油酸诱导鹅肝细胞脂肪变性对细胞内脂质代谢平衡相关基因表达的影响
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摘要
动物肝脏脂代谢平衡的破坏会导致大量三酰甘油(TG)沉积于肝脏,从而引起肝脂肪变性。本实验首先扩增出鹅脂质代谢的关键基因DGAT1、DGAT2、FOXO1、MTP、PLIN和CPT-1的序列,然后以30日龄四川白鹅原代肝细胞为材料,添加不同浓度油酸诱导鹅肝细胞脂肪变性,构建肝细胞体外脂肪变性模型,检测细胞内、外TG和细胞外apoB的变化情况,并应用实时荧光定量PCR检测DGAT1、DGAT2、PPARα、PPARγ、FOXO1、MTP、PLIN和CPT-1 mRNA表达的变化情况,获得以下主要结果:
(1)首次克隆得到了四川白鹅MTP、DGAT1、DGAT2、CPT-1、FOXO1和PLIN六个基因的部分CDS,它们的大小分别为663bp、874bp、409bp、551bp、1087bp和277bp。
(2)通过序列BLAST比对发现,鹅MTP、DGAT1、DGAT2、CPT-1、FOXO1和PLIN等基因与原鸡或斑胸草雀的同源性都在90%以上,而与其它脊椎动物的同源性在70%-80%之间。
(3)0.5 mmol/L、1.0 mmol/L和1.5 mmol/L油酸处理细胞24 h,都能显著增加鹅肝细胞内TG的合成,与对照相比分别上升3倍(P＜0.01)、3.43倍(P＜0.01)和2.86倍(P＜0.05)。
(4)通过油红O染色,经油酸处理细胞后,对照组细胞内脂滴极少,而实验组明显沉积了大量的脂滴,并且随着油酸浓度的增加,细胞内脂滴聚集程度增加。
(5)用MTT法对细胞活性进行检测,油酸处理细胞24 h后,0.5 mmol/L油酸显著增强细胞活性(P＜0.01),而1.5 mmol/L油酸对细胞活性有抑制作用(P＜0.01),1.0 mmol/L油酸对细胞活性没有显著影响。
(6)处理细胞24 h后,与对照组相比,0.5 mmol/L、1.0 mmol/L和1.5 mmol/L油酸都能减少鹅肝细胞脂蛋白apoB的分泌,3个油酸处理组分泌到胞外的apoB分别是对照的0.96倍、0.83倍(P＜0.05)和0.72倍(P＜0.01),同时分泌到胞外的TG也逐渐减少,分别是对照的4.69倍(P＜0.01)、2倍(P＜0.05)和1.3倍。
(7)油酸处理细胞24 h后,对脂质代谢相关基因的表达有明显影响,与对照相比,0.5 mmol/L、1.0 mmol/L和1.5 mmol/L三个处理组的PPARγ的表达量分别为对照的1.893倍(P＜0.05)、1.297倍和0.909倍;PLIN的表达量分别为对照的5.706倍(P＜0.01)、5.31倍(P＜0.01)和4.145倍(P＜0.01),且与胞内TG含量呈显著正相关;DGAT1的表达量分别为对照的1.531倍(P＜0.05)、1.244倍和0.936倍,与胞外TG的变化显著正相关;DGAT2的表达量分别为对照的2.651倍(P＜0.01)、2.054倍(P＜0.01)和1.814倍(P＜0.05);PPARα和CPT-1的变化趋势一致,随着油酸浓度的增加表达上升;而FOXO1和MTP的表达量随着油酸浓度增加显著降低,且与apoB的分泌显著相关。
The disorder of Lipid metabolism could lead to the massive triglycerides (TG) accumulation in the liver,then result in hepatic steatosis.At present,the molecular mechanism of hepatic steatosis caused by lipid metabolism disorder in goose hepatocytes has not been reported.In this study,DGAT1,DGAT2,FOXO1, MTP,PLIN and CPT-1 partial sequences of goose were cloned for the first time, which are related with goose lipid metabolism balance.Then the hepatic steatosis model in vitro was constructed by culturing the goose primary hepatocytes with different concentration oleic acid to induce goose hepatocytic steatosis.TG concentration intracellular and extracellular of steatosis hepatocytes,and the change of apoB concentration were measured.Meanwhile,the mRNA expression of DGAT1、DGAT2、PPARα、PPARγ、FOXO1、MTP、PLIN and CPT-1 was dedected by Real-Time RT-PCR.The results were as follows:
(1) The partial CDS of MTP、DGAT1、DGAT2、CPT-1、FOXO1 and PLIN genes were amplificationed for the first time,and the length of their cloned sequences were 663bp、874bp、409bp、551bp、1087bp and 277bp, respectively.
