鄰苯二甲酸二（2-乙基）己酯加重油酸誘導(dǎo)的HepG2細(xì)胞內(nèi)脂滴的蓄積的相關(guān)研究
發(fā)布時(shí)間：2018-09-09 10:24
【摘要】：目的(1)采用油酸刺激Hep G2細(xì)胞建立肝細(xì)胞脂肪蓄積模型,模擬非酒精性脂肪肝(NAFLD)肝臟脂肪蓄積狀態(tài),觀察鄰苯二甲酸二(2-乙基)己酯(DEHP)對(duì)細(xì)胞模型的影響,并對(duì)其部分機(jī)制進(jìn)行探討。(2)利用表面增強(qiáng)拉曼散射(SERS)技術(shù)建立快速、便捷、靈敏度高的檢測(cè)DEHP的方法。方法用0.5 m M油酸刺激Hep G2細(xì)胞建立肝細(xì)胞脂肪蓄積模型,油紅O染色和脂肪分化相關(guān)蛋白(ADRP)免疫組化對(duì)細(xì)胞模型進(jìn)行評(píng)價(jià);試劑盒檢測(cè)細(xì)胞超氧化物歧化酶(SOD)活性和丙二醛(MDA)以及甘油三酯(TG)的含量;Western blot檢測(cè)細(xì)胞內(nèi)過(guò)氧化物酶體增殖劑物活受體-γ(PPAR-γ)蛋白表達(dá)。運(yùn)用微波輔助法合成尺寸均一的納米銀顆粒,采用旋涂法制備銀基底膜,表面增強(qiáng)拉曼光譜檢測(cè)水溶液中極低濃度的DEHP。結(jié)果1.DEHP對(duì)油酸刺激Hep G2細(xì)胞建立的肝細(xì)胞脂肪蓄積模型的影響1.1細(xì)胞模型的建立油紅O染色結(jié)果顯示:對(duì)照組細(xì)胞邊緣清晰,細(xì)胞內(nèi)少見(jiàn)著色顆粒;模型組細(xì)胞內(nèi)可見(jiàn)大量染色脂滴,脂滴大多圍繞細(xì)胞膜內(nèi)側(cè)區(qū)域,使整個(gè)細(xì)胞呈“印戒”狀。以上結(jié)果說(shuō)明本研究成功建立了肝細(xì)胞脂肪蓄積模型。1.2油酸刺激Hep G2細(xì)胞對(duì)細(xì)胞內(nèi)ADRP蛋白表達(dá)的影響ADRP的免疫組化結(jié)果顯示,模型組與正常組細(xì)胞相比,ADRP的表達(dá)顯著增加,說(shuō)明油酸刺激Hep G2細(xì)胞顯著增加了細(xì)胞內(nèi)ADRP的蛋白表達(dá),從而佐證了模型的成功建立。1.3 DEHP對(duì)模型細(xì)胞內(nèi)脂滴的影響油紅O染色結(jié)果顯示:與模型組相比,DEHP(5 m M)處理細(xì)胞后細(xì)胞內(nèi)脂滴明顯增加;TG含量檢測(cè)顯示:模型組細(xì)胞內(nèi)TG含量較正常組顯著增加,DEHP處理后細(xì)胞內(nèi)TG的含量較模型組進(jìn)一步增加,結(jié)果有統(tǒng)計(jì)學(xué)差異。1.4 DEHP對(duì)模型細(xì)胞內(nèi)SOD活性和MDA水平的影響與正常對(duì)照組相比,模型組細(xì)胞內(nèi)SOD酶活性顯著降低,結(jié)果有統(tǒng)計(jì)學(xué)差異;與模型組相比,DEHP組(DEHP:5 m M)細(xì)胞內(nèi)的SOD酶活性顯著降低,結(jié)果有統(tǒng)計(jì)學(xué)差異。由結(jié)果可知,DEHP通過(guò)降低油酸誘導(dǎo)的Hep G2細(xì)胞內(nèi)SOD酶活性,降低了細(xì)胞的抗氧化性。模型組Hep G2細(xì)胞內(nèi)MDA水平顯著高于正常對(duì)照組,結(jié)果有統(tǒng)計(jì)學(xué)差異;與模型組相比,DEHP組(DEHP:5 m M)細(xì)胞內(nèi)的MDA水平顯著增加,結(jié)果有統(tǒng)計(jì)學(xué)差異。由結(jié)果可知,DEHP加重了Hep G2細(xì)胞內(nèi)的脂質(zhì)過(guò)氧化水平。1.5 DEHP對(duì)模型細(xì)胞內(nèi)PPAR-γ蛋白表達(dá)的影響與正常對(duì)照組相比,油酸誘導(dǎo)的Hep G2細(xì)胞內(nèi)PPAR-γ表達(dá)水平顯著增加,結(jié)果有統(tǒng)計(jì)學(xué)差異;與模型組相比,DEHP組(DEHP:5 m M)細(xì)胞內(nèi)的PPAR-γ表達(dá)水平明顯增加,結(jié)果有統(tǒng)計(jì)學(xué)差異。2.表面增強(qiáng)拉曼散射法檢測(cè)DEHP方法的建立2.1納米銀顆粒的表征通過(guò)透射電子顯微鏡觀察檸檬酸鈉水熱還原法,微波法以及改進(jìn)的微波氨基酸還原法三種方法制備出來(lái)的納米銀顆粒,結(jié)果顯示前兩種方法制備的銀顆粒形貌上均做不到粒徑尺寸均一,而改進(jìn)后的微波氨基酸還原法得到的銀顆粒不僅形貌好,粒徑均勻,而且產(chǎn)量可觀,能夠滿足后續(xù)實(shí)驗(yàn)需要。2.2 DEHP的SERS結(jié)果DEHP在1729、1600、1578、1439、1393、1293、1161、1121、1080、1038、956、891、842、648、396 cm-1處有明顯的拉曼特征峰。DEHP銀基底膜的表面增強(qiáng)光譜與其拉曼光譜在主要波數(shù)位置基本相同。微波輔助制備的納米銀顆組裝的基底膜對(duì)DEHP的拉曼光譜有顯著的增強(qiáng)效果,對(duì)塑化劑含量的檢測(cè)能夠達(dá)到10-9mol/L。結(jié)論1.DEHP可加重油酸刺激Hep G2細(xì)胞建立肝細(xì)胞脂肪蓄積模型細(xì)胞內(nèi)脂質(zhì)的蓄積,其機(jī)制可能與DEHP加重氧化應(yīng)激和脂質(zhì)過(guò)氧化有關(guān)和增加PPAR-γ蛋白表達(dá)有關(guān)。2.微波輔助旋涂法制備的納米銀基底膜能檢測(cè)低濃度的DEHP,檢測(cè)限能夠達(dá)到10-9 mol/l。該方法靈敏度高,檢測(cè)速度快,具有廣闊的市場(chǎng)應(yīng)用前景。
[Abstract]:Objective (1) To establish a hepatocyte adipose accumulation model by oleic acid stimulating Hep G2 cells, and to observe the effect of di (2-ethyl) hexyl phthalate (DEHP) on hepatocyte adipose accumulation in NAFLD, and to explore its mechanism. (2) To establish a rapid and convenient method by surface enhanced Raman scattering (SERS). Methods Hep G2 cells were stimulated by 0.5 m oleic acid to establish a hepatocyte adipose accumulation model. The cell model was evaluated by oil red O staining and adipose differentiation associated protein (ADRP) immunohistochemistry. The activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) and triglyceride (TG) were detected by kit. Western blot was used to detect the expression of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) protein in Hep G2 cells.Silver nanoparticles with uniform size were synthesized by microwave-assisted method.Silver basement membrane was prepared by spin-coating method.DEHP in very low concentration