亚硫酸钠对HL-7702肝细胞极低密度脂蛋白组装途径的影响
发布时间：2019-06-10 18:52
【摘要】：目的： 动物实验显示亚硫酸钠接触可引起肝细胞内脂肪沉积，为探究其原因，观察了食品添加剂亚硫酸钠(Na_2SO_3)对人肝HL-7702细胞甘油三酯(TG)含量和与肝细胞极低密度脂蛋白(VLDL)组装密切相关的因子在转录、翻译水平的影响，以期找到亚硫酸盐影响肝细胞TG或VLDL分泌的关键分子和环节，确定亚硫酸盐是否通过影响极低密度脂蛋白的包装过程从而影响其最终的分泌外排。 方法： 以人正常二倍体肝细胞株HL-7702(简称L-02)为研究对象。L-02细胞以含10%小牛血清(FBS)的高糖DMEM培养基培养，经不同浓度Na_2SO_3及阳性对照1mM油酸作用24h、48h后，采用CCK-8试剂盒检测细胞活性；油红O染色法观察肝细胞脂肪变性；甘油三酯甘油磷酸氧化酶法(GPO-POD酶法)测定试剂盒检测细胞内TG含量；Western Blotting检测细胞外培养上清中VLDL的分泌；荧光定量PCR(Q-PCR)法检测肝细胞内微粒体甘油三酯转移蛋白(MTP)、二脂酰甘油酰基转移酶2(DGAT2)、甘油三酯水解酶(TGH)基因的mRNA表达。 结果： 1、不同浓度Na_2SO_3对L-02细胞活性的影响：当接触染毒24h、48h后，Na_2SO_3呈剂量依赖性的降低细胞存活率(r24h=-0.941,p0.01; r48h=-0.915, p0.01)，其中10mM、2.5mM、0.625mM、0.156mM Na_2SO_3作用组细胞活性与正常对照组相比，差异具有统计学意义(P0.05)。说明Na_2SO_3可直接引起肝细胞活性的降低。 2、不同浓度Na_2SO_3对L-02细胞内脂肪沉积的影响：经油红O染色观察发现，阴性对照组细胞间结合紧密，呈多边形，单层排列，无重叠生长，细胞界限清晰，贴壁良好。随着Na_2SO_3染毒剂量的增加，可见细胞数量有所减少，细胞间隙增加。染毒24h、48h后，各Na_2SO_3染毒组均未见橘红色脂肪着染颗粒，只有阳性对照油酸组可见明显红色脂滴。说明Na_2SO_3在本实验条件下未能引起胞内明显脂肪沉积。 3、不同浓度Na_2SO_3对L-02细胞内TG含量的影响：接触染毒24h和48h后，10mM Na_2SO_3组细胞内TG含量增加，，与阴性对照组比较有显著性差异(P0.05)，阳性对照1mM油酸组与阴性对照组比较，细胞内TG水平明显增高(P0.05)。说明一定剂量Na_2SO_3可引起肝细胞TG含量增加。 4、不同浓度Na_2SO_3对L-02细胞VLDL分泌的影响：染毒24h后，10mM Na_2SO_3处理组培养上清中VLDL水平与阴性对照组相比明显增加，经检验，差异有统计学意义(P0.05)；染毒48h后，各处理组培养上清中VLDL水平与阴性对照组相比无明显变化，经检验，差异无统计学意义(P0.05)。 5、不同浓度Na_2SO_3对L-02细胞MTP、DGAT2、TGHmRNA表达的影响：接触染毒24h、48h，各处理组细胞MTP、DGAT2mRNA的表达量与阴性对照组相比均无明显变化，差异无统计学意义(P0.05)；接触染毒24h，10mM Na_2SO_3、0.5mM Na_2SO_3处理组肝细胞内TGH mRNA表达升高，与阴性对照组相比差异有统计学意义(P0.05)。接触染毒48h，10mM Na_2SO_3组及阳性对照1mM油酸组肝细胞内TGH mRNA表达明显降低，与阴性对照组相比差异有统计学意义(P0.05)。而2.5mM Na_2SO_3、0.1mM Na_2SO_3处理组肝细胞内TGH mRNA表达升高，与阴性对照组相比差异有统计学意义(P0.05)。 结论： 1、Na_2SO_3对L-02肝细胞活性的抑制作用呈剂量依赖性，说明Na_2SO_3具有肝细胞毒性作用。 2、高浓度Na_2SO_3可增加肝细胞内TG含量，引起肝细胞脂肪代谢紊乱。 3、一定条件下Na_2SO_3可使L-02肝细胞内TG累积进而促进VLDL分泌。 4、Na_2SO_3染毒24h引起VLDL分泌增多可能与TGH基因mRNA表达上调有一定关联；染毒48h引起L-02肝细胞内TG含量增多可能与下调TGH基因mRNA表达有关。
[Abstract]:Purpose: The animal experiment shows that the contact of sodium sulfite can cause the fat deposition in the liver cells, and to explore its origin The effect of sodium sulfite (Na _ 2SO _ 3) on the content of triglyceride (TG) of human liver HL-7702 and its association with the assembly of low-density lipoprotein (VLDL) in human hepatocytes were observed. In response, it is expected to find the key molecules and links of sulfite to influence the secretion of TG or VLDL in the liver cells, to determine whether the sulfite influences its final secretion by affecting the packaging process of the very low-density lipoprotein. Row. Method: Human normal diploid liver cell line HL-7702 (L-02) The cells were cultured with a high-glucose DMEM medium containing 10% calf serum (FBS). After 24 h and 48 h of Na _ 2SO _ 3 and 1 mM oleic acid, the activity of cells was detected by CCK-8 kit. Cell fat degeneration; Triglyceride glycerophosphate oxidase method (GPO-POD enzyme method) assay kit for detecting the content of TG in cells; Western Blotting to detect the secretion of VLDL in the supernatant of the cells; and the fluorescence quantitative PCR (Q-PCR) method for the detection of microsomal triglyceride transfer protein (MTP), dilipids, and triglycerides 2 (D) in the liver cells. GAT2), Triglyceride Hydrolases (TGH) Genes m RNA The results were as follows:1. The effect of Na _ 2SO _ 3 on the activity of L-02 cells: After 24 h and 48 h exposure, Na _ 2SO _ 3 showed a dose-dependent decrease in cell survival rate (r24h =-0.941, p0.01; r48h =-0.915, p0.01), in which 10 mM, 2.5 mM, 0.625 mM, 0.156 mM Na _ 2SO _ 3 acted as a group of cells. The difference was statistically significant compared to the normal control group. Significance (P0.05). It is indicated that Na _ 2SO _ 3 can be directly cited. The effect of Na _ 2SO _ 3 on the lipid deposition in L-02 cells was observed. The results showed that the cells of the negative control group were closely