核黃素缺乏對(duì)HepG2細(xì)胞蛋白質(zhì)表達(dá)譜影響的研究
發(fā)布時(shí)間：2018-07-24 11:39
【摘要】：目的建立核黃素缺乏的細(xì)胞模型,用含不同濃度核黃素的培養(yǎng)基培養(yǎng)細(xì)胞,觀測可使細(xì)胞維持正常狀態(tài)的適宜核黃素水平,用蛋白質(zhì)組學(xué)技術(shù)檢測核黃素缺乏對(duì)HepG2蛋白質(zhì)表達(dá)譜的影響,進(jìn)一步分析驗(yàn)證蛋白質(zhì)組學(xué)結(jié)果,為今后深入研究核黃素缺乏對(duì)人體影響的機(jī)制提供科學(xué)依據(jù)。方法1.胎牛血清(FBS)中核黃素的去除:無菌條件下用紫外線照射方法去除FBS中核黃素,采用高效液相色譜(HPLC)法測定經(jīng)不同照射時(shí)間后FBS中核黃素的含量,并用不同照射時(shí)間后的血清培養(yǎng)細(xì)胞,觀測細(xì)胞活力。選擇使FBS中核黃素顯著減少且可維持細(xì)胞正常狀態(tài)的紫外線照射時(shí)間,作為進(jìn)一步模型建立的前處理方法。2.核黃素缺乏的模型建立與適宜干預(yù)濃度探討:通過定制無核黃素培養(yǎng)基、紫外線清除FBS中核黃素建立核黃素缺乏培養(yǎng)條件,在此基礎(chǔ)上,以不同濃度核黃素(0.76、3.76、6.76、12.76、24.76、48.76nmol/L)培養(yǎng)HepG2細(xì)胞96小時(shí),期間于不同時(shí)間點(diǎn)取樣測定細(xì)胞活力、細(xì)胞凋亡率,于72小時(shí)測定培養(yǎng)上清液的丙二醛(MDA)含量、丙氨酸轉(zhuǎn)氨酶(ALT)和天冬氨酸轉(zhuǎn)氨酶(AST)活性,以及細(xì)胞中谷胱甘肽還原酶(GR)、谷胱甘肽過氧化物酶(GSH-Px)與超氧化物歧化酶(SOD)活性,細(xì)胞內(nèi)還原型谷胱甘肽(GSH)與氧化型谷胱甘肽(GSSG)含量。3.蛋白質(zhì)組學(xué)技術(shù)檢測核黃素缺乏對(duì)HepG2細(xì)胞蛋白表達(dá)譜的影響:運(yùn)用非標(biāo)記(labelfree)定量蛋白質(zhì)組學(xué)技術(shù)分析比較在核黃素缺乏(0.76nmol/l)和核黃素適宜(12.76nmol/l)培養(yǎng)基中培養(yǎng)4天后的hepg2細(xì)胞中蛋白質(zhì)差異表達(dá)情況,并進(jìn)行了生物信息學(xué)分析(包括go富集分析、kegg通路分析,差異蛋白質(zhì)互相作用網(wǎng)絡(luò)分析),然后對(duì)篩選出的5個(gè)關(guān)鍵差異蛋白(ndufs1,ndufv2,sdha,sqstm1,ero1a)進(jìn)行蛋白免疫印跡(westernbolt)驗(yàn)證。4.核黃素缺乏對(duì)hepg2細(xì)胞載脂蛋白b100折疊及其表達(dá)通路的影響:因蛋白質(zhì)組學(xué)結(jié)果顯示核黃素缺乏導(dǎo)致了可能影響載脂蛋白(apo)b100二硫鍵折疊的關(guān)鍵蛋白(ero1a)顯著下調(diào),故采用酶聯(lián)免疫(elisa)法定量檢測細(xì)胞內(nèi)和細(xì)胞外分泌apob100的含量,用westernbolt和逆轉(zhuǎn)錄實(shí)時(shí)熒光定量(rt-qpcr)技術(shù)分別檢測核黃素缺乏對(duì)apob100表達(dá)通路基因和蛋白的影響,隨后用ellman法測定細(xì)胞中蛋白結(jié)合巰基以間接反映apob100的二硫鍵形成情況。結(jié)果1.fbs中的核黃素含量隨紫外線照射時(shí)間的延長而逐漸減少,fbs經(jīng)照射30min后核黃素含量較未照射組極顯著下降(p0.01),繼續(xù)延長照射時(shí)間核黃素含量下降趨勢逐漸減弱;用照射30min血清培養(yǎng)細(xì)胞,我們發(fā)現(xiàn)其活力與未照射組無顯著差異,而照射時(shí)間大于30min的血清組細(xì)胞活力顯著下降(p0.05)。