3T3-L1前脂肪细胞的体外培养及胰岛素样生长因子1对其增殖、分化的作用

Effect of insulin-like growth factor-1 on in vitro proliferation and differentiation of 3T3-L1 preadipocytes

背景:前脂肪细胞是一类具有增殖和向脂肪细胞分化潜力的特异化的前体细胞,如果能够找到可以强力促进前脂肪细胞增殖和分化的因子或药物,在脂肪组织移植的同时使用,则有希望提高脂肪组织的移植效果。目的:摸索体外培养经典的3T3-L1前脂肪细胞的最佳培养环境并积累培养经验,观察胰岛素样生长因子1对3T3-L1前脂肪细胞增殖、分化的影响,并探求胰岛素样生长因子1的最佳作用剂量。设计、时间及地点:以细胞为对象的对照观察实验,于2007-10/2008-02在辽宁医学院科学实验中心完成。材料:3T3-L1前脂肪细胞株购于上海中科院生命科学院;胰岛素样生长因子1购于美国Biological公司。方法:根据3T3-L1前脂肪细胞的体外培养条件分为6组:空白对照组应用含体积分数为0.1的胎牛血清的标准高糖DMEM培养基培养;各实验组分别应用含有5,10,20,50,100μg/L胰岛素样生长因子1的含体积分数为0.1的胎牛血清的高糖DMEM培养基培养。主要观察指标:倒置显微镜下观察各组3T3-L1前脂肪细胞的生长、增殖及分化情况并拍照记录。待细胞铺满瓶底达到单层汇合时,应用流式细胞仪检测细胞周期,应用四甲基偶氮唑盐比色法测定3T3-L1前脂肪细胞的增殖率;应用油红O染色提取法测定3T3-L1前脂肪细胞内脂肪含量的增殖率。结果:①倒置显微镜下观察分化前3T3-L1前脂肪细胞呈梭形,胞浆内无脂滴,分化后3T3-L1前脂细胞呈圆形,胞浆内含有大量的脂滴,脂滴聚集于核周,被油红O染成橙红色,形成"戒环"样形态。②四甲基偶氮唑盐比色法和油红O染色提取法检测结果均显示,不同剂量的胰岛素样生长因子1组中3T3-L1前脂肪细胞的增殖率及细胞内脂肪含量的增殖率均高于空白对照组(P<0.05),胰岛素样生长因子120μg/L组对3T3-L1前脂肪细胞的增殖、分化作用最为明显(P<0.05);流式细胞仪的分析结果与四甲基偶氮唑盐比色法及油红O染色提取法的检测结果基本一致。结论:体外细胞培养实验表明,胰岛素样生长因子1对3T3-L1前脂肪细胞的增殖、分化有明显促进作用,其促进作用与浓度并非成正比,而是达到一定剂量后,其促进作用不再增加,而这个剂量接近本实验的最佳剂量值,即20μg/L。

BACKGROUND： Preadipocyte is a kind of specific precursor cells with the potential of proliferation and differentiation into adipocytes. If factor or drug with strong promotion of preadipocyte proliferation and differentiation is obtained and used in adipose tissue transplantation, which could elevate transplantation outcome of adipose tissues. OBJECTIVE： To find an optimal culture environment and to accumulate culture experience of in vitro culture 3T3-L1 preadipocytes, and to observe the effect of insulin-like growth factor （IGF）- 1 on the proliferation and differentiation of 3T3-L1 preadipocytes, and to explore the optimal concentration of IGF- 1. DESIGN, TIME AND SETTING： The cell control observation was performed at the Science Experiment Center, Liaoning University of Traditional Chinese Medicine from October 2007 to February 2008. MATERIALS： 3T3-L1 preadipocytes were purchased from Shanghai Institutes For Biological Sciences. IGF-1 was bought from Biological, USA. METHODS： According to in vitro culture direction of 3T3-L1 preadipocytes, 3T3-L1 preadipocytes were divided into 6 groups. 3T3-L1 preadipocytes in the blank control group were inoculated in high-glucose DMEM supplemented with 0.1 volume fraction of fetal bovine serum. 3T3-L1 preadipocytes in each experimental groups were respectively treated with high-glucose DMEM containing 5, 10, 20, 50, 100 u g/L IGF-1 and 0.1 volume fraction of fetal bovine serum. MAIN OUTCOME MEASURES： Growth, proliferation and differentiation of 3T3-L 1 preadipocytes in each experimental groups under the inverted microscope, and photographed. When cells were confluence, cell cycles were detected by flow cytometry. Growth rate of 3T3-L1 preadipocytes was measured by MTT essay. Fat growth rate in 3T3-L1 preadipocytes were examined by using oil red O stained-extracted assay. RESULTS： Inverted microscope showed that 3T3-L1 preadipocytes before differentiation were fusiform shape, with little fat in their endochylemas. 3T3-L1 preadipocytes after differentiation were spherical shape, with much fat in their endochylemas. Lipid droplet gathered in the perinuclear space, and was stained orange by oil red O stained-extracted assay, forming ring-shape. Results from MTT essay and oil redo stained-extracted assay demonstrated that growth rate of 3T3-L1 preadipocytes and fat growth rate in 3T3-L1 preadipocytes were higher in each experimental groups treated with different doses of IGF-1 compared with the blank control group （P 〈 0.05）. 20 u g/L IGF-1 had a significant effect on proliferation and differentiation of 3T3-L1 preadipocytes （P 〈 0.05）. Results from flow cytometry analysis were mostly coincided with that of MTT essay and oil red O stained-extracted assay. CONCLUSION： IGF- Ⅰ has conspicuous effect on promoting proliferation and differentiation of 3T3-L1 preadipocytes. The effect is not positively associated with concentration, but till a certain concentration, the effect does not increase, which can be the optimal concentration, that is nearly 20 u g/L.