新西兰兔肌内和皮下前体脂肪细胞原代培养与诱导分化的研究
|
摘要
为建立新西兰兔前体脂肪细胞的体外培养模型,比较新西兰兔肌内和皮下前体脂肪细胞分化过程中相关基因的差异表达,实验采集1日龄新西兰兔背最长肌和皮下脂肪组织,采用胶原酶消化方法,分别从2种组织中分离前体脂肪细胞,进行细胞原代培养,绘制细胞生长曲线,并对肌内和皮下前体脂肪细胞诱导分化,利用RT-PCR检测相关基因的表达变化。结果表明:细胞在分离后2 h已经贴壁,24 h时呈现出短梭形的细胞形态,第2天细胞变成长梭形,第3天进入对数生长期。肌内和皮下前体脂肪细胞在诱导分化后均被油红O染色,皮下前体脂肪细胞的脂质积累在诱导分化的第2天显著高于肌内前体脂肪细胞(P<0.05);荧光定量结果表明,肌内和皮下前体脂肪细胞CCAAT增强子结合蛋白(C/EBPα)基因的表达趋势在诱导分化过程中相同,肌内前体脂肪细胞过氧化物酶体增殖激活受体(PPARγ)第4天的表达量显著高于第6天,而皮下前体脂肪细胞PPARγ表达量差异不显著;肌内前体脂肪细胞脂蛋白脂肪酶(LPL)表达量在第0天和第2天差异不显著,而皮下前体脂肪细胞第2天显著高于第0天(P<0.05);肌内前体脂肪细胞脂肪酸合酶(FAS)基因第0、2、4天的表达量差异不显著,而皮下前体脂肪细胞第2、4天显著高于第0天(P<0.05)。本研究成功构建了新西兰兔肌内和皮下前体脂肪细胞的体外培养和诱导分化模型,并发现皮下前体脂肪细胞分化早于肌内前体脂肪细胞,为进一步研究新西兰兔前体脂肪细胞的分化机制和脂肪沉积奠定基础。
In order to establish the culture model of preadipocytes in vitro of New Zealand rabbits, and to compare the differential expression of the differentiation related genes of intramuscular and subcutaneous preadipocytes. The longissimus dorsi muscle and subcutaneous adipose tissues of the 1-day-old New Zealand rabbits were collected, and the preadipocytes were isolated respectively from these two tissues through the digestion of collagenase, and the cells was primarily cultured.Cell growth curve was drawn, and intramuscular and subcutaneous preadipocytes were induced to differentiate. The expression of related genes was detected by RT-PCR. The results showed that the cells had been attached to the bottle wall at 2 h, and the cells showed a short shuttle shaped cell morphology at 24 h. The cells turned into a long spindle shape on the 2nd day, and the cells entered the logarithmic growth period of 3rd days. After induction of differentiation, both intramuscular and subcutaneous adipocytes can be stained with oil red O. Lipid accumulation in subcutaneous preadipocytes was significantly higher(P<0.05) than that in intramuscular preadipocytes on the 2nd day. The RT-PCR results showed that the expression trend of CEBPα gene in intramuscular and subcutaneous preadipocytes was the same in the induction of differentiation. The expression of PPARγ in intramuscular preadipocytes on the 4th day was significantly higher than that on the 6 th day(P<0.05),but the expression of PPARγ in subcutaneous preadipocytes was not significantly different. LPL expression in intramuscular preadipocytes was not significantly different on day 0 and 2, but significantly higher on 2th day than on d 0(P<0.05) in subcutaneous preadipocytes. The expression of FAS gene in intramuscular preadipocytes was not significantly different on d 0,2 and 4, but that in subcutaneous preadipocytes on d 2 and 4 was significantly higher than that on d 0(P<0.05). In this study,we successfully constructed the in vitro culture and differentiation models of intramuscular and subcutaneous preadipocytes from New Zealand rabbits, and found that subcutaneous preadipocytes differentiated earlier than intramuscular preadipocytes.This study laid the basis for further researching New Zealand rabbit adipocyte differentiation and fat deposition.
