花生四烯酸对日本沼虾肝胰腺细胞脂质代谢基因表达的影响

摘要

This experiment was conducted to determine the effects of arachidonic acid (ARA) concentration in culture medium on cell viability and lipid metabolism-related gene expressions of hepatopancreas cells isolated from juvenile oriental river prawn, Macrobrachium nipponense.The hepatopancreas cells were dissected from prawns and were cultured with complete culture medium for 5 days.After that, cultured cells were incubated in medium supplemented with graded levels [0 (ARA1) , 50 (ARA2), 100 (ARA3) , 200 (ARA4) and1 000 μmol/L (ARA5) of ARA.Cell viability at 24 h and gene expressions of lipid metabolism-related genes at 12 and 24 h were examined.The results showed as follows: the hepatopancreas cells showed well growth in complete culture medium, and could survive for 15 days;cell viability was significantly decreased by incubation with higher level (1 000 μmol/L) of ARA (ARA5 group) compared with ARA1 and ARA2 groups (P<0.05) after 24 h;higher level (1 000 μmol/L) of ARA (ARA5 group) caused significant decreases of gene expressions of delta-4 fatty acyl desaturase (Δ4 FAD), delta-6 fatty acyl desaturase (Δ6 FAD), very-longchain fatty acids-6 (Elovl6), scavenger receptor class B type Ⅰ (SR-B Ⅰ), fatty acid-binding protein 10(FABP10) and acyl-CoA binding protein (ACBP) of hepatopancreas cells incubation for both 12 and 24 h;after incubation with ARA for 12 h, the gene expression of SR-B Ⅰ of ARA2 group was significantly higher than that of other groups (P<0.05), FABP10 gene expression of ARA2 and ARA3 groups was significantly higher than that of ARA1 and ARA5 groups (P<0.05), and ACBP gene expression of ARA3 group was significantly higher than that of other groups (P<0.05);after incubation with ARA for 24 h, the highest expressions of SR-B Ⅰ , FABP10 and ACBP were observed in ARA2 group, which was significantly higher than those of other groups (P<0.05).These findings suggest that ARA can influence cell viability and lipid metabolism-related gene expressions of hepatopancreas cell isolated from Macrobrachium nipponense.Cell viability can be decreased by incubation with higher level of ARA (1 000 μmol/L).Appropriate levels of ARA (50 to100 μmol/L) can promote the expressions of genes related to fatty acyl desaturase, elongases of very-longchain fatty acids and fatty acid transport.[Chinese Journal of Animal Nutrition, 2017, 29 (2) : 536-546]%本试验旨在评价细胞培养液中花生四烯酸(arachidonic acid,ARA)浓度对日本沼虾肝胰腺细胞活力及脂质代谢相关基因表达的影响.分离日本沼虾肝胰腺细胞,使用M199完全培养液培养5d后换成含ARA的培养液,ARA浓度分别为0(ARA1)、50(ARA2)、100(ARA3)、200(ARA4)和1000μmol/L (ARA5),测定12和24 h时脂质代谢相关基因的表达水平,以及24 h时细胞活力.结果表明:原代肝胰腺细胞使用完全培养液时,生长状况良好,能存活15d左右;ARA5组24 h时细胞活力显著低于ARA1和ARA2组(P<0.05);高浓度的ARA降低了12和24 h时△4脱饱和酶(A4 FAD)、Δ6脱饱和酶(△6FAD)、碳链延长酶6(Elovl6)、B类Ⅰ型清道夫受体(SR-B Ⅰ)、脂肪酸结合蛋白10 (FABP10)、乙酰辅酶A结合蛋白(ACBP)基因表达水平;ARA作用12 h时,ARA2组SR-BⅠ基因表达水平显著高于其余各组(P<0.05),ARA2和ARA3组FABP10基因表达水平显著高于ARA1和ARA5组(P<0.05),ARA3组ACBP基因表达水平显著高于其余各组(P<0.05);ARA作用24 h时,ARA2组SR-BⅠ、FABP10和ACBP基因表达水平显著高于其余各组(P<0.05).由此可见,细胞培养液中ARA浓度会影响日本沼虾肝胰腺细胞活力及脂质代谢相关基因的表达,过高的ARA浓度(1000 μmol/L)会降低细胞的活力,适宜的ARA浓度(50～ 100 μmol/L)可促进脂肪酸脱饱和酶、碳链延长酶及脂肪酸转运相关基因的表达.