combined, polygonal, single-layer and non-overlapping. The cell line is clear and the adhesion is good. With the increase of the dose of Na _ 2SO _ 3, the number of cells can be seen. There was a decrease in cell gap. After 24 h and 48 h of exposure, no orange-red fat-stained particles were found in all Na _ 2SO _ 3 groups, only positive control. The effect of Na _ 2SO _ 3 on the experimental conditions was not found in the oleic acid group. The effect of Na _ 2SO _ 3 on the content of TG in L-02 cells was induced by different concentrations of Na _ 2SO _ 3. The content of TG increased in 10 mM Na _ 2SO _ 3 group after 24 h and 48 h exposure, and there was a significant difference with the negative control group (P0.05). The positive control was compared with that of the negative control group. The level of Na _ 2SO _ 4 in a certain dose was significantly higher (P <0.05). 3. The content of TG increased.4. The effect of Na _ 2SO _ 3 on the secretion of VLDL in L-02 cells: after 24 h of exposure, the level of VLDL in the 10 mM Na _ 2SO _ 3 treatment group was significantly increased compared with that of the negative control group. (P0.05); after 48 h of exposure, the level of VLDL in the culture supernatant of each treatment group did not change significantly compared with that of the negative control group, and tested, The effect of Na _ 2SO _ 3 on the expression of MTP, DGAT2 and TGHmRNA in L-02 cells was not statistically significant (P0.05). Significance (P0.05); the expression of TGH mRNA in the hepatocytes of the treated group of 24 h,10 mM Na _ 2SO _ 3 and 0.5 mM Na _ 2SO _ 3 was increased and the expression of TGH mRNA in the liver of the treated group was higher than that of the negative control group. Compared with the control group, the expression of TGH mRNA in the hepatocyte of the 10 mM Na _ 2SO _ 3 group and the positive control 1mM oleic acid group was significantly lower than that of the negative control group. Compared with the negative control group, the expression of TGH mRNA in the liver of the treated group was higher than that of the negative control group. in contrast to poor Conclusion:1. The inhibitory effect of Na _ 2SO _ 3 on the activity of L-02 hepatocytes was dose-dependent. sex, indicating that Na _ 2SO _ 3 has hepatotoxicity.2. High concentration of Na _ 2SO _ 3 The content of TG in the liver cells can be increased, and the metabolic disorder of the liver cell is caused. Under certain conditions, Na _ 2SO _ 4 can be increased. 3. The accumulation of TG in L-02 hepatocytes could further promote the secretion of VLDL.4. The increase of VLDL induced by Na _ 2SO _ 3 was associated with the up-regulation of the expression of the mRNA of the TGH gene.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
[Abstract]:Purpose: The animal experiment shows that the contact of sodium sulfite can cause the fat deposition in the liver cells, and to explore its origin The effect of sodium sulfite (Na _ 2SO _ 3) on the content of triglyceride (TG) of human liver HL-7702 and its association with the assembly of low-density lipoprotein (VLDL) in human hepatocytes were observed. In response, it is expected to find the key molecules and links of sulfite to influence the secretion of TG or VLDL in the liver cells, to determine whether the sulfite influences its final secretion by affecting the packaging process of the very low-density lipoprotein. Row. Method: Human normal diploid liver cell line HL-7702 (L-02) The cells were cultured with a high-glucose DMEM medium containing 10% calf serum (FBS). After 24 h and 48 h of Na _ 2SO _ 3 and 1 mM oleic acid, the activity of cells was detected by CCK-8 kit. Cell fat degeneration; Triglyceride glycerophosphate oxidase method (GPO-POD enzyme method) assay kit for detecting