本文選題：環(huán)氧合酶-2 + 非酒精性脂肪性肝病��； 參考：《南華大學(xué)》2014年碩士論文

【摘要】：目的：研究COX-2對(duì)脂變肝細(xì)胞BRL-3A株甘油三酯代謝及Acsl1、Acsl6表達(dá)的影響。 方法： (1)篩選最佳誘導(dǎo)濃度，誘導(dǎo)細(xì)胞脂肪變性：體外培養(yǎng)大鼠肝細(xì)胞BRL-3A株，不同濃度醫(yī)用脂肪乳誘導(dǎo)大鼠肝細(xì)胞成脂肪變肝細(xì)胞，油紅O染色及蘇木素復(fù)染方法觀察各組細(xì)胞脂肪變狀態(tài)，MTT檢測(cè)細(xì)胞增殖活性及ELISA檢測(cè)誘導(dǎo)前后細(xì)胞內(nèi)TG含量確定最佳誘導(dǎo)濃度。 (2)實(shí)驗(yàn)分為正常BRL-3A組、脂變BRL-3A組，pYr-1.1-hU6-EGFP-COX-2shRNA組，pYr-1.1-hU6-EGFP-HK組，pcDNA3.1（+）-r-COX-2組，pcDNA3.1（+）-r-HK組，尼美舒利（200ug/ml）陽(yáng)性對(duì)照組，除正常BRL-3A組外，其余各組均以最佳誘導(dǎo)濃度6%脂肪乳培養(yǎng)24h后，瞬時(shí)轉(zhuǎn)染各組對(duì)應(yīng)的質(zhì)粒，ELISA檢測(cè)轉(zhuǎn)染后各組細(xì)胞內(nèi)TG含量，RT-PCR檢測(cè)各組COX-2mRNA、Acsl1mRNA、Acsl6mRNA的表達(dá)。 結(jié)果：（1）醫(yī)用脂肪乳培養(yǎng)細(xì)胞24h誘導(dǎo)細(xì)胞脂變，油紅染色示肝細(xì)胞內(nèi)空泡脂滴增加，，提示細(xì)胞脂肪變。ELISA結(jié)果表明細(xì)胞內(nèi)TG含量增加。結(jié)合油紅染色、MTT檢測(cè)的OD值及ELISA結(jié)果確定本實(shí)驗(yàn)最佳誘導(dǎo)濃度為6%醫(yī)用脂肪乳。 （2）pYr-1.1-hU6-EGFP-COX-2shRNA轉(zhuǎn)染至脂變BRL-3A細(xì)胞培養(yǎng)24h后熒光顯微鏡下觀察，細(xì)胞質(zhì)中可見(jiàn)綠色熒光，轉(zhuǎn)染效率達(dá)70%以上。 （3）ELISA檢測(cè)轉(zhuǎn)染后各組細(xì)胞內(nèi)TG含量，pYr-1.1-hU6-EGFP-COX-2shRNA組與尼美舒利組TG含量均減少(p0.05)，以pYr-1.1-hU6-EGFP-COX-2shRNA組減少明顯，均低于其余6組(p0.01)，而pcDNA3.1（+）-r-COX-2組高于其余6組(p0.05)。尼美舒利組低于pcDNA3.1（+）-r-COX-2組但高于pYr-1.1-hU6-EGFP-COX-2shRNA組，pYr-1.1-hU6-EGFP-HK組和pcDNA3.1（+）-r-HK組無(wú)明顯變化，無(wú)統(tǒng)計(jì)學(xué)意義（p0.05）。 （4）7個(gè)組別COX-2、Acsl1、Acsl6基因表達(dá)情況：COX-2mRNA表達(dá)情況：與正常肝細(xì)胞組相比，脂變肝細(xì)胞組中COX-2mRNA表達(dá)增高(p0.05)。與脂變肝細(xì)胞組相比， pYr-1.1-hU6-EGFP-COX-2shRNA組及尼美舒利組中COX-2mRNA表達(dá)降低(p0.05)，以pYr-1.1-hU6-EGFP-COX-2shRNA組降低明顯(p0.01)，而pcDNA3.1（+）-r-COX-2組中COX-2mRNA表達(dá)增高(p0.05)。pYr-1.1-hU6-EGFP-HK組和pcDNA3.1（+）-r-HK組COX-2mRNA無(wú)明顯增高，無(wú)統(tǒng)計(jì)學(xué)意義（p0.05）。脂代謝基因Acsl1mRNA、Acsl6mRNA表達(dá)情況：與正常肝細(xì)胞組相比，脂變肝細(xì)胞中Acsl1mRNA、Acsl6mRNA表達(dá)增高(p0.05)。與脂變肝細(xì)胞組相比，pYr-1.1-hU6-EGFP-COX-2shRNA及尼美舒利組中Acsl1mRNA、Acsl6mRNA表達(dá)均降低(p0.05)。以pYr-1.1-hU6-EGFP-COX-2shRNA組下調(diào)明顯(p0.01)，pcDNA3.1（+）-r-COX-2組中Acsl1、Acsl6表達(dá)則明顯增高(p0.05)。而pYr-1.1-hU6-EGFP-HK組和pcDNA3.1（+）-r-HK組中Acsl1mRNA、Acsl6mRNA無(wú)明顯改變，無(wú)統(tǒng)計(jì)學(xué)意義(p＞0.05)。 結(jié)論： （1）沉默COX-2表達(dá)，肝細(xì)胞脂肪變性減輕；脂變肝細(xì)胞內(nèi)TG含量降低；同時(shí)，脂代謝基因Acsl1、Acsl6表達(dá)減少。 （2）COX-2過(guò)表達(dá)，肝細(xì)胞脂肪變性加重；脂變肝細(xì)胞內(nèi)TG含量增加；同時(shí)，脂代謝基因Acsl1、Acsl6表達(dá)增加。
[Abstract]:Aim: to study the effects of COX-2 on triglyceride metabolism and the expression of Acsl1 / Acsl6 in adipogenic hepatocytes BRL-3A. Methods: (1) to select the best inductive concentration and induce adipocytic degeneration: rat hepatocyte BRL-3A strain was cultured in vitro, and different concentrations of medical fat milk were used to induce adipogenic hepatocytes. Oil red O staining and hematoxylin restaining were used to observe the cell proliferation activity by MTT assay and to determine the optimal concentration of TG by Elisa before and after induction. (2) the experiment was divided into two groups: normal BRL-3A group. PYr-1.1-hU6-EGFP-COX-2shRNA group pYr-1.1-hU6-EGFP-HK group, pcDNA3.1 () -r-COX-2 group pcDNA3.1 () -r-HK group, nimesulide positive control group (200ug/ml) positive control group, with the exception of normal BRL-3A group, all other groups were cultured with the best induced concentration of 6% fat milk for 24 hours. After