Retinoic acid, testosterone or their combination affects the cell cycle of adipose-derived stem cells

doi:10.3969/j.issn.2095-4344.2014.41.022

Abstract

Abstract:

BACKGROUND: The researches about the effect of retinoic acid on the proliferation of adipose-derived stem cells are rare, and the researches on the testosterone are mainly on the inhibition of cell aging.

OBJECTIVE: To study the effects of retinoic acid and testosterone or combination on the cell cycle of adipose derived stem cells.

METHODS: Adipose derived stem cells were isolated from adult female Sprague Dawley rats with 2 months age and cultured in vitro till passage 3 adipose derived stem cells, and then the 3^rd passage adipose-derived stem cells were performed with adipogenic induction, osteogenic induction and surface marker identification. The cells were divided into six groups: (1) Control group; (2) 10^-⁵ mol/L retinoic acid group; (3) Retinoic acid group; (4) 10^-⁵ mol/L retinoic acid+testosterone group; (5) 10^-⁶ mol/L retinoic acid+testosterone group; (6) Testosterone group. The adipose-derived stem cells in the control group were cultured with Dulbecco’s modified Eagle’s medium+10% fetal bovine serum culture medium, and the adipose-derived stem cells in the other five groups were induced with corresponding dose of retinoic acid and testosterone on the basis of control group. After cultured for 36 hours, the flow cytometry was used to detect the changes of cell cycle.

RESULTS AND CONCLUSION: Compared with the control group, cell proportions in phase G₁ of 10^-⁵ mol/L retinoic acid group and 10^-⁶ mol/L retinoic acid group were increased significantly, and the cell proportions in phase S were decreased. Compared with control group, the cell proportion in phase G₁ of testosterone group was significantly reduced, and the cell proportion in phase S was increased. Compared with 10^-⁵ mol/L retinoic acid group and 10^-⁶ mol/L retinoic acid group, cell proportions in phase G₁ of 10^-⁵ mol/L retinoic acid+testosterone group and 10^-⁶ mol/L retinoic acid+testosterone group were reduced significantly and the cell proportions in phase S were increased. Retinoic acid can inhibit the cell cycle of adipose-derived stem cells in phase G₁, and delay the process of the cell cycle from phase G₁ to phase S; while testosterone can promote the cell cycle of adipose-derived stem cells from phase G₁ to phase S; the combination induction of retinoic acid and testosterone can accelerate the process of the cell cycle of adipose-derived stem cells from phase G₁ to phase S.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: stem cells, cell cycle, tretinoin, testosterone

CLC Number:

R394.2

Duan Fu-hua, Zeng Wen-qin, Yang Chun, Yang Hui-ying, Yu Mei-chun, Tao Hui, Dai Jing-xing, Yuan Lin. Retinoic acid, testosterone or their combination affects the cell cycle of adipose-derived stem cells[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(41): 6684-6688.

Figures/Tables 4

2.1 大鼠脂肪源干细胞的鉴定形态学鉴定：原代脂肪源干细胞细胞呈单细胞悬液状态，体积较红细胞大、椭圆形，胞核稍暗，观察原代接种 3 h后开始贴壁在48 h内开始伸展。伸展后，细胞呈多角形，有小的胞浆突起，胞核位于中央胞浆对比明显，严格按贴附方式生长，10 d后，细胞呈长梭形、多角形等，成纤维细胞样，胞浆丰富、核仁明显，呈漩涡状排列，细胞以集落形式增殖，7至8 d后细胞生长融合至80%，传至1代(图1A)。流式细胞仪检测结果：脂肪源干细胞高表达CD29，阳性率大于90%，CD90呈阳性，阳性率达90%以上，CD49d呈阴性，阴性率达90%以上，CD45亦表现阴性，阴性率90%以上(图1B)。成骨诱导：诱导后的细胞形态开始发生变化，由梭形逐渐转化为方形、鳞片形、三角形或多角形等，细胞外基质分泌增多，胞质中及胞质外均可见较多细小黑色颗粒，随时间延长，细胞呈铺路石样。大量细胞常围绕集落中央呈岛状分布，并可重叠生长，分泌大量细胞基质，形成大小不一的颗粒状结节。茜素红染色表现为红色的致密结节(图2A，B)。成脂诱导：诱导后的细胞逐渐变为圆形、椭圆形。第5天起光镜下可见胞质内零星出现黄色折光性强的圆形小脂滴，逐渐增多融合成大脂肪滴，细胞核被脂滴挤于一侧。有少部分大脂滴破裂，油红O染色显示特异性的红色脂滴(图2C，D)。 2.2 大鼠脂肪源干细胞经维甲酸和睾酮诱导后的形态学变化 3代脂肪源干细胞用10-5 mol/L 浓度维甲酸诱导3 d后，细胞胞浆突起呈丝状伸长(图2E)。3代脂肪源干细胞用 10-6 mol/L 浓度维睾酮诱导3 d后，细胞呈鹅卵石样变化(图2F)。 2.3 维甲酸与睾酮单独和联合诱导对脂肪源干细胞周期的影响见图3，表1。结果表明维甲酸可将脂肪源干细胞细胞周期阻滞于G1期，延缓细胞周期由G1期向S期的进程；睾酮可加速脂肪源干细胞的细胞周期由G1期向S期的进程；维甲酸睾酮联合诱导与维甲酸单独诱导相比，联合诱导可加速脂肪源干细胞的细胞周期由G1期向S期的进程，同样表明睾酮可加速脂肪源干细胞的细胞周期由G1期向S期的进程。"

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