鸡原代肝细胞培养及叶酸对脂质代谢相关基因表达的影响

doi:10.3864/j.issn.0578-1752.2017.21.015

摘要/Abstract

摘要： 【目的】腹脂沉积是目前家禽养殖过程中的一个普遍现象。与哺乳动物不同，家禽的脂质代谢主要发生在肝脏，前人很多研究发现叶酸和IGF2均参与调控动物的脂质代谢，因此，试验旨在建立鸡原代肝细胞培养条件，并研究叶酸对鸡原代肝细胞脂质代谢相关基因表达的影响，为体外探究叶酸调控家禽脂质代谢机制提供依据。【方法】采用胶原酶消化配合鸡淋巴细胞分离液纯化法分离得到鸡原代肝细胞，用高碘酸希夫氏染色方法进行肝细胞鉴定；同时分离得到的肝细胞用正常培养基培养36 h后，更换含不同叶酸浓度的培养基（0，1，5，10，15，和20 mg·L^-1），每个叶酸浓度6个重复，处理12 h后收集上清液，并收集细胞提取总RNA，用RT-qPCR法检测基因相对表达量，MTT法检测细胞增殖，商业试剂盒方法检测上清液中LDH含量。并分析基因表达之间的相关性以及叶酸剂量与基因表达之间的回归关系。【结果】采用胶原酶消化配合鸡淋巴细胞分离液纯化法可以获得分离效果好且纯度高的鸡肝细胞；不同剂量的叶酸没有影响肝细胞培养液中的LDH活性（P＞0.05），也没有对肝细胞的增殖产生影响（P＞0.05）；与1 mg·L^-1剂量的叶酸对照组相比，10、15和20 mg·L^-1的叶酸显著降低了肝细胞IGF2以及与脂质合成代谢有关的ACC和FAS基因的表达（P＜0.05），但叶酸并没有对鸡肝细胞中与脂质分解代谢有关的CPT1和PPARα基因的表达产生影响（P＞0.05）；并且鸡肝细胞中ACC和FAS基因的表达与IGF2存在一定的正相关（P＜0.05）；除此之外，鸡肝细胞IGF2，FAS和ACC的表达与叶酸剂量也存在着线性和二次曲线的回归关系（P＜0.05）。【结论】叶酸的添加可降低鸡原代肝细胞中与脂肪酸合成相关基因的表达，并且FAS和ACC基因的表达可能与IGF2具有正相关关系；本试验中叶酸的最适剂量为15 mg·L^-1。

关键词: 叶酸, IGF2, 肝细胞, 脂质代谢, 鸡

Abstract: 【Objective】Abdominal fat deposition is a universal phenomenon at present in poultry industry. Different from mammals, 90%-95% of lipid metabolism in poultry occurs in the liver. Previous studies have demonstrated folic acid and IGF2 were involved in animals lipid metabolism. The study was conducted to establish the method for primary culture of chicken hepatocytes and investigate the effects of folic acid on IGF2 and genes expression associated with lipid metabolism in primary chicken hepatocytes, further providing a basis for exploring lipid metabolism of poultry in vitro. 【Method】Chicken hepatocytes were isolated by collagenase digestion combined with purification of chicken lymphocyte separation fluid, and evaluated by PAS staining, respectively. After 36 h culture of hepatocytes, cells were treated with different concentration of folic acid for 12 h. Hepatocytes proliferation and injury was detected by MTT method and LDH activity in culture medium respectively. Cells were collected to get total RNA for gene expression analysis by RT-PCR. Later, correlation analysis was carried out between IGF2 and lipid metabolism related genes expression. Regression analysis was performed between folic acid concentration and genes expression.【Result】The results showed that hepatocytes were isolated with high purity. Folic acid didn’t affect cell proliferation and cellular LDH activity (P＞0.05).When compared with the control group (1 mg·L^-1 folic acid), 10, 15 and 20 mg·L^-1 folic acid significantly reduced IGF2 expression in chicken hepatocytes (P＜0.05), and same phenomenon was observed in FAS and ACC expression. However, folic acid had no effects on CPT-1 and PPARα expression (P＞0.05) . In addition, FAS and ACC expression had positive correlation with IGF2 mRNA level in chicken hepatocytes (P＜0.05). There existed linear and quadratic regression between genes expression and folic acid concentration (P＜0.05). 【Conclusion】 In conclusion, folic acid could reduce gene expression associated with fatty acid synthesis in chicken hepatocytes. In addition, FAS and ACC mRNA level might have positive connection with IGF2 expression. Optimum treatment dose of folic acid was 15 mg·L^-1 in this study.

Key words: folic acid, IGF2, hepatocytes, lipid metabolism, chicken

刘艳利，党燕娜，段玉兰，杨小军. 鸡原代肝细胞培养及叶酸对脂质代谢相关基因表达的影响[J]. 中国农业科学, 2017, 50(21): 4205-4211.

LIU YanLi, DANG YanNa, DUAN YuLan, YANG XiaoJun. Effect of Folic Acid on Lipid Metabolism Associated Gene Expression in Primarily Cultured Chickens Hepatocytes[J]. Scientia Agricultura Sinica, 2017, 50(21): 4205-4211.

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