COX-2对脂变BRL-3A肝细胞甘油三酯代谢的影响及Acsl1、Acsl6表达的调节

发布时间：2018-06-23 11:47

本文选题：环氧合酶-2 + 非酒精性脂肪性肝病　；参考：《南华大学》2014年硕士论文

【摘要】：目的：研究COX-2对脂变肝细胞BRL-3A株甘油三酯代谢及Acsl1、Acsl6表达的影响。方法： (1)筛选最佳诱导浓度，诱导细胞脂肪变性：体外培养大鼠肝细胞BRL-3A株，不同浓度医用脂肪乳诱导大鼠肝细胞成脂肪变肝细胞，油红O染色及苏木素复染方法观察各组细胞脂肪变状态，MTT检测细胞增殖活性及ELISA检测诱导前后细胞内TG含量确定最佳诱导浓度。 (2)实验分为正常BRL-3A组、脂变BRL-3A组，pYr-1.1-hU6-EGFP-COX-2shRNA组，pYr-1.1-hU6-EGFP-HK组，pcDNA3.1（+）-r-COX-2组，pcDNA3.1（+）-r-HK组，尼美舒利（200ug/ml）阳性对照组，除正常BRL-3A组外，其余各组均以最佳诱导浓度6%脂肪乳培养24h后，瞬时转染各组对应的质粒，ELISA检测转染后各组细胞内TG含量，RT-PCR检测各组COX-2mRNA、Acsl1mRNA、Acsl6mRNA的表达。结果：（1）医用脂肪乳培养细胞24h诱导细胞脂变，油红染色示肝细胞内空泡脂滴增加，，提示细胞脂肪变。ELISA结果表明细胞内TG含量增加。结合油红染色、MTT检测的OD值及ELISA结果确定本实验最佳诱导浓度为6%医用脂肪乳。（2）pYr-1.1-hU6-EGFP-COX-2shRNA转染至脂变BRL-3A细胞培养24h后荧光显微镜下观察，细胞质中可见绿色荧光，转染效率达70%以上。（3）ELISA检测转染后各组细胞内TG含量，pYr-1.1-hU6-EGFP-COX-2shRNA组与尼美舒利组TG含量均减少(p0.05)，以pYr-1.1-hU6-EGFP-COX-2shRNA组减少明显，均低于其余6组(p0.01)，而pcDNA3.1（+）-r-COX-2组高于其余6组(p0.05)。尼美舒利组低于pcDNA3.1（+）-r-COX-2组但高于pYr-1.1-hU6-EGFP-COX-2shRNA组，pYr-1.1-hU6-EGFP-HK组和pcDNA3.1（+）-r-HK组无明显变化，无统计学意义（p0.05）。（4）7个组别COX-2、Acsl1、Acsl6基因表达情况：COX-2mRNA表达情况：与正常肝细胞组相比，脂变肝细胞组中COX-2mRNA表达增高(p0.05)。与脂变肝细胞组相比， pYr-1.1-hU6-EGFP-COX-2shRNA组及尼美舒利组中COX-2mRNA表达降低(p0.05)，以pYr-1.1-hU6-EGFP-COX-2shRNA组降低明显(p0.01)，而pcDNA3.1（+）-r-COX-2组中COX-2mRNA表达增高(p0.05)。pYr-1.1-hU6-EGFP-HK组和pcDNA3.1（+）-r-HK组COX-2mRNA无明显增高，无统计学意义（p0.05）。脂代谢基因Acsl1mRNA、Acsl6mRNA表达情况：与正常肝细胞组相比，脂变肝细胞中Acsl1mRNA、Acsl6mRNA表达增高(p0.05)。与脂变肝细胞组相比，pYr-1.1-hU6-EGFP-COX-2shRNA及尼美舒利组中Acsl1mRNA、Acsl6mRNA表达均降低(p0.05)。以pYr-1.1-hU6-EGFP-COX-2shRNA组下调明显(p0.01)，pcDNA3.1（+）-r-COX-2组中Acsl1、Acsl6表达则明显增高(p0.05)。而pYr-1.1-hU6-EGFP-HK组和pcDNA3.1（+）-r-HK组中Acsl1mRNA、Acsl6mRNA无明显改变，无统计学意义(p＞0.05)。结论：（1）沉默COX-2表达，肝细胞脂肪变性减轻；脂变肝细胞内TG含量降低；同时，脂代谢基因Acsl1、Acsl6表达减少。（2）COX-2过表达，肝细胞脂肪变性加重；脂变肝细胞内TG含量增加；同时，脂代谢基因Acsl1、Acsl6表达增加。
[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.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575.5

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