胰岛素、高血糖素和神经肽Y对初生犊牛原代培养肝细胞中硬脂酰CoA去饱和酶mRNA表达的影响
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摘要
选用健康初生犊牛，采用双灌流的方法，成功分离犊牛肝脏细胞，通过测定培养液中尿素合成能力、白蛋白分泌能力和乳酸脱氢酶的泄漏量等指标，检测细胞在分离后损伤和修复情况，寻找神经内分泌激素处理的恰当时机。以含有目的基因的质粒为模板，采用梯度稀释的方法，以SYBR greenⅠ为荧光染料，检测β-actin和SCD两个基因的扩增效率。在培养到72h时，用0、5、10、20、50IU／ml的胰岛素，0、50、100、500、1000pg/ml高血糖素，0、50、100、500、1000pg／ml神经肽Y分别处理肝细胞，以β-actin为内参基因，用荧光PCR方法检测肝细胞内硬脂酰CoA去饱和酶(SCD)mRNA的相对变化。结果表明：1．离体双灌流法可以成功的分离犊牛肝细胞，成活率均在90％以上，细胞合成尿素的能力、分泌白蛋白的能力随着培养时间先减少后增加，在培养到3～4天时乳酸脱氢酶泄漏明显减少，表明培养3～4天后肝细胞能够修复胶原酶带来的损伤，因此分离的肝细胞培养72h后，用作实验材料比较适合。2．通过质粒浓度对数与CT值，求得Y=34.14-2.95*X(Ⅰ)和Y=35.46-2.97*X(Ⅱ)，两个基因的扩展效率相同，可以用比较CT法计算SCDmRNA相对表达量。3．胰岛素能显著的促进肝细胞内SCDmRNA表达，不同浓度组间差异均显著(P＜0.05)，并呈现剂量依赖性促进效应。4．胰高血糖素显著地抑制了肝细胞SCDmRNA表达水平，不同浓度组差异极显著(P＜0.01)，并呈现剂量依赖性抑制效应。5．神经肽Y促进肝细胞内SCDmRNA表达，添加不同浓度神经肽Y组与对照组比较差异显著(P＜0.05)，除50pg／ml处理组和500pg／ml处理组之间差异不显著外，其他处理组之间差异显著(P＜0.05)。6．胰岛素和神经肽Y在体外培养的肝细胞中可通过促进SCDmRNA表达，促进肝脏脂肪沉积。而高血糖素可通过抑制SCDmRNA表达，减少肝脏脂肪沉积。
The express of SCD mRNA was effected by insulin, glucagon and NPY in the hepatocytesof new born calf. Healthy new born calf were chosen and the hepatocytes were dissociated withperfusion succesefully, The ability of synthetized urea, the ability of secreted albumen and lactic aciddehydrogenase spillage were detected in the culture solution. The damage and recovery of hepatocyteswas detected. The amplication rate ofβ-actin and SCD was detected by fluorescence PCR withSRBR greenⅠand plasmid. The hepatocytes were treated with insulin (0、5、10、20、50IU/m),glucagons(0、50、100、500、1000pg/ml) and NPY (0、50、100、500、1000pg/ml) after cultured 72h.ThemRNA was extractd and correspondencet change of SCD mRNA was detected by fluorescence PCRaccording to change ofβ-actin. The results indicate that:1.The perfusion can dissociatehepatocytes as well as can,the survival rate of hepatocytes maintain up to 90%. The ability ofsynthetized urea, the ability of secreted albumen begin to descend after 3 d, lactic acid dehydrogenase islower after 3d.That show the damage of hepatocytes are recoverd after 3d. 2. Twoequations(Y=34.14-2.95*X (Ⅰ) and Y=35.46-2.97*X (Ⅱ)) were getten according to plasmidconcentration log and CT. The two equations show that SCD andβ-actin have same amplicationrate. Relative amplication of SCDmRNA can be computed using 2~(-ΔΔCt) method. 3 The SCD mRNAexpresss of hepatocytes is dose-dependently increased by insulin. There is significant difference amongall group.4 The SCD mRNA expresss of hepatocytes is dose-dependently depressed by glucagons Thereis extremely significant difference among all group.5 The SCD mRNA expresss of hepatocytes isincreased by NPY There is significant difference between all group except treated with 50pg/ml and500pg/ml.6 Insulin and NPY increase SCDmRNA express and increase liver fat storing.Glucagondepress SCDmRNA express and inh inhibite liver fat storing.