in aqueous solution was detected by surface-enhanced Raman spectroscopy.Results 1.DEHP stimulated Hep G2 cells by oleic acid. Effect of hepatocyte fat accumulation model 1.1 Cell model Establishment Oil red O staining results showed that: the control group cells edge clear, few staining particles; model group cells can see a large number of staining lipid droplets, lipid droplets mostly around the inner region of the cell membrane, so that the whole cell was "ring" shape. The adipose accumulation model of hepatocytes was established. 3. Effect of DEHP on intracellular lipid droplets in model cells by oil red O staining showed that compared with the model group, intracellular lipid droplets were significantly increased after DEHP (5m M) treatment; TG content detection showed that intracellular TG content in model group was significantly increased compared with the normal group, and the intracellular TG content was further increased after DEHP treatment compared with the model group, the results were statistically different. 4. The effect of DEHP on SOD activity and M DA level in the model cells was significantly lower than that in the normal control group, and the results were statistically different. Compared with the model group, the activity of SOD in the DEHP group (DEHP: 5m M) was significantly lower, the results were statistically different. The level of MDA in Hep G2 cells of model group was significantly higher than that of normal control group, and the level of MDA in DEHP group (DEHP:5m M) was significantly higher than that of model group, the results showed that DEHP aggravated lipid peroxidation in Hep G2 cells. Level 1.5 DEHP on the expression of PPAR-gamma protein in model cells compared with the normal control group, oleic acid-induced PPAR-gamma expression in Hep G2 cells significantly increased, the results were statistically significant; compared with the model group, DEHP: 5m M cell PPAR-gamma expression level increased significantly, the results were statistically significant. 2. Characterization of silver nanoparticles prepared by sodium citrate hydrothermal reduction method, microwave method and improved microwave amino acid reduction method were observed by transmission electron microscopy. The results showed that silver nanoparticles prepared by the two methods could not be uniform in size and morphology. 1. The silver particles obtained by the improved microwave amino acid reduction method not only have good morphology, uniform particle size, but also can meet the needs of subsequent experiments. 2.2 DEHP SERS results showed that DEHP had obvious Raman characteristic peaks at 1729, 1600, 1578, 1439, 1393, 1293, 1161, 1080, 1038, 956, 891, 842, 648, 396 cm-1. The Raman spectra of DEHP were almost the same as those of its Raman spectra at the main wavenumber position. The results showed that the nano-silver-coated substrate prepared by microwave-assisted method could significantly enhance the Raman spectra of DEHP, and the content of plasticizer could reach 10-9 mol/L. Conclusion 1. DEHP could aggravate the stimulation of oleic acid on Hep G2 cells to establish hepatocyte fat accumulation model cells. The mechanism of lipid accumulation may be related to DEHP aggravating oxidative stress and lipid peroxidation and increasing PPAR-gamma protein expression. 2. Silver nanoparticles basement membrane prepared by microwave-assisted spin-coating method can detect low concentration of DEHP, and the detection limit can reach 10-9 mol/l. This method has high sensitivity, fast detection speed, and broad market prospects.
