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
发布时间：2019-01-21 19:29
【摘要】：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PPARγ2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7 d时呈长梭形,未见透明脂滴;14 d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:B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(P0.05),C组无明显变化(P0.05);(6)MTT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(P0.05);(7)Western blot实验:B组细胞PPARAγ2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(P0.05);(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。
[Abstract]:Background: co-culture of cells is to co-culture cells with another cell in vitro. For the lateral differentiation of osteoblasts and adipocytes, osteoblasts were induced by adipogenic medium in vitro, but the factors of cell interaction in vivo were neglected. Aim: to investigate the ability of adipocytes to induce adipogenic transversal differentiation of osteoblasts in Transwell indirect co culture. Methods: (1) Mouse fibroblast-like 3t3-l1 precursor adipocytes were selected for adipogenic induction to mature adipocytes. (2) experiment group: group A was inoculated with mature adipocytes (), B group was inoculated with mc3t3-e1 of mouse osteoblast in Transwell indirect co-culture system) (mc3t3-e1 was inoculated into the upper chamber of Transwell indirect co-culture system at 1:4). Mc3t3-e1; of osteoblast-like cells isolated in group C (3) Indexes: the morphological changes of the cells were observed under the inverted phase contrast microscope at day 714 and 21, respectively. The relative content of triglyceride and the expression of PPAR 纬 2 in each group were detected, and the oil red O staining and alizarin red staining were carried out. The cell proliferation inhibition rate of group B and group C was measured at 24 h. Results and conclusion: (1) after 2 weeks of culture, 3t3-l1 cells changed from long fusiform to round, and the increase of lipid droplets in the cytoplasm was bright and round. (2) Cell morphology: in group B, the cells were long fusiform at 7 d, no hyaline lipid droplets, black particles and small circular lipid droplets in the cytoplasm at 14 d, and changed from long fusiform to elliptic or round at 21 d, and the lipid droplets became larger. (3) alizarin red staining: group B cells showed a decreasing trend of staining area at day 21, while group C cells showed no obvious changes in large areas of staining; (4) Oil red O staining: the staining area of cells in group B showed an increasing trend and was proportional to the time at day 21, but the staining of cells in group C was negative and had no obvious change. (5) triacylglycerol: the accumulation of triglyceride in group B showed an increasing trend and was in direct proportion to the time, and there was a significant difference between group B and group A (P0.05) in), C group (P0.05); (6) inhibition rate of MTT: the inhibition rate of cell proliferation in group B was in direct proportion to time, and there was significant difference between group B and group C (P0.05). (7) Western blot test: the expression of PPARA 纬 2 protein in group B showed an increasing trend and was in direct proportion to time, and there was significant difference compared with group C (P0.05). (8) the results suggested that in Transwell indirect co-culture system, osteoblasts showed a tendency of adipogenic transversal differentiation induced by mature adipocytes, and adipocyte metabolites and cytokines could significantly inhibit the proliferation of osteoblasts. Whether it can be completely transversely differentiated into adipocytes remains to be further studied.
【作者单位】： 遵义医学院第五附属(珠海)医院骨科二病区;
【分类号】：R318.08;R68
本文编号：2412963
[Abstract]:Background: co-culture of cells is to co-culture cells with another cell in vitro. For the lateral differentiation of osteoblasts and adipocytes, osteoblasts were induced by adipogenic medium in vitro, but the factors of cell interaction in vivo were neglected. Aim: to investigate the ability of adipocytes to induce adipogenic transversal differentiation of osteoblasts in Transwell indirect co culture. Methods: (1) Mouse fibroblast-like 3t3-l1 precursor adipocytes were selected for adipogenic induction to mature adipocytes. (2) experiment group: group A was inoculated with mature adipocytes (), B group was inoculated with mc3t3-e1 of mouse osteoblast in Transwell indirect co-culture system) (mc3t3-e1 was inoculated into the upper chamber of Transwell indirect co-culture system at 1:4). Mc3t3-e1; of osteoblast-like cells isolated in group C (3) Indexes: the morphological changes of the cells were observed under the inverted phase contrast microscope at day 714 and 21, respectively. The relative content of triglyceride and the expression of PPAR 纬 2 in each group were detected, and the oil red O staining and alizarin red staining were carried out. The cell proliferation inhibition rate of group B and group C was measured at 24 h. Results and conclusion: (1) after 2 weeks of culture, 3t3-l1 cells changed from long fusiform to round, and the increase of lipid droplets in the cytoplasm was bright and round. (2) Cell morphology: in group B, the cells were long fusiform at 7 d, no hyaline lipid droplets, black particles and small circular lipid droplets in the cytoplasm at 14 d, and changed from long fusiform to elliptic or round at 21 d, and the lipid droplets became larger. (3) alizarin red staining: group B cells showed a decreasing trend of staining area at day 21, while group C cells showed no obvious changes in large areas of staining; (4) Oil red O staining: the staining area of cells in group B showed an increasing trend and was proportional to the time at day 21, but the staining of cells in group C was negative and had no obvious change. (5) triacylglycerol: the accumulation of triglyceride in group B showed an increasing trend and was in direct proportion to the time, and there was a significant difference between group B and group A (P0.05) in), C group (P0.05); (6) inhibition rate of MTT: the inhibition rate of cell proliferation in group B was in direct proportion to time, and there was significant difference between group B and group C (P0.05). (7) Western blot test: the expression of PPARA 纬 2 protein in group B showed an increasing trend and was in direct proportion to time, and there was significant difference compared with group C (P0.05). (8) the results suggested that in Transwell indirect co-culture system, osteoblasts showed a tendency of adipogenic transversal differentiation induced by mature adipocytes, and adipocyte metabolites and cytokines could significantly inhibit the proliferation of osteoblasts. Whether it can be completely transversely differentiated into adipocytes remains to be further studied.
【作者单位】： 遵义医学院第五附属(珠海)医院骨科二病区;
【分类号】：R318.08;R68
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