The express of SCD mRNA was effected by insulin, glucagon and NPY in the hepatocytesof new born calf. Healthy new born calf were chosen and the hepatocytes were dissociated withperfusion succesefully, The ability of synthetized urea, the ability of secreted albumen and lactic aciddehydrogenase spillage were detected in the culture solution. The damage and recovery of hepatocyteswas detected. The amplication rate ofβ-actin and SCD was detected by fluorescence PCR withSRBR greenⅠand plasmid. The hepatocytes were treated with insulin (0、5、10、20、50IU/m),glucagons(0、50、100、500、1000pg/ml) and NPY (0、50、100、500、1000pg/ml) after cultured 72h.ThemRNA was extractd and correspondencet change of SCD mRNA was detected by fluorescence PCRaccording to change ofβ-actin. The results indicate that:1.The perfusion can dissociatehepatocytes as well as can,the survival rate of hepatocytes maintain up to 90%. The ability ofsynthetized urea, the ability of secreted albumen begin to descend after 3 d, lactic acid dehydrogenase islower after 3d.That show the damage of hepatocytes are recoverd after 3d. 2. Twoequations(Y=34.14-2.95*X (Ⅰ) and Y=35.46-2.97*X (Ⅱ)) were getten according to plasmidconcentration log and CT. The two equations show that SCD andβ-actin have same amplicationrate. Relative amplication of SCDmRNA can be computed using 2~(-ΔΔCt) method. 3 The SCD mRNAexpresss of hepatocytes is dose-dependently increased by insulin. There is significant difference amongall group.4 The SCD mRNA expresss of hepatocytes is dose-dependently depressed by glucagons Thereis extremely significant difference among all group.5 The SCD mRNA expresss of hepatocytes isincreased by NPY There is significant difference between all group except treated with 50pg/ml and500pg/ml.6 Insulin and NPY increase SCDmRNA express and increase liver fat storing.Glucagondepress SCDmRNA express and inh inhibite liver fat storing.
引文
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[2]Storlien LH, Jenkins AB, Chisholm DJ, et al. Influence of dietary fat composition on development of insulin resistance in rats. Relationship to muscle triglyceride and omega-3 fatty acids in muscle phospholipid. Diabetes,1991,40:280-289.
[3] Jones BH, Maher MA, Banz WJ, et al,.Adipose tissue stearoyl-CoA desaturase mRNA is increased by obesity and decreased by polyunsaturated fatty acids. Am J Physiol, 1996,271: E44-E49.
[4] Agatha G, Hafer R, Zintl F. Fatty acid composition oflymphocyte membrane phospholipids in children with acuteleukemia. Cancer Lett,2001,173:139—144.
[5] Ntambi JM. Regulation of stearoyl-CoA desaturase by polyunsaturated fatty acids and cholesterol. J Lipid Res 1999,40:1549-58.
[6] Enoch H.G, Catala A.,Strittmatter P.. Mechanism of rat liver microsomal stearyl-CoA desaturase. J. Biol. Chem, 1976 ,251:5095-5103.
[7] Shanklin J, Achim C, Schmidt H, et al,.Mossbauer studies of alkane ω-hydroxylase: Evidence for a diiron. cluster in an integral membrane enzyme. Proc Nat. Acad. Sci, 1997,94:2981-2986.
[8] JE Stukey, VM McDonough, CE Martin. The OLE1 gene of Saccharomyces cerevisiae encodes the delta 9 fatty acid desaturase and can be functionally replaced by the rat stearoyl- CoA desaturase gene J. Biol. Chem. 1990, 265: 20144-20149
[9] Watts JL, Browse JA . palmitoyl-CoA-specific D9 fatty acid desaturase from Caenorhabditis elegans. Biochem Biophys Res. Commun ,2000,272:263-269
[10] Ward RJ, Travers MT, Vernon RG, Salter AM, Buttery PJ, Barber MC. The ovine stearyl-CoA desaturase gene: cloning and determination of gene number within the ovine genome. Biochem Soc Trans. 1997. 25:S673-S680
[11] Ideta R, Seki T, Adachi K, et al. The isolation and characterization of androgen-dependent genes in the flank organs of golden Syrian hamsters. Dermatology, 1998: 196:47-50.
[12] Mihara K. Structure and regulation of rat liver microsomal stearoyl-. CoA desaturase gene. J. Biochem, 1990,108:1022-1029.
[13] Kaestner K. H., Ntambi J. M, Kelly T. J.,et al. Differentiation-induced gene expression in 3T3-L1 preadipocytes. J. Biol. Chem ,1989,264: 14755-14761
[14] Zhang L., Ge L., Parimoo S., Stenn, K.,et al. Human stearoyl-CoA desaturase: alternative transcripts generated from a single gene by usage of tandem polyadenylation sites. Biochem. J. 1999,340:255-264.
[15] Ideta R, Seki T, Adachi K, et al. The isolation and characterization of androgen-dependent genes in the flank organs of golden Syrian hamsters. Dermatology. 1998. 196:47-50.
[16] Zheng Y, Prouty SM, Harmon A, etal. Scd3-a novel gene of the stearoyl-coA desaturase family with restricted expression in skin. Genomics 2001, 71:182-91.
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