In order to establish the culture model of preadipocytes in vitro of New Zealand rabbits, and to compare the differential expression of the differentiation related genes of intramuscular and subcutaneous preadipocytes. The longissimus dorsi muscle and subcutaneous adipose tissues of the 1-day-old New Zealand rabbits were collected, and the preadipocytes were isolated respectively from these two tissues through the digestion of collagenase, and the cells was primarily cultured.Cell growth curve was drawn, and intramuscular and subcutaneous preadipocytes were induced to differentiate. The expression of related genes was detected by RT-PCR. The results showed that the cells had been attached to the bottle wall at 2 h, and the cells showed a short shuttle shaped cell morphology at 24 h. The cells turned into a long spindle shape on the 2nd day, and the cells entered the logarithmic growth period of 3rd days. After induction of differentiation, both intramuscular and subcutaneous adipocytes can be stained with oil red O. Lipid accumulation in subcutaneous preadipocytes was significantly higher(P<0.05) than that in intramuscular preadipocytes on the 2nd day. The RT-PCR results showed that the expression trend of CEBPα gene in intramuscular and subcutaneous preadipocytes was the same in the induction of differentiation. The expression of PPARγ in intramuscular preadipocytes on the 4th day was significantly higher than that on the 6 th day(P<0.05),but the expression of PPARγ in subcutaneous preadipocytes was not significantly different. LPL expression in intramuscular preadipocytes was not significantly different on day 0 and 2, but significantly higher on 2th day than on d 0(P<0.05) in subcutaneous preadipocytes. The expression of FAS gene in intramuscular preadipocytes was not significantly different on d 0,2 and 4, but that in subcutaneous preadipocytes on d 2 and 4 was significantly higher than that on d 0(P<0.05). In this study,we successfully constructed the in vitro culture and differentiation models of intramuscular and subcutaneous preadipocytes from New Zealand rabbits, and found that subcutaneous preadipocytes differentiated earlier than intramuscular preadipocytes.This study laid the basis for further researching New Zealand rabbit adipocyte differentiation and fat deposition.
引文
[1]Fox K E,Fankell D M,Erickson P F,et al.Depletion of cAMP-response element-binding protein/ATF1 inhibits adipogenic conversion of 3T3-L1 cells ectopically expressing CCAAT/enhancer-binding protein(C/EBP)alpha,C/EBP beta,or PPARgamma 2[J].J Biol Chem,2006,281(52):40341-40353.
[2]Kawai M,Mushiake S,Bessho K,et al.Wnt/Lrp/β-catenin signaling suppresses adipogenesis by inhibiting mutual activation of PPARγand C/EBPα[J].Biochem Bioph Res Co,2007,363(2):276-282.
[3]Liu H,Wang J,Liu M,et al.Antiobesity Effects of Ginsenoside Rg1 on 3T3-L1 Preadipocytes and High Fat Diet-Induced Obese Mice Mediated by AMPK[J].Nutrients,2018,10(7):830-844.
[4]胡艳霞,曾勇庆,崔志峰,等.莱芜猪前体脂肪细胞的原代培养及诱导分化研究[J].畜牧兽医学报,2012,43(9):1346-1352.
[5]Ma X,Hou Y Q,Dahanayaka S,et al.Technical note:Isolation and characterization of ovine brown adipocyte precursor cells[J].J Anim Sci,2015,93(5):2094.
[6]Ma X,Ding W,Wang J,et al.LOC66273 isoform 2,a novel protein highly expressed in white adipose tissue,induces adipogenesis in 3T3-L1 cells[J].J Nutr,2012,142(3):448.
[7]孙成娟,许厚强,赵佳福,等.从江香猪肌内和皮下脂肪前体细胞分化过程中相关基因的表达[J].农业生物技术学报,2017(12):1979-1988.
[8]Ma X,Han M,Li D,et al.L-Arginine promotes protein synthesis and cell growth in brown adipocyte precursor cells via the mTOR signal pathway[J].Amino Acids,2017,49(5):1-8.
[9]Xie L,Yin A,Nichenko A S,et al.Transient HIF2A inhibition promotes satellite cell proliferation and muscle regeneration[J].J Clin Invest,2018,128(6):2339-2355.
[10]Miller M.Interactions of CCAAT/enhancer-binding proteinβwith transcriptional coregulators[J].Postepy Biochemii,2016,62(3):343.
[11]Noh J R,Kim Y H,Hwang J H,et al.Scoparone inhibits adipocyte differentiation through down-regulation of peroxisome proliferators-activated receptorγin 3T3-L1 preadipocytes[J].Food Chem,2013,141(2):723-730.
[12]Ming G F,Li X,Yin J Y,et al.JAZF1 regulates visfatin expression in adipocytes via PPARαand PPARβ/δsignaling[J].Metabolism,2014,63(8):1012-1021.
[13]李优磊.PPAR信号通路在调控猪皮下脂肪与肌内脂肪差异沉积中的作用及机制研究[D].杨凌:西北农林科技大学,2018.
