Trans-differentiation from osteoblasts to adipocytes in indirect co-culture

doi:10.3969/j.issn.2095-4344.2017.28.001

Abstract

Abstract:

BACKGROUND: Co-culture technique makes different kinds of cells 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 receptor γ2 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 cells 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 cell 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 receptor γ2 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.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Coculture Techniques, Adipocytes, Osteoblasts, Tissue Engineering

CLC Number:

R318

Qi Xin-wen, An Rong-ze, Li Yi-fei, Yuan Xiao-hong, Chen Jun-ping, Tan Wei-yuan, Ye Yan-bin. Trans-differentiation from osteoblasts to adipocytes in indirect co-culture[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(28): 4429-4435.

Figures/Tables 2

2.1 3t3-l1前体脂肪细胞的成脂诱导成脂诱导2 d后，前体脂肪细胞变得更为细长，圆形，多角形的细胞消失；成脂诱导4 d时前体脂肪细胞胞质内开始出现小的脂滴，呈亮圆形，多少大小不一，胞核被推向一侧，细胞形态仍呈长梭形(见图3A)；诱导6 d，细胞胞质内的脂滴数目增加，脂滴出现融合，约30%细胞变为圆形(见图3B)；诱导培养8 d后，脂滴融合现象更加明显，并且超过细胞本身的体积，80%的前体脂肪细胞胞质内出现脂滴(见图3C)；成脂诱导第14天时，出现变化的细胞数目未见明显增加(可能与细胞状态及老化有关)，脂滴体积进一步增加，形成大的脂滴空泡(见图3D)，细胞变为圆形或者类圆形，呈典型成熟脂肪细胞的形态和特征。 2.2 细胞油红O脂肪染色鉴定倒置相差显微镜下，小鼠3t3-l1前体脂肪细胞成脂诱导10 d后油红O染色结果显示，90%以上胞质红染(见图4)，可鉴定小鼠3t3-l1细胞分化为成熟脂肪细胞。 Transwell共培养B组、A组及C组油红O染色结果显示：①B组接种于Transwell上室的小鼠MC3T3-E1成骨样细胞呈成纤维样或三角形；共培养7 d后于倒置相差显微镜下见细胞形态开始变化为圆形，但胞质内未见明显脂滴，油红O染色未见明显红染(见图5a)；共培养14 d后，胞质内开始出现少许脂滴，偶见脂滴融合，油红O染色少量细胞出现红染；共培养21 d后，细胞变圆且膨胀，胞质内脂滴数量明显增多，仍然有细胞呈梭形及三角形，油红O染色可见细胞大量红染，但无成熟脂肪细胞明显(见图5b)；②C组单独培养的MC3T3-E1小鼠成骨样细胞形态也未见明显改变，细胞数量未见明显减少，行油红O染色未见明显的红染(见图5c)。本镜下图像选取无偏向性，选取图片为多个图片及多个视野的平均中性图片。该处本应该量化比较(即应用灰度值进行量化)，这样更客观及更加容易和科学选取平均值作为结局指标。由于图片阳性阴性对照较明显，固本实验未采取量化比较(茜素红染色试验的比较为量化，原因同上)。 2.3 茜素红染色结果共培养组B组7 d时茜素红染色明显，14 d时染色区开始出现减少，21 d时染色区明显减少；C组采在培养7，14，21 d后采用茜素红染色检测未见染色区明显变化(见图6)。 2.4 Transwell 间接共培养条件下细胞增殖抑制率的检测(MTT法) 采用t 检验对 0 h B、C组间比较，差异无显著性意义(P > 0.05)；24 h时，B组抑制率为(64.917±0.002)%，C组抑制率为(23.333±0.034)%)，差异有显著性意义(P < 0.05)；36 h时，B组抑制率为(71.000±0.004)%，C组抑制率为(56.333±0.003)%，差异有显著性意义(P < 0.05)，见图7。 2.5 三酰甘油相对含量的测定分别取7，14，21 d为截点终止细胞生长，BCA法测量三酰甘油相对含量，B组与C组相比采用t 检验，14，21 d两组细胞三酰甘油相对含量以及B组间14 d和21 d 的三酰甘油相对含量比较，差异有显著性意义(P < 0.05)；7 d 时B组与C组及C组组间7，14，21 d三酰甘油相对含量比较，差异无显著性意义(P > 0.05)，见图8。B组细胞14 d左右出现形态变化，胞质内出现透明圆形脂滴，21 d时细胞大部分由长梭形变为圆形、椭圆形，胞质内脂滴变大变亮。"

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