山羊Kruppel样转录因子家族在前体脂肪细胞分化中的表达模式及相关性分析

【目的】 优化山羊前体脂肪细胞体外培养体系,揭示Kruppel样转录因子家族（KLFs）成员在前体脂肪细胞分化过程中的表达模式。 【方法】 于四川省简阳大哥大牧业有限公司随机选取3只7日龄简州大耳羊为研究对象。放血致死并清洗后,在无菌环境中取其皮下脂肪组织。利用Ⅰ型胶原酶消化法获得其皮下前体脂肪细胞,待细胞长至细胞密度为80%时传代至6孔培养板中（F₃代）,以“150 μmol·L ^-1油酸+10 mg·L ^-1胰岛素”前体脂肪细胞进行成脂诱导,并在诱导分化后0 d、3 d、5 d、7 d收集细胞,Trizol法提取细胞总RNA,利用实时荧光定量PCR（RT-qPCR）检测KLF2-10, KLF 12和KLF15共11个KLF转录因子家族成员在不同分化阶段细胞中的表达水平,并利用皮尔逊系数分析各个成员mRNA总体表达水平之间的相关性。 【结果】 150 μmol·L ^-1油酸+10 mg·L ^-1胰岛素处理前体脂肪细胞24 h后细胞中开始出现小脂滴,48 h后脂滴数量增多、体积增大,诱导120 h后脂肪细胞分化程度显著增加。RT-qPCR结果显示,脂肪分化标志基因过氧化物酶体增殖物活化受体γ（PPARγ）基因随着诱导分化的进行其表达水平持续上升,其中在第7天时其表达水平极显著高于其他各组（P＜0.01）。KLF2、KLF3、KLF4、KLF6、KLF7和KLF15在脂肪细胞中表达水平显著（P＜0.05）或极显著（P＜0.01）高于前体脂肪细胞中表达水平,其中KLF3、KLF6和KLF15在分化5 d时表达水平最高,KLF2和KLF4表达水平在7 d时最高,而KLF7则在3 d时表达水平最高;KLF5、KLF8、KLF9、KLF10和KLF12在前体脂肪细胞中的表达水平极显著高于分化后脂肪细胞中的表达水平（P＜0.01）,其中KLF8、KLF10和KLF12的表达水平均在5 d时达到最低,而KLF5和KLF9则在7 d时表达水平最低;相关性分析结果显示KLF家族各成员mRNA在山羊前体脂肪细胞分化过程中总体表达水平存在相关性,KLF12与6个基因具有较强的相关性,其次为KLF4和KLF9,分别与5个成员具有较强的相关性,而KLF2与其他基因均没有较强的相关性。 【结论】 明确了KLF家族在山羊前体脂肪细胞分化过程中的表达模式,解析了各个成员mRNA表达之间的相关性,为阐明山羊前体脂肪细胞分化的分子机制提供重要的基础数据。

Expression Profile and Correlations of Kruppel Like Factors During Caprine (Capra Hircus) Preadipocyte Differentiation

【Objective】 The aim of this study was to optimize the culture system of goat preadipocytes in vitro and elucidate the expression patterns of Kruppel like factors (KLFs) during preadipocytes differentiation. 【Method】 Three 7-day-old Jianzhou Big-Eared goats were selected randomly from Sichuan Jianyang DAGEDA Animal Husbandry Co. Ltd. as experimental animal. After bloodletting death of goats, their subcutaneous fat tissues were collected in a sterile room. The goat subcutaneous preadipocytes were collected using collagenase I digestion method. The cells at Generation Three were then passaged to a 6-well plate when the cells were grown to 80% density. The combined use of were used for preadipocytes differentiate inducing. The cells were then collected after cultivation for 0 d, 3 d, 5 d and 7 d. The total RNA was extracted by using Trizol method. Real-time fluorescent quantitative PCR (RT-qPCR) was performed to determine the expression levels of 11 members of KLFs, including KLF2-10, KLF12 and KLF15, during goat preadipocytes differentiation. Pearson correlation coefficient was used for expression correlation analysis between KLF individuals. 【Result】 The results showed that the combined use of 150 μmol·L ^-1oleic acid and 10 mg·L ^-1 insulin promoted the formation of small lipid droplets after 24 h cultivation. After 48 h, the lipid droplets became bigger and much more. The differentiation of the preadipocytes were enhanced significantly after 120 h comparing with preadipocytes at 0 h. The results of RT-qPCR showed that the combined use of oleic acid and insulin stimulated the expression of peroxisome proliferator-activated receptor-gamma (PPARγ) continuously companying the differentiation of preadipocytes, and toped at 7 d which significantly higher than other groups (P＜0.01). The expression levels of KLF2, KLF3, KLF4, KLF6, KLF7 and KLF15 in adipocytes were significantly (P＜0.05) or extremely significantly (P＜0.01) higher than those in preadipocytes. While the expression of KLF3, KLF6 and KLF15 were topped at 5 d, the KLF2 and KLF4 toped at 7 d. However, the KLF7 expressed most at 3 d. For the other KLF individuals, the expression levels of KLF5, KLF8, KLF9, KLF10 and KLF12 in preadipocytes were extremely significantly higher (P＜0.01) than those in adipocytes. The expression of KLF8, KLF10 and KLF12 bottomed at 5 d, while that of the KLF5 and KLF9 bottomed at 7 d. Correlation analysis showed that KLF individual significantly (P＜0.05) correlated with each other across cell differentiation. KLF12 was strongly correlated with 6 individuals, followed by KLF4 and KLF9 both of which were significantly correlated with 5 individuals. However, KLF2 was not significantly correlated with other individuals. 【Conclusion】 In this study, the expression patterns of KLFs during goat preadipocytes differentiation were clarified, and also the correlations among KLF individuals. These results provided important basic data for elucidating the molecular mechanism of goat preadipocytes differentiation.