【摘要】：目的:通過建立游離脂肪酸體外誘導(dǎo)的LO2細(xì)胞非酒精脂肪肝模型,探討不同劑量的清肝降脂方對(duì)肝臟脂肪變LO2細(xì)胞HO-1、Egr-1基因及蛋白表達(dá)水平的影響。方法:建立游離脂肪酸誘導(dǎo)脂肪變性LO2肝細(xì)胞體外模型。人肝LO2細(xì)胞采用含10%胎牛血清的RPMI 1640培養(yǎng)基培養(yǎng),待細(xì)胞均勻分布,隨機(jī)分為6組:正常對(duì)照組、模型組、清肝降脂方高劑量組、中劑量組、低劑量組、水林佳對(duì)照組,每組6瓶。除正常對(duì)照組外,其余5組細(xì)胞均以0.5 mmol/L的游離脂肪酸誘導(dǎo)LO2非酒精脂肪肝細(xì)胞模型,誘導(dǎo)24 h后,各組隨機(jī)抽取1瓶進(jìn)行油紅O染色,觀察LO2細(xì)胞的脂肪化程度。確定模型誘導(dǎo)成功后,正常對(duì)照組和模型組分別加入10%正常大鼠血清,低、中、高劑量清肝降脂方藥物血清組及水林佳藥物血清組添加10%含相對(duì)應(yīng)藥物的大鼠血清,培養(yǎng)24h后,收集細(xì)胞;采用RT-PCR及Western blot法檢測(cè)血紅素氧化酶-1(HO-1)、早期生長(zhǎng)反應(yīng)因子-1(Egr-1)的基因及蛋白表達(dá)水平。結(jié)果:與正常對(duì)照組比較,模型對(duì)照組LO2細(xì)胞HO-1、Egr-1 mRNA和蛋白表達(dá)有所增加;與模型對(duì)照組比較,自擬清肝降脂方三劑量組和對(duì)照藥物組HO-1 mRNA及蛋白表達(dá)有所增高,Egr-1mRNA和蛋白表達(dá)明顯降低;與對(duì)照藥物組比較,清肝降脂方高劑量組HO-1 mRNA表達(dá)有所增高,Egr-1 mRNA表達(dá)有所降低。結(jié)論:Egr-1高表達(dá)可能會(huì)導(dǎo)致肝損傷機(jī)制之一;HO-1高表達(dá)可能是保護(hù)肝損傷機(jī)制之一;兩者可能都是通過調(diào)節(jié)TNF-α表達(dá)來調(diào)節(jié)NAFLD細(xì)胞的損傷進(jìn)展。清肝降脂方可明顯改善LO2細(xì)胞Egr-1、HO-1 mRNA及蛋白的表達(dá),可能為其治療機(jī)制、臨床診斷和判斷進(jìn)展參考指標(biāo)。
[Abstract]:Aim: to study the effect of Qinggan Jiangzhi recipe (QQQQF) on the expression of HO-1,Egr-1 gene and protein in LO2 cells induced by free fatty acids in vitro. Methods: the model of LO2 hepatocytes induced by free fatty acids was established in vitro. Human liver LO2 cells were cultured on RPMI 1640 medium containing 10% fetal bovine serum and were randomly divided into 6 groups: normal control group, model group, high dose group, middle dose group, low dose group and Shuilingjia control group. Six bottles per group. In addition to the normal control group, the other five groups were treated with free fatty acids for 0.5 mmol/L to induce LO2 non-alcoholic fatty liver cells. After 24 h of induction, 1 bottle was randomly selected for oil red O staining in each group to observe the fatty degree of LO2 cells. After the model was induced successfully, the normal control group and the model group were added with 10% normal rat serum, the low, middle and high doses of Qinggan Jiangzhi prescription drug serum group and the water linjia drug serum group supplemented with 10% rat serum containing corresponding drugs, respectively. After 24 hours of culture, the cells were collected. The gene and protein expression of heme oxygenase-1 (HO-1) and early growth response factor-1 (Egr-1) were detected by RT-PCR and Western blot. Results: compared with the normal control group, the expression of HO-1,Egr-1 mRNA and protein in LO2 cells in the model control group was increased. Compared with the model control group, the expression of HO-1 mRNA and protein was increased and the expression of Egr-1mRNA and protein was significantly decreased in the three dose group and the control group. Compared with the control group, the expression of HO-1 mRNA was increased and the expression of Egr-1 mRNA was decreased in the high dose group of Qinggan Jiangzhi recipe. Conclusion: the high expression of Egr-1 may lead to one of the mechanisms of liver injury, the high expression of HO-1 may be one of the mechanisms of protecting liver injury, and both of them may regulate the progression of NAFLD cell injury by regulating the expression of TNF- 偽. Qinggan Jiangzhi decoction can obviously improve the expression of Egr-1,HO-1 mRNA and protein in LO2 cells, which may be a reference index for its therapeutic mechanism, clinical diagnosis and judgement of progress.
【作者單位】： 遼寧中醫(yī)藥大學(xué);遼寧中醫(yī)藥大學(xué)附屬醫(yī)院;
【基金】：沈陽市科技創(chuàng)新專項(xiàng)基金-生物與制藥科技攻關(guān)專項(xiàng)項(xiàng)目(F13-208-9-00)
【分類號(hào)】：R285

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