(2) Gene sequence of MTP、DGAT1、DGAT2、CPT-1、FOXO1 and PLIN of goose has high homology with Gallus gallus or Poephila castanotis above 90%,with other vertebrate by 70%-80%through sequence BLAST.
(3) Compared with control group,0.5,1.0 and 1.5 mmol/L oleic oil cultured with hapetocytes for 24h all could increase intracellular TG accumulation by 3 (P＜0.01),3.43(P＜0.01),2.86 times(P＜0.05),respectively.
(4) The sesult of Oil Red O staining showed,the lipid droplets in hepatocytes of control group were much less than that in experimental group,and the degree of lipid droplet accumulation increased intra hepatocytes with the increasing of oleic oil concentration.
(5) The MTT assay was used for the quantification of cytoactive.After 24h cultured with oleic oil,1.5 mmol/L(P＜0.01) oleic oil had an inhibiting effect on the cytoactive,0.5 mmol/L(P＜0.01)oleic oil could induce cytoactive,and 1.0 mmol/L oleic oil had no effect on cell viability.
(6) Compared with control group,0.5,1.0 and 1.5 mmol/L oleic oil cultured with goose hepatocytes for 24h could all decreased secretion of apoB-containing lipoproteins by 0.96,0.83(P＜0.05) and 0.72 times(P＜0.01),respectively.The extracellular TG concentration decreased by 4.69(P＜0.01),2(P＜0.05) and 1.3 times,gradually.
(7) Different concentration of oleic oil cultured with hepatocytes for 24h had evident effect on some genes expression related to lipid metabolism. Compared with control group,the mRNA level of PPARγin the experimental groups which were treated with 0.5,1.0 and 1.5 mmol/L oleic oil was increased by 1.893(P＜0.05),1.297 and 0.909 times,respectively;The mRNA level of PLIN was increased by 5.706(P＜0.01),5.31(P＜0.01) and 4.145 times (P＜0.01),respectively.The mRNA level of FOXO1 was decreased by 0.593(P＜0.05),0.402(P＜0.01) and 0.314 times(P＜0.01),respectively; The mRNA level of MTP was decreased by 0.913,0.806(P＜0.05) and 0.543 times(P＜0.01),respectively;The mRNA level of PPARαwas increased by 2.26(P＜0.05),2.957(P＜0.01) and 10.302 times(P＜0.01),respectively; The mRNA level of CPT-1 was increased by 2.107(P＜0.01),2.033(P＜0.01) and 1.631 times(P＜0.05),respectively.The mRNA level of DGAT1 was increased by 1.531(P＜0.05),1.244 and 0.936 times(P＜0.05),respectively. The mRNA level of DGAT2 was increased by 2.651(P＜0.01),2.054 (P＜0.01) and 1.814(P＜0.05) times(P＜0.05),respectively.
(1)首次克隆得到了四川白鹅MTP、DGAT1、DGAT2、CPT-1、FOXO1和PLIN六个基因的部分CDS,它们的大小分别为663bp、874bp、409bp、551bp、1087bp和277bp。
(2)通过序列BLAST比对发现,鹅MTP、DGAT1、DGAT2、CPT-1、FOXO1和PLIN等基因与原鸡或斑胸草雀的同源性都在90%以上,而与其它脊椎动物的同源性在70%-80%之间。
(3)0.5 mmol/L、1.0 mmol/L和1.5 mmol/L油酸处理细胞24 h,都能显著增加鹅肝细胞内TG的合成,与对照相比分别上升3倍(P＜0.01)、3.43倍(P＜0.01)和2.86倍(P＜0.05)。
(4)通过油红O染色,经油酸处理细胞后,对照组细胞内脂滴极少,而实验组明显沉积了大量的脂滴,并且随着油酸浓度的增加,细胞内脂滴聚集程度增加。
(5)用MTT法对细胞活性进行检测,油酸处理细胞24 h后,0.5 mmol/L油酸显著增强细胞活性(P＜0.01),而1.5 mmol/L油酸对细胞活性有抑制作用(P＜0.01),1.0 mmol/L油酸对细胞活性没有显著影响。
(6)处理细胞24 h后,与对照组相比,0.5 mmol/L、1.0 mmol/L和1.5 mmol/L油酸都能减少鹅肝细胞脂蛋白apoB的分泌,3个油酸处理组分泌到胞外的apoB分别是对照的0.96倍、0.83倍(P＜0.05)和0.72倍(P＜0.01),同时分泌到胞外的TG也逐渐减少,分别是对照的4.69倍(P＜0.01)、2倍(P＜0.05)和1.3倍。
(7)油酸处理细胞24 h后,对脂质代谢相关基因的表达有明显影响,与对照相比,0.5 mmol/L、1.0 mmol/L和1.5 mmol/L三个处理组的PPARγ的表达量分别为对照的1.893倍(P＜0.05)、1.297倍和0.909倍;PLIN的表达量分别为对照的5.706倍(P＜0.01)、5.31倍(P＜0.01)和4.145倍(P＜0.01),且与胞内TG含量呈显著正相关;DGAT1的表达量分别为对照的1.531倍(P＜0.05)、1.244倍和0.936倍,与胞外TG的变化显著正相关;DGAT2的表达量分别为对照的2.651倍(P＜0.01)、2.054倍(P＜0.01)和1.814倍(P＜0.05);PPARα和CPT-1的变化趋势一致,随着油酸浓度的增加表达上升;而FOXO1和MTP的表达量随着油酸浓度增加显著降低,且与apoB的分泌显著相关。