2.培養(yǎng)72至96小時(shí),隨著核黃素濃度的降低,hepg2細(xì)胞活力顯著下降、細(xì)胞凋亡率顯著升高(p0.05),培養(yǎng)液中ast、alt活性顯著升高、mda含量顯著升高(p0.05),細(xì)胞內(nèi)gr活性顯著下降、而gsh-px的活性顯著上升、gssg含量顯著上升、gsh含量顯著降低、gsh/gssg顯著下降(p0.05)。上述大部分指標(biāo)的核黃素劑量效應(yīng)拐點(diǎn)在12.76nmol/l左右。證明核黃素缺乏細(xì)胞模型建立成功,維持細(xì)胞正常狀態(tài)的核黃素濃度應(yīng)高于12.76nmol/l。3.本研究共鑒定到3730個(gè)蛋白質(zhì),其中在核黃素缺乏組鑒定到2830個(gè)蛋白質(zhì),在對(duì)照組鑒定到3020個(gè)蛋白質(zhì),即85個(gè)蛋白是核黃素缺乏組特有、275個(gè)蛋白是對(duì)照組特有,在兩組共有的2745個(gè)蛋白質(zhì)中經(jīng)差異性比較后獲得37個(gè)差異表達(dá)倍數(shù)大于2的蛋白,其中,與核黃素適宜組相比,核黃素缺乏組有13個(gè)蛋白表達(dá)顯著上調(diào),24個(gè)蛋白顯著下調(diào)。采用GO富集分析發(fā)現(xiàn)37個(gè)差異蛋白在在線粒體氧化呼吸鏈有較高富集,分子功能主要為氧化還原活性,主要參與了電子轉(zhuǎn)移,氧化還原,能量代謝等生物學(xué)過程,隨后我們采用KEGG信號(hào)通路富集分析發(fā)現(xiàn)這些差異蛋白主要參與了18個(gè)信號(hào)通路,其中富集度較高的通路為帕金森病、脂肪酸代謝、內(nèi)質(zhì)網(wǎng)應(yīng)激等信號(hào)通路。Western bolt結(jié)果顯示:與核黃素適宜組相比,核黃素缺乏組的NDUFS1、NDUFV2、SDHA、ERO1A表達(dá)顯著上調(diào),SQSTM1表達(dá)顯著下調(diào)。驗(yàn)證結(jié)果與蛋白質(zhì)組學(xué)結(jié)果基本一致。4.與核黃素適宜組相比,核黃素缺乏組ApoB100細(xì)胞內(nèi)含量及細(xì)胞外分泌量均顯著下降(P0.05),二硫鍵異構(gòu)酶(PDI)蛋白表達(dá)量顯著降低、內(nèi)質(zhì)網(wǎng)應(yīng)激標(biāo)志性蛋白(GRP78,GADD153)表達(dá)量顯著升高(P0.05),細(xì)胞內(nèi)總蛋白結(jié)合巰基含量顯著下降(P0.05)。結(jié)論核黃素缺乏顯著影響人肝癌HepG2細(xì)胞的正常狀態(tài),維持該細(xì)胞狀態(tài)的培養(yǎng)液中核黃素濃度應(yīng)高于12.76nmol/L。核黃素缺乏顯著影響了HepG2細(xì)胞蛋白質(zhì)表達(dá)譜,受影響的相關(guān)蛋白可使能量代謝和脂質(zhì)代謝下調(diào),且可促進(jìn)內(nèi)質(zhì)網(wǎng)應(yīng)激和凋亡的發(fā)生等,進(jìn)一步針對(duì)ApoB100的通路研究發(fā)現(xiàn)核黃素缺乏可導(dǎo)致ApoB100二硫鍵形成障礙從而影響脂質(zhì)轉(zhuǎn)運(yùn)。此研究為深入探索核黃素乏對(duì)人體影響的分子機(jī)制提供線索和實(shí)驗(yàn)基礎(chǔ)。
[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.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R151
本文編號(hào)：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.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R151
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