3种方法诱导猪去分化脂肪细胞成肌分化效果的比较

doi:10.16098/j.issn.0529-1356.2018.03.009

摘要/Abstract

摘要：

目的采用3种方法比较诱导猪去分化脂肪细胞（DA）成肌分化的效果，旨在筛选诱导猪DA成肌分化的较优方法。方法采用糖皮质激素、半乳糖凝集素-1（galectin-1）法、5-氮胞苷（5-aza）分别诱导猪DA成肌分化，于培养6 d、15 d和21 d后，并分别用倒置显微镜观察分化的细胞形态；诱导21 d后用间接免疫荧光法检测成肌特异蛋白结蛋白（desmin）和肌球蛋白重链（MyHC）的表达，用Real-time PCR检测肌细胞生成素（MyoG） mRNA的表达,每种方法诱导3组，每组重复检测3次。结果诱导细胞的形态学观察显示，galectin-1法诱导的肌管形态最佳，多核细胞数量也最多；5-aza法次之，但诱导过程中细胞死亡率较高；糖皮质激素法诱导的细胞形态不规则，多形成放射状突起，而且出现较多成脂分化细胞。间接免疫荧光分析显示，galectin-1法和糖皮质激素法诱导的细胞Desmin、MyHC和MyoG mRNA都呈强阳性表达，而在5-aza法诱导的细胞这两种蛋白和基因都呈弱表达。结论从诱导细胞的肌管形态，分化的纯度，生存能力，特异蛋白和基因的表达量等综合判断，诱导猪DA成肌分化，galectin-1法优于糖皮质激素法和5-aza法。

关键词: 去分化脂肪细胞, 成肌转分化, 糖皮质激素法, 半乳糖凝集素-1, 5-氮胞苷, 免疫荧光法, 猪

Abstract:

Objective To compare the three methods on the effect of myogenic differentiation of porcine dedifferentiated adipocytes (DA), in order to screen the better method for inducing myogenic differentiation of porcine DA. Methods The glucocorticoid, galectin-1 and 5-azacytidine(5-aza) were used to induce myogenic differentiation of porcine DA for 6 days, 15 days and 21 days respectively, and then the morphology of cell differentiation was observed by inverted microscope respectively. After 21 days of induction, the expression of muscle specific protein which desmin and myosin heavy chain(MyHC) was detected by indirect immunofluorescence, and the expression of myogenin(MyoG) mRNA was detected by Real-time PCR. Each method was used for detecting induction effect in 3 groups and repeat three times in each group. Results The morphological observation of induced cell showed that the best myotube morphology and the largest number of multinucleated cells was found via galectin-1 induced, followed by 5-aza, however the higher percent of cell death was occurred during 5-aza induction. The irregular cell morphology, multiform radial protrusion, and some cells of adipogenic differentiation were observed by glucocorticoid induction. The indirect immunofluorescence showed that the strong positive of Desmin and MyHC as well as a high expression of MyoG mRNA was observed in induced cells via galectin-1 method and glucocorticoid method, while the weak positive expression of these two kinds of protein with a low MyoG mRNA level was found in induced cells by 5-aza induction. Conclusion Judged from myotube morphology, differentiation of purity and viability, specific protein and gene expression, we suggest that galectin-1 method is superior to the glucocorticoid and 5-aza method for inducing myogenic differentiation of porcine DA.

Key words: Dedifferentiated adipocyte, Myogenic differentiation, Glucocorticoid, Galectin-1, 5-azacytidine, Immunofluorescence, Porcine

胡晓丽隋丽虹李方正郇延军姜忠玲蒋南宋学雄. 3种方法诱导猪去分化脂肪细胞成肌分化效果的比较[J]. 解剖学报, 2018, 49(3): 324-329.

HU Xiao-li SUI Li-hong LI Fang-zheng HUAN Yan-jun JIANG Zhong-ling JIANG Nan SONG Xue-xiong. Comparaison of three methods on the effect of myogenic differentiation of porcine dedifferentiated adipocytes[J]. Acta Anatomica Sinica, 2018, 49(3): 324-329.

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