The disorder of Lipid metabolism could lead to the massive triglycerides (TG) accumulation in the liver,then result in hepatic steatosis.At present,the molecular mechanism of hepatic steatosis caused by lipid metabolism disorder in goose hepatocytes has not been reported.In this study,DGAT1,DGAT2,FOXO1, MTP,PLIN and CPT-1 partial sequences of goose were cloned for the first time, which are related with goose lipid metabolism balance.Then the hepatic steatosis model in vitro was constructed by culturing the goose primary hepatocytes with different concentration oleic acid to induce goose hepatocytic steatosis.TG concentration intracellular and extracellular of steatosis hepatocytes,and the change of apoB concentration were measured.Meanwhile,the mRNA expression of DGAT1、DGAT2、PPARα、PPARγ、FOXO1、MTP、PLIN and CPT-1 was dedected by Real-Time RT-PCR.The results were as follows:
(1) The partial CDS of MTP、DGAT1、DGAT2、CPT-1、FOXO1 and PLIN genes were amplificationed for the first time,and the length of their cloned sequences were 663bp、874bp、409bp、551bp、1087bp and 277bp, respectively.
(2) Gene sequence of MTP、DGAT1、DGAT2、CPT-1、FOXO1 and PLIN of goose has high homology with Gallus gallus or Poephila castanotis above 90%,with other vertebrate by 70%-80%through sequence BLAST.
(3) Compared with control group,0.5,1.0 and 1.5 mmol/L oleic oil cultured with hapetocytes for 24h all could increase intracellular TG accumulation by 3 (P＜0.01),3.43(P＜0.01),2.86 times(P＜0.05),respectively.
(4) The sesult of Oil Red O staining showed,the lipid droplets in hepatocytes of control group were much less than that in experimental group,and the degree of lipid droplet accumulation increased intra hepatocytes with the increasing of oleic oil concentration.
(5) The MTT assay was used for the quantification of cytoactive.After 24h cultured with oleic oil,1.5 mmol/L(P＜0.01) oleic oil had an inhibiting effect on the cytoactive,0.5 mmol/L(P＜0.01)oleic oil could induce cytoactive,and 1.0 mmol/L oleic oil had no effect on cell viability.
(6) Compared with control group,0.5,1.0 and 1.5 mmol/L oleic oil cultured with goose hepatocytes for 24h could all decreased secretion of apoB-containing lipoproteins by 0.96,0.83(P＜0.05) and 0.72 times(P＜0.01),respectively.The extracellular TG concentration decreased by 4.69(P＜0.01),2(P＜0.05) and 1.3 times,gradually.
(7) Different concentration of oleic oil cultured with hepatocytes for 24h had evident effect on some genes expression related to lipid metabolism. Compared with control group,the mRNA level of PPARγin the experimental groups which were treated with 0.5,1.0 and 1.5 mmol/L oleic oil was increased by 1.893(P＜0.05),1.297 and 0.909 times,respectively;The mRNA level of PLIN was increased by 5.706(P＜0.01),5.31(P＜0.01) and 4.145 times (P＜0.01),respectively.The mRNA level of FOXO1 was decreased by 0.593(P＜0.05),0.402(P＜0.01) and 0.314 times(P＜0.01),respectively; The mRNA level of MTP was decreased by 0.913,0.806(P＜0.05) and 0.543 times(P＜0.01),respectively;The mRNA level of PPARαwas increased by 2.26(P＜0.05),2.957(P＜0.01) and 10.302 times(P＜0.01),respectively; The mRNA level of CPT-1 was increased by 2.107(P＜0.01),2.033(P＜0.01) and 1.631 times(P＜0.05),respectively.The mRNA level of DGAT1 was increased by 1.531(P＜0.05),1.244 and 0.936 times(P＜0.05),respectively. The mRNA level of DGAT2 was increased by 2.651(P＜0.01),2.054 (P＜0.01) and 1.814(P＜0.05) times(P＜0.05),respectively.
引文
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