牦牛前体脂肪细胞的分离培养及成脂诱导分化

Isolation and culture of yak precursor adipocyte and induced differentiation by lipogenesis

本文旨在建立牦牛前体脂肪细胞的体外分离及原代培养体系,为牦牛脂肪细胞的相关研究提供试验材料和原代细胞培养模型。以高原型牦牛皮下脂肪组织为材料,通过酶消化法和组织块培养法分离前体脂肪细胞,再使用分化培养基诱导其分化为成熟的脂肪细胞,并采用CCK-8检测细胞增殖期间的生长曲线,实时荧光定量PCR(qRT-PCR)检测DLK-1和PPAR-γ的表达量。结果表明:酶消化法获得的牦牛前体脂肪细胞在24 h后开始贴壁,贴壁细胞形态多样;组织块培养法在第4天才呈现出游离的前体脂肪细胞,且其生长速度较缓慢。前体脂肪细胞的生长曲线整体呈现“S”形。前体脂肪细胞诱导分化后,细胞内部积累大量脂滴,并逐渐分化为成熟的脂肪细胞;相较于原代脂肪细胞,传代后的脂肪细胞生长速度较快。qRT-PCR检测发现,脂肪细胞分化后,DLK-1的表达量显著降低,PPAR-γ的表达量显著升高。综上,无论采用酶消化法还是组织块培养法,均能成功获得牦牛前体脂肪细胞,并可通过诱导使其分化为成熟的脂肪细胞。

The aim of this paper is to establish an in vitro isolation and primary culture system of yak precursor adipocyte, so as to provide test materials and primary cell culture models for yak adipocyte related research.Taking the subcutaneous adipose tissue of high-altitude yak as the materials, precursor adipocytes are isolated by enzymatic digestion and tissue block culture methods, and then are induced to differentiate into mature adipocytes by using differentiation medium.The growth curve during cell proliferation is detected by CCK-8.The expression levels of DLK-1 and PPAR-γ are detected by using real-time fluorescence quantitative PCR(qRT-PCR).The results are as follows: The yak precursor adipocytes obtained by enzyme digestion start to adhere to the wall after 24 h, and the morphology of adherent cells is diverse.The tissue block culture method shows free precursor adipocytes only on the 4th day, and their groeth rate is slow.The growth curve of precursor adipocytes is S-shaped.After the induction of differentiation of precursor adipocytes, adipocytes accumulate a large number of lipid droplets and gradually differentiate into mature ones.Compared with the primary adipocytes, the growth rate of the passaged adipocytes is faster.The detection results of qRT-PCR show that the expression of DLK-1 decrease and PPAR-γ increase significantly after adipocytes differentiation.In conclusion, by using both enzymatic digestion and tissue block culture methods, precursor adipocytes of yak can be obtained and induced to differentiate into mature adipocytes.