the content of TG in cells; Western Blotting to detect the secretion of VLDL in the supernatant of the cells; and the fluorescence quantitative PCR (Q-PCR) method for the detection of microsomal triglyceride transfer protein (MTP), dilipids, and triglycerides 2 (D) in the liver cells. GAT2), Triglyceride Hydrolases (TGH) Genes m RNA The results were as follows:1. The effect of Na _ 2SO _ 3 on the activity of L-02 cells: After 24 h and 48 h exposure, Na _ 2SO _ 3 showed a dose-dependent decrease in cell survival rate (r24h =-0.941, p0.01; r48h =-0.915, p0.01), in which 10 mM, 2.5 mM, 0.625 mM, 0.156 mM Na _ 2SO _ 3 acted as a group of cells. The difference was statistically significant compared to the normal control group. Significance (P0.05). It is indicated that Na _ 2SO _ 3 can be directly cited. The effect of Na _ 2SO _ 3 on the lipid deposition in L-02 cells was observed. The results showed that the cells of the negative control group were closely combined, polygonal, single-layer and non-overlapping. The cell line is clear and the adhesion is good. With the increase of the dose of Na _ 2SO _ 3, the number of cells can be seen. There was a decrease in cell gap. After 24 h and 48 h of exposure, no orange-red fat-stained particles were found in all Na _ 2SO _ 3 groups, only positive control. The effect of Na _ 2SO _ 3 on the experimental conditions was not found in the oleic acid group. The effect of Na _ 2SO _ 3 on the content of TG in L-02 cells was induced by different concentrations of Na _ 2SO _ 3. The content of TG increased in 10 mM Na _ 2SO _ 3 group after 24 h and 48 h exposure, and there was a significant difference with the negative control group (P0.05). The positive control was compared with that of the negative control group. The level of Na _ 2SO _ 4 in a certain dose was significantly higher (P <0.05). 3. The content of TG increased.4. The effect of Na _ 2SO _ 3 on the secretion of VLDL in L-02 cells: after 24 h of exposure, the level of VLDL in the 10 mM Na _ 2SO _ 3 treatment group was significantly increased compared with that of the negative control group. (P0.05); after 48 h of exposure, the level of VLDL in the culture supernatant of each treatment group did not change significantly compared with that of the negative control group, and tested, The effect of Na _ 2SO _ 3 on the expression of MTP, DGAT2 and TGHmRNA in L-02 cells was not statistically significant (P0.05). Significance (P0.05); the expression of TGH mRNA in the hepatocytes of the treated group of 24 h,10 mM Na _ 2SO _ 3 and 0.5 mM Na _ 2SO _ 3 was increased and the expression of TGH mRNA in the liver of the treated group was higher than that of the negative control group. Compared with the control group, the expression of TGH mRNA in the hepatocyte of the 10 mM Na _ 2SO _ 3 group and the positive control 1mM oleic acid group was significantly lower than that of the negative control group. Compared with the negative control group, the expression of TGH mRNA in the liver of the treated group was higher than that of the negative control group. in contrast to poor Conclusion:1. The inhibitory effect of Na _ 2SO _ 3 on the activity of L-02 hepatocytes was dose-dependent. sex, indicating that Na _ 2SO _ 3 has hepatotoxicity.2. High concentration of Na _ 2SO _ 3 The content of TG in the liver cells can be increased, and the metabolic disorder of the liver cell is caused. Under certain conditions, Na _ 2SO _ 4 can be increased. 3. The accumulation of TG in L-02 hepatocytes could further promote the secretion of VLDL.4. The increase of VLDL induced by Na _ 2SO _ 3 was associated with the up-regulation of the expression of the mRNA of the TGH gene.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
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