COX-2通过AA对HSC-T6细胞增殖及Acsl家族基因表达的调控

发布时间：2018-03-23 08:38

本文选题：COX-2　切入点：AA　出处：《南华大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的本实验拟从COX-2代谢底物花生四烯酸这一视角，，阐明COX-2调控HSC细胞增殖及ACSL家族基因表达的机制。实验一方法 1、利用MTT比色法检测大鼠HSC-T6细胞在花生四烯酸20μM、40μM、60μM24h、48h的增殖情况。 2、利用RT-PCR检测AA20μM、AA40μM、AA60μM组时HSC的COX-2、ACSL家族基因的表达情况。结果 1、花生四烯酸20μM、40μM、60μM时HSC细胞生存率增加（p0.05），在40μM浓度下作用48h时增加显著（p0.05）。 2、COX-2基因在AA20μM、AA40μM表达增高(p0.05)，ACSL1基因在AA60μM表达相对增高(p0.05)，ACSL3、ACSL6基因在AA40μM、AA60μM表达增高(p0.05)，ACSL4、ACSL5基因在各组表达无显著变化(p0.05）。实验二方法 1、向HSC-T6转染构建的pYr-1.1-hU6-EGFP-COX-2shRNA1，荧光显微镜下评估其转染效率。分组为①HSC组；②AA40μM组；③pYr-1.1HK+AA组；④COX-2shRNA1+AA组。AA组浓度为MTT筛选的最佳反应浓度40μM。 2、利用MTT比色法检测转染后各组HSC细胞增殖情况。 3、利用WB技术检测转染48h后各组COX-2、ACSL家族基因的表达情况。结果 1、AA40μM组较HSC组OD值增加（p0.05），COX-2shRNA1+AA组与pYr-1.1HK+AA组比较OD值降低（p0.05），COX-2shRNA1+AA组较HSC组OD值无显著差异。 2、 AA组COX-2、 ACSL1、 ACSL6基因表达较HSC组增加(p0.05），AA+COX-2shRNA转染组较pYr-1.1HK+AA空质粒组COX-2、ACSL1、ACSL6蛋白表达降低(p0.05），而对照组COX-2、ACSL1、ACSL6表达无显著差异(p0.05）。结论 1、COX-2可以通过代谢底物花生四烯酸促进肝星状细胞的增殖作用。 2、COX-2可以通过代谢底物花生四烯酸调控HSC细胞ACSL1、ACSL6基因表达。
[Abstract]:objective
This study aims to elucidate the mechanism of COX-2 regulation of HSC cell proliferation and ACSL gene expression from the perspective of COX-2 metabolite groundnut four enoic acid.
Experiment 1
Method
1, MTT colorimetric assay was used to detect the proliferation of HSC-T6 cells in peanut four enoic acid 20 mu M, 40 mu M, 60 mu M24h, and 48h.
2, the expression of the COX-2 and ACSL family of HSC in the group of AA20 M, AA40, M and AA60 mu M was detected by RT-PCR.
Result
1, the survival rate of HSC cells increased (P0.05) when the peanut four enoic acid 20 mu M, 40 mu M, 60 micron M, and increased significantly at the concentration of 40 mu M (P0.05).
2, the expression of COX-2 gene increased at AA20, M, AA40 and M (P0.05), ACSL1 gene expression was increased at AA60 (M), P0.05 (ACSL3), and the expression of M gene increased in the two groups.
Experiment two
Method
1, transfection of pYr-1.1-hU6-EGFP-COX-2shRNA1 constructed into HSC-T6, and the transfection efficiency under fluorescent microscope. It was grouped as follows: HSC group; AA40 AA40 M group; pYr-1.1HK+AA group; COX-2shRNA1+AA group.AA group concentration was MTT screening for the best reaction concentration of M..
2, the proliferation of HSC cells after transfection was detected by MTT colorimetric assay.
3, WB technique was used to detect the expression of COX-2 and ACSL family genes in each group after transfection of 48h.
Result
1, the AA40 M group than in HSC group was increased (P0.05), COX-2shRNA1+AA group and pYr-1.1HK+AA group was lower than that in group HSC (P0.05), OD group COX-2shRNA1+AA had no significant difference.
2, the expression of COX-2, ACSL1 and ACSL6 genes in group AA increased compared with that in HSC group (P0.05), and the expression of COX-2, ACSL1 and ACSL6 protein in AA+COX-2shRNA transfection group was lower than that in pYr-1.1HK+AA empty plasmid group.
conclusion
1, COX-2 can promote the proliferation of hepatic stellate cells by metabolic substrates, arachidic acid (arachidic acid).
2, COX-2 can regulate the expression of ACSL1 and ACSL6 gene in HSC cells by metabolizing the substrate, arachidic acid.

【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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