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

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。

Comparative Study on Primary Culture and Induced Differentiation of Intramuscular and Subcutaneous Preadipocytes of New Zealand Rabbits

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.