Björntorp P, Karlsson M, Pertoft H, Pettersson P, Sjöström L, Smith U
J Lipid Res. 1978 Mar;19(3):316-24.
To identify cells developing into adipocytes by accumulation of triglyceride, rat epididymal fat pad cells from small rats were exposed to (3)H-labeled chylomicron fatty acids in vivo and then liberated with collagenase. Tissue remnants were removed by filtration and mature fat cells by flotation. Aggregating cells were then removed by filtration through a 25- micro m nylon screen. Further purification of cells labeled in vivo was obtained by removing floating cells from those adhering to the bottom of a culture dish. The adhering cells multiplied to a confluent monolayer when cultured in Medium 199 containing serum, glucose, insulin, and a triglyceride emulsion. The cells then gradually enlarged due to granulation of the cytoplasm by a lipid-staining material. After about 2 weeks these granules had coalesced forming mature adipocytes of typical signet-ring appearance. Free adipocytes could then be recovered from the cultures by collagenase treatment. After about 2 weeks of culture these cells had the same size (about 30 micro m) as adipocytes recovered in the original collagenase preparation of the rat epididymal fat pad. They contained triglyceride lipase activity and incorporated glucose into triglycerides to the same extent as cells developed in vivo but had higher lipoprotein lipase activity. In vitro, heparin in a low concentration, prostaglandin E(1), isobutylmethylxanthine, and cholera toxin markedly promoted the development of these cells into adipocytes. This could be shown to occur almost completely indicating that this fraction of cells was homogeneous and consisted of cells with the capacity to form adipocytes. The duplication time was about 2 days and did not change with subculturing. Preadipocytes could be obtained by density gradient centrifugation, isolating triglyceride-containing cells either directly from the pad or after 3 days in culture. All of these cells developed into adipocytes as described above but did not multiply as readily. It was concluded that cells from the epididymal fat pad from small rats can be isolated in a homogenous fraction that develops in culture into cells of identical morphology and function as adipocytes formed in vivo. The differentiation of these cells into adipocytes may be manipulated in vitro.
为了通过甘油三酯的积累来鉴定发育为脂肪细胞的细胞，将来自幼鼠的大鼠附睾脂肪垫细胞在体内暴露于³H标记的乳糜微粒脂肪酸，然后用胶原酶释放出来。通过过滤去除组织残余物，通过浮选去除成熟脂肪细胞。然后通过25微米尼龙筛过滤去除聚集细胞。通过从粘附在培养皿底部的细胞中去除漂浮细胞，进一步纯化体内标记的细胞。当在含有血清、葡萄糖、胰岛素和甘油三酯乳液的199培养基中培养时，粘附细胞增殖形成汇合的单层。然后，由于细胞质被脂质染色物质颗粒化，细胞逐渐增大。大约2周后，这些颗粒融合形成典型印戒外观的成熟脂肪细胞。然后可以通过胶原酶处理从培养物中回收游离脂肪细胞。培养约2周后，这些细胞的大小（约30微米）与在大鼠附睾脂肪垫原始胶原酶制剂中回收的脂肪细胞相同。它们含有甘油三酯脂肪酶活性，并且将葡萄糖掺入甘油三酯的程度与体内发育的细胞相同，但具有更高的脂蛋白脂肪酶活性。在体外，低浓度的肝素、前列腺素E₁、异丁基甲基黄嘌呤和霍乱毒素显著促进这些细胞向脂肪细胞的发育。这几乎可以完全证明发生了这种情况，表明这部分细胞是同质的，并且由具有形成脂肪细胞能力的细胞组成。倍增时间约为2天，传代培养时不变。前脂肪细胞可以通过密度梯度离心获得，直接从脂肪垫或培养3天后分离含甘油三酯的细胞。所有这些细胞都如上所述发育为脂肪细胞，但增殖不那么容易。得出的结论是，来自幼鼠附睾脂肪垫的细胞可以分离成同质部分，在培养中发育成与体内形成的脂肪细胞形态和功能相同的细胞。这些细胞向脂肪细胞的分化可以在体外进行调控。
