间接共培养条件下成熟脂肪细胞诱导成骨细胞成脂横向分化的能力

摘要

BACKGROUND:Co-culture technique makes different kinds of ceils cultured in the same system.The trans-differentiation from osteoblasts to adipocytes is usually analyzed under the action of adipogenesis inducers in vitro,but the cellular interactions in vivo are neglected.OBJECTIVE:To investigate the trans-differentiation from osteoblasts to adipocytes in indirect co-culture using Transwell system.METHODS:Mouse preadipocytes 3t3-l1 were induced to adipocytes.There were three groups:group A:mature adipocytes in the lower chamber of Transwell system;group B:mouse osteoblasts MC3T3-E1 in the upper chamber of Transwell system according to a ratio of 1:4 (MC3T3-E1:3t3-l1);group C:mouse osteoblasts MC3T3-E1 alone.At 7,14,and 21 days the cell morphology was observed under inverted phase contrast microscope,the relative level of triglyceride,expression of peroxisome proliferator-activated recoptor y2 in each group were detected,and red oil O staining and alizarin red staining were performed.The cell proliferation inhibition rate in the groups B and C were detected at 0,24 and 36 hours.RESULTS AND CONCLUSION:At 2 weeks after culture,spindle-shaped 3t3-l1 coils changed into round,the light and round lipid droplets in the cytoplasm were increased and were reserved after identified by oil red O staining.In the group B,the cells presented with spindle shape with no transparent lipid droplets after 7-day co-culture until black granules and small round lipid droplets appeared on day 14;and the cells changed from spindle shape to oval or round,and larger lipid droplets were found on day 21.Alizarin red staining results:the staining region in the group B was on a decline with time,while the group C showed no significant changes at each time point and all appeared with large staining region.Oil red O staining results:the staining region in the group B increased gradually in a time-dependant manner,while the group C was negative for oil red O staining and showed no significant changes at different time points.The relative level of triacylglycerol in the group B was increased with time,and there was significant difference between groups A and B (P ＜0.05),and the group C showed no significant differences (P ＞ 0.05).The coll proliferation inhibiting rate in the group B was increased with time,which showed significant difference from the group C (P ＜ 0.05).The expression level of peroxisome proliferator-activated recoptor y2 in the group B was on a rise with time,which had significant difference compared with the groups A and C (P ＜ 0.05).These results indicate that the trans-differentiation from osteoblasts to adipocytes appears in the Transwell system,and metabolic products and cytokines of adipocytes obviously inhibit the proliferation of osteoblasts,but all above conclusions need to be studied in depth.%背景:细胞共培养技术是在体外条件下将细胞与另一种细胞共同培养.对于成骨细胞和脂肪细胞横向分化研究多采用体外条件下成脂诱导液诱导成骨细胞发生横向分化,而忽略了体内环境中细胞互相作用的因素.目的:探讨在Transwell间接共培养条件下成熟脂肪细胞诱导成骨细胞成脂横向分化的能力.方法:①选取小鼠成纤维样3t3-l1前体脂肪细胞进行成脂诱导为成熟脂肪细胞;②实验分组:A组接种成熟脂肪细胞(Transwell间接共培养体系下室),B组接种小鼠成骨样细胞mc3t3-e1(以1:4比例接种到Transwell间接共培养体系上室),C组单独培养的小鼠成骨样细胞mc3t3-e1;③检测指标:分别在7,14,21 d倒置相差显微镜下观察细胞形态变化,检测各组细胞三酰甘油相对含量、成脂目的蛋白PPAR γ 2表达,并进行油红O染色以及茜素红染色观察;以0,24,36 h为截点检测B组与C组细胞增殖抑制率的变化.结果与结论:①培养2周时,3t3-l1细胞由长梭形变为圆形,胞质内脂滴增多呈亮圆形,油红O染色鉴定后备用;②细胞形态:B组共培养7d时呈长梭形,未见透明脂滴:14d时胞质内出现黑色颗粒及小圆形脂滴;21 d细胞形态由长梭形变为椭圆形或圆形,脂滴变大;③茜素红染色:7,14,21 d时B组细胞呈染色区减少的趋势,C组细胞无明显变化均呈大片着色区;④油红O染色:7,14,21 d时,B组细胞染色区呈递增趋势且与时间成正比,C组细胞染色阴性且无明显变化;⑤三酰甘油:6组细胞三酰甘油聚集量呈递增趋势且与时间成正比,B组组间及与A组相比差异有显著性意义(P＜0.05),C组无明显变化(P＞0.05);⑥MTT抑制率:B组细胞增殖抑制率与时间成正比,与C组差异有显著性意义(P＜0.05);⑦Westem blot实验:B组细胞PPARA γ 2蛋白表达量呈递增趋势且与时间成正比,与A、C组相比,差异均有显著性意义(P＜0.05);⑧结果提示,在Transwell间接共培养体系中,成骨细胞在成熟脂肪细胞诱导下呈现出成脂横向分化趋势,其中脂肪细胞代谢产物及细胞因子可明显抑制成骨细胞的增殖能力,其是否可以完全横向分化为脂肪细胞有待进一步研究.