【摘要】：目的： 動物實(shí)驗(yàn)顯示亞硫酸鈉接觸可引起肝細(xì)胞內(nèi)脂肪沉積，為探究其原因，觀察了食品添加劑亞硫酸鈉(Na_2SO_3)對人肝HL-7702細(xì)胞甘油三酯(TG)含量和與肝細(xì)胞極低密度脂蛋白(VLDL)組裝密切相關(guān)的因子在轉(zhuǎn)錄、翻譯水平的影響，以期找到亞硫酸鹽影響肝細(xì)胞TG或VLDL分泌的關(guān)鍵分子和環(huán)節(jié)，確定亞硫酸鹽是否通過影響極低密度脂蛋白的包裝過程從而影響其最終的分泌外排。 方法： 以人正常二倍體肝細(xì)胞株HL-7702(簡稱L-02)為研究對象。L-02細(xì)胞以含10%小牛血清(FBS)的高糖DMEM培養(yǎng)基培養(yǎng)，經(jīng)不同濃度Na_2SO_3及陽性對照1mM油酸作用24h、48h后，采用CCK-8試劑盒檢測細(xì)胞活性；油紅O染色法觀察肝細(xì)胞脂肪變性；甘油三酯甘油磷酸氧化酶法(GPO-POD酶法)測定試劑盒檢測細(xì)胞內(nèi)TG含量；Western Blotting檢測細(xì)胞外培養(yǎng)上清中VLDL的分泌；熒光定量PCR(Q-PCR)法檢測肝細(xì)胞內(nèi)微粒體甘油三酯轉(zhuǎn)移蛋白(MTP)、二脂酰甘油酰基轉(zhuǎn)移酶2(DGAT2)、甘油三酯水解酶(TGH)基因的mRNA表達(dá)。 結(jié)果： 1、不同濃度Na_2SO_3對L-02細(xì)胞活性的影響：當(dāng)接觸染毒24h、48h后，Na_2SO_3呈劑量依賴性的降低細(xì)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作用組細(xì)胞活性與正常對照組相比，差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。說明Na_2SO_3可直接引起肝細(xì)胞活性的降低。 2、不同濃度Na_2SO_3對L-02細(xì)胞內(nèi)脂肪沉積的影響：經(jīng)油紅O染色觀察發(fā)現(xiàn)，陰性對照組細(xì)胞間結(jié)合緊密，呈多邊形，單層排列，無重疊生長，細(xì)胞界限清晰，貼壁良好。隨著Na_2SO_3染毒劑量的增加，可見細(xì)胞數(shù)量有所減少，細(xì)胞間隙增加。染毒24h、48h后，各Na_2SO_3染毒組均未見橘紅色脂肪著染顆粒，只有陽性對照油酸組可見明顯紅色脂滴。說明Na_2SO_3在本實(shí)驗(yàn)條件下未能引起胞內(nèi)明顯脂肪沉積。 3、不同濃度Na_2SO_3對L-02細(xì)胞內(nèi)TG含量的影響：接觸染毒24h和48h后，10mM Na_2SO_3組細(xì)胞內(nèi)TG含量增加，，與陰性對照組比較有顯著性差異(P0.05)，陽性對照1mM油酸組與陰性對照組比較，細(xì)胞內(nèi)TG水平明顯增高(P0.05)。說明一定劑量Na_2SO_3可引起肝細(xì)胞TG含量增加。 4、不同濃度Na_2SO_3對L-02細(xì)胞VLDL分泌的影響：染毒24h后，10mM Na_2SO_3處理組培養(yǎng)上清中VLDL水平與陰性對照組相比明顯增加，經(jīng)檢驗(yàn)，差異有統(tǒng)計(jì)學(xué)意義(P0.05)；染毒48h后，各處理組培養(yǎng)上清中VLDL水平與陰性對照組相比無明顯變化，經(jīng)檢驗(yàn)，差異無統(tǒng)計(jì)學(xué)意義(P0.05)。 5、不同濃度Na_2SO_3對L-02細(xì)胞MTP、DGAT2、TGHmRNA表達(dá)的影響：接觸染毒24h、48h，各處理組細(xì)胞MTP、DGAT2mRNA的表達(dá)量與陰性對照組相比均無明顯變化，差異無統(tǒng)計(jì)學(xué)意義(P0.05)；接觸染毒24h，10mM Na_2SO_3、0.5mM Na_2SO_3處理組肝細(xì)胞內(nèi)TGH mRNA表達(dá)升高，與陰性對照組相比差異有統(tǒng)計(jì)學(xué)意義(P0.05)。接觸染毒48h，10mM Na_2SO_3組及陽性對照1mM油酸組肝細(xì)胞內(nèi)TGH mRNA表達(dá)明顯降低，與陰性對照組相比差異有統(tǒng)計(jì)學(xué)意義(P0.05)。而2.5mM Na_2SO_3、0.1mM Na_2SO_3處理組肝細(xì)胞內(nèi)TGH mRNA表達(dá)升高，與陰性對照組相比差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論： 1、Na_2SO_3對L-02肝細(xì)胞活性的抑制作用呈劑量依賴性，說明Na_2SO_3具有肝細(xì)胞毒性作用。 2、高濃度Na_2SO_3可增加肝細(xì)胞內(nèi)TG含量，引起肝細(xì)胞脂肪代謝紊亂。 3、一定條件下Na_2SO_3可使L-02肝細(xì)胞內(nèi)TG累積進(jìn)而促進(jìn)VLDL分泌。 4、Na_2SO_3染毒24h引起VLDL分泌增多可能與TGH基因mRNA表達(dá)上調(diào)有一定關(guān)聯(lián)；染毒48h引起L-02肝細(xì)胞內(nèi)TG含量增多可能與下調(diào)TGH基因mRNA表達(dá)有關(guān)。
[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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329

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