邻苯二甲酸二（2-乙基）己酯加重油酸诱导的HepG2细胞内脂滴的蓄积的相关研究
发布时间：2018-09-09 10:24
【摘要】：目的(1)采用油酸刺激Hep G2细胞建立肝细胞脂肪蓄积模型,模拟非酒精性脂肪肝(NAFLD)肝脏脂肪蓄积状态,观察邻苯二甲酸二(2-乙基)己酯(DEHP)对细胞模型的影响,并对其部分机制进行探讨。(2)利用表面增强拉曼散射(SERS)技术建立快速、便捷、灵敏度高的检测DEHP的方法。方法用0.5 m M油酸刺激Hep G2细胞建立肝细胞脂肪蓄积模型,油红O染色和脂肪分化相关蛋白(ADRP)免疫组化对细胞模型进行评价;试剂盒检测细胞超氧化物歧化酶(SOD)活性和丙二醛(MDA)以及甘油三酯(TG)的含量;Western blot检测细胞内过氧化物酶体增殖剂物活受体-γ(PPAR-γ)蛋白表达。运用微波辅助法合成尺寸均一的纳米银颗粒,采用旋涂法制备银基底膜,表面增强拉曼光谱检测水溶液中极低浓度的DEHP。结果1.DEHP对油酸刺激Hep G2细胞建立的肝细胞脂肪蓄积模型的影响1.1细胞模型的建立油红O染色结果显示:对照组细胞边缘清晰,细胞内少见着色颗粒;模型组细胞内可见大量染色脂滴,脂滴大多围绕细胞膜内侧区域,使整个细胞呈“印戒”状。以上结果说明本研究成功建立了肝细胞脂肪蓄积模型。1.2油酸刺激Hep G2细胞对细胞内ADRP蛋白表达的影响ADRP的免疫组化结果显示,模型组与正常组细胞相比,ADRP的表达显著增加,说明油酸刺激Hep G2细胞显著增加了细胞内ADRP的蛋白表达,从而佐证了模型的成功建立。1.3 DEHP对模型细胞内脂滴的影响油红O染色结果显示:与模型组相比,DEHP(5 m M)处理细胞后细胞内脂滴明显增加;TG含量检测显示:模型组细胞内TG含量较正常组显著增加,DEHP处理后细胞内TG的含量较模型组进一步增加,结果有统计学差异。1.4 DEHP对模型细胞内SOD活性和MDA水平的影响与正常对照组相比,模型组细胞内SOD酶活性显著降低,结果有统计学差异;与模型组相比,DEHP组(DEHP:5 m M)细胞内的SOD酶活性显著降低,结果有统计学差异。由结果可知,DEHP通过降低油酸诱导的Hep G2细胞内SOD酶活性,降低了细胞的抗氧化性。模型组Hep G2细胞内MDA水平显著高于正常对照组,结果有统计学差异;与模型组相比,DEHP组(DEHP:5 m M)细胞内的MDA水平显著增加,结果有统计学差异。由结果可知,DEHP加重了Hep G2细胞内的脂质过氧化水平。1.5 DEHP对模型细胞内PPAR-γ蛋白表达的影响与正常对照组相比,油酸诱导的Hep G2细胞内PPAR-γ表达水平显著增加,结果有统计学差异;与模型组相比,DEHP组(DEHP:5 m M)细胞内的PPAR-γ表达水平明显增加,结果有统计学差异。2.表面增强拉曼散射法检测DEHP方法的建立2.1纳米银颗粒的表征通过透射电子显微镜观察柠檬酸钠水热还原法,微波法以及改进的微波氨基酸还原法三种方法制备出来的纳米银颗粒,结果显示前两种方法制备的银颗粒形貌上均做不到粒径尺寸均一,而改进后的微波氨基酸还原法得到的银颗粒不仅形貌好,粒径均匀,而且产量可观,能够满足后续实验需要。2.2 DEHP的SERS结果DEHP在1729、1600、1578、1439、1393、1293、1161、1121、1080、1038、956、891、842、648、396 cm-1处有明显的拉曼特征峰。DEHP银基底膜的表面增强光谱与其拉曼光谱在主要波数位置基本相同。微波辅助制备的纳米银颗组装的基底膜对DEHP的拉曼光谱有显著的增强效果,对塑化剂含量的检测能够达到10-9mol/L。结论1.DEHP可加重油酸刺激Hep G2细胞建立肝细胞脂肪蓄积模型细胞内脂质的蓄积,其机制可能与DEHP加重氧化应激和脂质过氧化有关和增加PPAR-γ蛋白表达有关。2.微波辅助旋涂法制备的纳米银基底膜能检测低浓度的DEHP,检测限能够达到10-9 mol/l。该方法灵敏度高,检测速度快,具有广阔的市场应用前景。
[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.
【学位授予单位】：安徽中医药大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R96
本文编号：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.
【学位授予单位】：安徽中医药大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R96
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