【學(xué)位授予單位】：安徽中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R96
本文編號(hào)：2232114
[Abstract]:Objective (1) To establish a hepatocyte adipose accumulation model by oleic acid stimulating Hep G2 cells, and to observe the effect of di (2-ethyl) hexyl phthalate (DEHP) on hepatocyte adipose accumulation in NAFLD, and to explore its mechanism. (2) To establish a rapid and convenient method by surface enhanced Raman scattering (SERS). Methods Hep G2 cells were stimulated by 0.5 m oleic acid to establish a hepatocyte adipose accumulation model. The cell model was evaluated by oil red O staining and adipose differentiation associated protein (ADRP) immunohistochemistry. The activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) and triglyceride (TG) were detected by kit. Western blot was used to detect the expression of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) protein in Hep G2 cells.Silver nanoparticles with uniform size were synthesized by microwave-assisted method.Silver basement membrane was prepared by spin-coating method.DEHP in very low concentration in aqueous solution was detected by surface-enhanced Raman spectroscopy.Results 1.DEHP stimulated Hep G2 cells by oleic acid. Effect of hepatocyte fat accumulation model 1.1 Cell model Establishment Oil red O staining results showed that: the control group cells edge clear, few staining particles; model group cells can see a large number of staining lipid droplets, lipid droplets mostly around the inner region of the cell membrane, so that the whole cell was "ring" shape. The adipose accumulation model of hepatocytes was established. 3. Effect of DEHP on intracellular lipid droplets in model cells by oil red O staining showed that compared with the model group, intracellular lipid droplets were significantly increased after DEHP (5m M) treatment; TG content detection showed that intracellular TG content in model group was significantly increased compared with the normal group, and the intracellular TG content was further increased after DEHP treatment compared with the model group, the results were statistically different. 4. The effect of DEHP on SOD activity and M DA level in the model cells was significantly lower than that in the normal control group, and the results were statistically different. Compared with the model group, the activity of SOD in the DEHP group (DEHP: 5m M) was significantly lower, the results were statistically different. The level of MDA in Hep G2 cells of model group was significantly higher than that of normal control group, and the level of MDA in DEHP group (DEHP:5m M) was significantly higher than that of model group, the results showed that DEHP aggravated lipid peroxidation in Hep G2 cells. Level 1.5 DEHP on the expression of PPAR-gamma protein in model cells compared with the normal control group, oleic acid-induced PPAR-gamma expression in Hep G2 cells significantly increased, the results were statistically significant; compared with the model group, DEHP: 5m M cell PPAR-gamma expression level increased significantly, the results were statistically significant. 2. Characterization of silver nanoparticles prepared by sodium citrate hydrothermal reduction method, microwave method and improved microwave amino acid reduction method were observed by transmission electron microscopy. The results showed that silver nanoparticles prepared by the two methods could not be uniform in size and morphology. 1. The silver particles obtained by the improved microwave amino acid reduction method not only have good morphology, uniform particle size, but also can meet the needs of subsequent experiments. 2.2 DEHP SERS results showed that DEHP had obvious Raman characteristic peaks at 1729, 1600, 1578, 1439, 1393, 1293, 1161, 1080, 1038, 956, 891, 842, 648, 396 cm-1. The Raman spectra of DEHP were almost the same as those of its Raman spectra at the main wavenumber position. The results showed that the nano-silver-coated substrate prepared by microwave-assisted method could significantly enhance the Raman spectra of DEHP, and the content of plasticizer could reach 10-9 mol/L. Conclusion 1. DEHP could aggravate the stimulation of oleic acid on Hep G2 cells to establish hepatocyte fat accumulation model cells. The mechanism of lipid accumulation may be related to DEHP aggravating oxidative stress and lipid peroxidation and increasing PPAR-gamma protein expression. 2. Silver nanoparticles basement membrane prepared by microwave-assisted spin-coating method can detect low concentration of DEHP, and the detection limit can reach 10-9 mol/l. This method has high sensitivity, fast detection speed, and broad market prospects.
【學(xué)位授予單位】：安徽中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R96
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