transfection, TG content in cells was detected by Elisa and RT-PCR was used to detect the expression of COX-2 mRNA-Acsl1mRNA-Acsl6 mRNA. Results: (1) lipids were induced by medical fat milk cultured cells for 24 hours, and lipid droplets in hepatocytes were increased by oil red staining, indicating that lipid droplets in hepatocytes were increased. Elisa showed that TG content in cells was increased. In combination with the OD value of MTT assay and Elisa results, the optimal concentration of this experiment was 6% medical fat emulsion. (2) pYr-1.1-hU6-EGFP-COX-2shRNA was transfected into adipogenic BRL-3A cells for 24 hours. Green fluorescence was observed in the cytoplasm of BRL-3A cells. The transfection efficiency was over 70%. (3) after transfection, the TG content in the cells of each group was detected by Elisa. The TG content of pYr-1.1-hU6-EGFP-COX-2shRNA group and nimesulide group decreased significantly (p0.05), and those of pYr-1.1-hU6-EGFP-COX-2shRNA group were significantly lower than those of the other 6 groups (p0.01), while the pcDNA3.1 () -r-COX-2 group was higher than the other six groups (p0.05). Nimesulide group was lower than pcDNA3.1 () -r-COX-2 group, but higher than pYr-1.1-hU6-EGFP-COX-2shRNA group. There was no significant difference between pYr-1.1-hU6-EGFP-HK group and pcDNA3.1 () -r-HK group. The expression of COX-2 mRNA was increased in adipogenic hepatocytes (p0.05). The expression of COX-2 mRNA in pYr-1.1-hU6-EGFP-COX-2shRNA group and nimesulide group was decreased (p0.05), the expression of COX-2 mRNA in pYr-1.1-hU6-EGFP-COX-2shRNA group was significantly lower than that in pYr-1.1-hU6-EGFP-COX-2shRNA group (p0.01), but the expression of COX-2 mRNA in pcDNA3.1 (p0.05) -r-COX-2 group was not significantly higher than that in pYr-1.1-hU6-EGFP-HK group and pcDNA3.1 (-r-HK) group (p0.05). The expression of COX-2 mRNA in pYr-1.1-hU6-EGFP-HK group and pcDNA3.1 (-rHK) group was not significantly increased (p0.05). The expression of Acsl1mRNA-Acsl6 mRNA: compared with normal hepatocytes, the expression of Acsl1mRNA-Acsl6 mRNA was increased in lipogenic hepatocytes (p0.05). The expression of Acsl1mRNA-Acsl6 mRNA was significantly decreased in both groups (p0.05), as compared with that in lipidized hepatocytes (p0.05). The expression of Acsl1mRNA-Acsl6 mRNA was significantly decreased in nimesulide group and hU6-EGFP-COX-2shRNA group (p0.05). In pYr-1.1-hU6-EGFP-COX-2shRNA group, the expression of Acsl1 + Acsl6 in pYr-1.1-hU6-EGFP-COX-2shRNA group was significantly increased (p0.05). In pYr-1.1-hU6-EGFP-HK group and pcDNA3.1 () -r-HK group, Acsl1 mRNA-Acsl6 mRNA did not change significantly (p > 0.05). Conclusion: (1) the expression of COX-2 was silenced, the fatty degeneration of hepatocytes was alleviated, the TG content in hepatocytes was decreased, and the expression of lipid metabolism gene Acsl1 / Acsl6 was decreased. (2) the overexpression of COX-2 was more serious than that of fatty degeneration in hepatocytes. The TG content and the expression of lipid metabolism gene Acsl1 + Acsl6 were increased in lipidized hepatocytes.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R575.5

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