,6.76,12.76,24.76,48.76nmol/L) for 96 hours. The cell viability and apoptosis rate were measured at different time points, and the content of malondialdehyde (MDA), alanine aminotransferase (ALT) and aspartate aminotransferase (AS) were measured at 72 hours. T) activity, as well as cell Nakatani Ka reductase (GR), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activity, intracellular reduced glutathione (GSH) and oxidized glutathione (GSSG) content.3. proteomics techniques to detect the effect of riboflavin deficiency on the protein expression profiles of HepG2 cells: using unlabeled (labelfr) EE) quantitative proteomics technology analysis and comparison of protein differential expression in HepG2 cells of 4 days after 4 days of riboflavin deficiency (0.76nmol/l) and riboflavin suitable (12.76nmol/l) culture medium, and bioinformatics analysis (including go enrichment analysis, KEGG pathway analysis, differential protein interaction network analysis), and then sieved. 5 key differential proteins (NDUFS1, ndufv2, sdhA, sqstm1, ero1a) were selected to carry out protein immunoblotting (westernbolt) to verify the effect of.4. riboflavin deficiency on the B100 folding and expression of apolipoprotein in HepG2 cells. The results of proteomics showed that the deficiency of riboflavin was responsible for the possible influence of the apolipoprotein (apo) B100 two sulfur bond folding. The bond protein (ero1a) was significantly downregulated, so the content of intracellular and extracellular secretory apoB100 was detected by the enzyme linked immunosorbent assay (ELISA). The effect of riboflavin deficiency on the gene and protein of apoB100 expression pathway was detected by westernbolt and reverse transcriptase real-time quantitative (RT-qPCR) technique. Then the protein binding sulfhydryl in cells was determined by Ellman method. The formation of the two sulfur bond of apoB100 was indirectly reflected. Results the riboflavin content in 1.fbs decreased gradually with the prolongation of ultraviolet irradiation time. After 30min irradiation, the riboflavin content of FBS decreased significantly (P0.01), and continued to decrease under the riboflavin content of the irradiation time. We found that there was no significant difference between the activity and the unirradiated group, but the cell vitality of the serum group of the serum group was significantly lower than 30min (P0.05) for 72 to 96 hours. With the decrease of the riboflavin concentration, the activity of HepG2 cells decreased significantly, the apoptosis rate increased significantly (P0.05), the activity of AST, ALT in the culture medium increased significantly, and the MDA content was significant. The activity of GR in cells decreased significantly (P0.05), while the activity of GSH-Px increased significantly, the content of GSSG increased significantly, the content of GSH decreased significantly, and gsh/gssg decreased significantly (P0.05). The riboflavin dose effect inflection point was around 12.76nmol/l. It proved that the riboflavin deficiency cell model was established successfully and maintained the normal state of the nuclear yellow. The element concentration should be higher than the 12.76nmol/l.3. study to identify 3730 proteins, of which 2830 proteins were identified in the riboflavin deficiency group and 3020 proteins were identified in the control group, that is, 85 proteins were endemic to the riboflavin deficiency group and 275 proteins were specific in the control group, and 3 of the 2745 proteins in the two groups were compared to 3. 7 proteins were expressed in more than 2, of which 13 protein expressions were significantly up-regulated in riboflavin deficiency group and 24 proteins were significantly downregulated in riboflavin deficiency group. GO enrichment analysis found that 37 differential proteins were enriched in mitochondrial oxidative respiration chain, and the molecular function was mainly redox activity, mainly involved in the electron. Biological processes such as metastasis, redox, energy metabolism, and so on, and then we use KEGG signal pathway enrichment analysis to find that these differential proteins are mainly involved in 18 signal pathways, of which the high concentration pathway is Parkinson's disease, fatty acid metabolism, endoplasmic reticulum stress and other signal passway.Western bolt results: compared with the riboflavin suitable group The expression of NDUFS1, NDUFV2, SDHA, ERO1A in the riboflavin deficiency group was significantly up-regulated, and the expression of SQSTM1 was down significantly. The results were in agreement with the results of the proteomics. The content of ApoB100 cells in the riboflavin deficiency group and the exocrine volume of the riboflavin deficiency group decreased significantly (P0.05), and the expression of two sulfur bond isomerase (PDI) protein was significant. The expression of endoplasmic reticulum stress marker protein (GRP78, GADD153) increased significantly (P0.05), and the total protein binding sulfhydryl content in the cells decreased significantly (P0.05). Conclusion riboflavin deficiency significantly affected the normal state of human hepatocellular carcinoma HepG2 cells. The concentration of riboflavin in the cultured cultured fluid of this cell should be higher than that of 12.76nmol/L. riboflavin deficiency. The protein expression profiles of HepG2 cells are affected. The related proteins can reduce the energy metabolism and lipid metabolism, and promote the occurrence of endoplasmic reticulum stress and apoptosis. Further research on ApoB100 pathway has found that riboflavin deficiency can lead to the formation of ApoB100 two sulfur bonds and thus affect lipid transport. Huang Sufa provides clues and experimental basis for the molecular mechanism of human impact.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R151
【参考文献】
相关期刊论文 前10条
1 辛中豪;高蔚娜;郭长江;;核黄素对脂质代谢影响研究进展[J];解放军预防医学杂志;2016年02期
2 顾清;周朋辉;张静姝;何宁;刘英华;李静;张大龙;;核黄素对高脂血症大鼠脂质代谢的影响[J];中国慢性病预防与控制;2015年01期
3 刘佳;李传飞;宁波;杨朝霞;;内质网应激通过SCAP/SREBP-1c调控L02肝细胞脂质合成代谢[J];第三军医大学学报;2015年05期
4 朱秀敏;;超氧化物歧化酶的生理活性[J];当代医学;2011年15期
5 王艳辉;王安;谢富;;维生素B_2对笼养蛋雏鸭生长性能、内分泌及抗氧化能力的影响[J];动物营养学报;2009年01期
6 郭长江;韦京豫;杨继军;吴健全;高蔚娜;安代志;;我国男性成年人血清游离核黄素浓度正常范围的探讨[J];营养学报;2008年06期
7 晏春根;任光圆;朱冬芳;倪培华;周惠娟;;同型半胱氨酸介导的内质网应激对肝细胞脂质代谢的影响[J];中国病理生理杂志;2007年12期
8 张嘉晴,郭莉群,黄秀峰,左保华,周志泳;核黄素对高脂血症所致内皮损伤的保护作用[J];皖南医学院学报;2003年03期
9 于长青;袁旭;付世新;张爱武;张丽萍;;麦胚黄酮对实验老鼠血脂水平和脂质抗氧化作用影响的研究[J];中国食品学报;2001年02期
10 左保华;周志泳;陆晓华;杨会杰;;核黄素对高血压合并症有关因素的影响[J];中国病理生理杂志;1997年04期
，本文编号：2141258
本文链接：https://www.wllwen.com/yixuelunwen/yufangyixuelunwen/2141258.html