[14]Kim J W,Monila H,Pandey A,et al.Upstream stimulatory factors regulate the C/EBPαgene during differentiation of 3T3-L1 preadipocytes[J].Biochem Bioph Res Co,2007,354(2):517-521.
[15]Ghosh C,Yang S H,Kim J G,et al.Zinc-chelated Vitamin CStimulates Adipogenesis of 3T3-L1 Cells[J].Asian Austral JAnim,2013,26(8):1189-1196.
[16]徐敏,许厚强,陈伟,等.从江香猪肌内前体脂肪细胞分化过程中相关基因的表达研究[J].中国畜牧兽医,2018,45(9):2492-2499.
[17]刘作华,陈代文,龙定彪,等.日粮能量水平对猪脂肪酸合成酶和激素敏感酯酶的影响[J].中国畜牧杂志,2009,45(5):31-33.
[18]颜新春,汪以真,许梓荣.动物脂肪酸合成酶(FAS)基因表达的调控[J].动物营养学报,2002,14(2):1-4.
[2]Kawai M,Mushiake S,Bessho K,et al.Wnt/Lrp/β-catenin signaling suppresses adipogenesis by inhibiting mutual activation of PPARγand C/EBPα[J].Biochem Bioph Res Co,2007,363(2):276-282.
[3]Liu H,Wang J,Liu M,et al.Antiobesity Effects of Ginsenoside Rg1 on 3T3-L1 Preadipocytes and High Fat Diet-Induced Obese Mice Mediated by AMPK[J].Nutrients,2018,10(7):830-844.
[4]胡艳霞,曾勇庆,崔志峰,等.莱芜猪前体脂肪细胞的原代培养及诱导分化研究[J].畜牧兽医学报,2012,43(9):1346-1352.
[5]Ma X,Hou Y Q,Dahanayaka S,et al.Technical note:Isolation and characterization of ovine brown adipocyte precursor cells[J].J Anim Sci,2015,93(5):2094.
[6]Ma X,Ding W,Wang J,et al.LOC66273 isoform 2,a novel protein highly expressed in white adipose tissue,induces adipogenesis in 3T3-L1 cells[J].J Nutr,2012,142(3):448.
[7]孙成娟,许厚强,赵佳福,等.从江香猪肌内和皮下脂肪前体细胞分化过程中相关基因的表达[J].农业生物技术学报,2017(12):1979-1988.
[8]Ma X,Han M,Li D,et al.L-Arginine promotes protein synthesis and cell growth in brown adipocyte precursor cells via the mTOR signal pathway[J].Amino Acids,2017,49(5):1-8.
[9]Xie L,Yin A,Nichenko A S,et al.Transient HIF2A inhibition promotes satellite cell proliferation and muscle regeneration[J].J Clin Invest,2018,128(6):2339-2355.
[10]Miller M.Interactions of CCAAT/enhancer-binding proteinβwith transcriptional coregulators[J].Postepy Biochemii,2016,62(3):343.
[11]Noh J R,Kim Y H,Hwang J H,et al.Scoparone inhibits adipocyte differentiation through down-regulation of peroxisome proliferators-activated receptorγin 3T3-L1 preadipocytes[J].Food Chem,2013,141(2):723-730.
[12]Ming G F,Li X,Yin J Y,et al.JAZF1 regulates visfatin expression in adipocytes via PPARαand PPARβ/δsignaling[J].Metabolism,2014,63(8):1012-1021.
[13]李优磊.PPAR信号通路在调控猪皮下脂肪与肌内脂肪差异沉积中的作用及机制研究[D].杨凌:西北农林科技大学,2018.
[14]Kim J W,Monila H,Pandey A,et al.Upstream stimulatory factors regulate the C/EBPαgene during differentiation of 3T3-L1 preadipocytes[J].Biochem Bioph Res Co,2007,354(2):517-521.
[15]Ghosh C,Yang S H,Kim J G,et al.Zinc-chelated Vitamin CStimulates Adipogenesis of 3T3-L1 Cells[J].Asian Austral JAnim,2013,26(8):1189-1196.
[16]徐敏,许厚强,陈伟,等.从江香猪肌内前体脂肪细胞分化过程中相关基因的表达研究[J].中国畜牧兽医,2018,45(9):2492-2499.
[17]刘作华,陈代文,龙定彪,等.日粮能量水平对猪脂肪酸合成酶和激素敏感酯酶的影响[J].中国畜牧杂志,2009,45(5):31-33.
[18]颜新春,汪以真,许梓荣.动物脂肪酸合成酶(FAS)基因表达的调控[J].动物营养学报,2002,14(2):1-4.