解剖学报 ›› 2020, Vol. 51 ›› Issue (4): 513-519.doi: 10.16098/j.issn.0529-1356.2020.04.007

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脂肪源性干细胞向成纤维细胞的分化

鞠晓军1 牛友芽2 饶利兵1 易黎明1 向莎1 张英俊3*   

  1. 1.湖南医药学院人体解剖学教研室; 2.细胞学与遗传学教研室; 3.影像诊断学教研室,湖南 怀化 418000
  • 收稿日期:2020-01-03 修回日期:2020-03-04 出版日期:2020-08-06 发布日期:2020-08-06
  • 通讯作者: 张英俊 E-mail:34873097@qq.com
  • 基金资助:
    湖南省教育厅科学研究项目;湖南省自然科学基金面上项目

Experiment of the differentiation of adipose-derived stem cells into fibroblasts

 JU Xiao-jun1 NIU You-ya2 RAO Li-bing1 YI Li-ming1 XIANG Sha1 ZHANG Ying-jun3*   

  1. 1. Department of Human Anatomy, Hunan University of Medicine; 2.Department of Cell and Genetics, Hunan University of Medicine; 3. Department of Imaging Diagnosis, Hunan University of Medicine, Hu’nan Huaihua 418000,China
  • Received:2020-01-03 Revised:2020-03-04 Online:2020-08-06 Published:2020-08-06
  • Contact: ZHANG Ying-jun E-mail:34873097@qq.com
  • Supported by:
    Scientific Research Project Of Hunan Provincial Department Of Education;General Project of Hunan Natural Science Foundation

摘要:

目的 探讨脂肪源性干细胞(ADSCs)向成纤维细胞分化的可能,为解决皮肤组织工程种子细胞提供有效途径。  方法 取健康成年SD大鼠10只,体重280~320 g,雌雄不限,麻醉、脱毛、消毒,于腹股沟处获取脂肪后进行ADSCs的体外分离、培养和传代,观察原代细胞的生长特点;取第3代细胞,成骨诱导后第16天行碱性磷酸酶(ALP)检测,成骨诱导后第23天行茜素红染色,成脂诱导后第12天行油红O染色;流式细胞术检测细胞表面标志;加入条件培养基向成纤维细胞诱导分化,于诱导后第2天、第4天、第6天、第8天摄片记录细胞形态变化过程,MTT检测各时相点的细胞活性;诱导后第6天和第8天行扫描电子显微镜检测;诱导后第8天行免疫细胞化学染色,检测成纤维细胞主要标志物波形蛋白(vimentin)的表达。  结果 原代ADSCs呈长梭形贴壁生长,成骨诱导后可见ALP强阳性表达,茜素红染色可见红色矿化结节;成脂诱导后可见胞内红染脂滴聚集;流式细胞术检测显示,间充质干细胞相关标志物CD90阳性表达,造血干细胞标记物CD45呈阴性表达;ADSCs向成纤维细胞诱导后第2天可见形态开始改变,部分细胞漂浮;诱导后第4天细胞呈水滴形或短棒状;诱导后第6天细胞呈有突起的多边形或三角形混杂;诱导后第8天细胞拥挤并铺满瓶底,呈细长纤维状; MTT检测表明,诱导后第2天的细胞活性要显著低于对照组(P<0.01);诱导后第4天、第6天和第8天细胞活性与对照组均无明显差异(P>0.05);扫描电子显微镜检测可见,诱导后第6天细胞呈三角形,表面纤毛较多;诱导后第8天细胞呈细长纤维状,有小突起,表面纤毛密集;诱导后第8天绝大多数细胞vimentin呈阳性表达。  结论 ADSCs在体外经诱导分化后可以具备成纤维细胞的形态特征;能表达成纤维细胞的标志蛋白。

关键词:  脂肪源性干细胞, 诱导分化, 组织工程, 流式细胞术, 扫描电子显微术, 大鼠

Abstract:

Objective To explore the possibility of adipose-derived stem cells(ADSCs)to differentiate into fibroblasts, and to provide an effective way for the effective solution of skin tissue engineering seed cells.   Methods Primary ADSCs were obtained from inguinal fat of ten healthy adult SD rats,weighing 280-320 g,and cultured in vitro and purified.When primary ADSCs expansion to the 3rd passages,the following experiments were performed alkaline phosphatase test on the 16th day after osteogenesis induction,staining of alizarin red mineralized nodules on day 23 after osteogenesis induction,oil red O staining on day 12 after adipogenic induction;Flow cytometry detection of cell surface markers;Addition of conditioned medium to induce differentiation into fibroblasts,Photograph the changes of cell morphology on the 2nd, 4th, 6th, and 8th days after induction,MTT to detect cell viability at various time points;Scanning electron microscopy on day 6 and day 8 after induction;Immunocytochemical staining on day 8 after induction,detect the expression of vimentin, the main marker of fibroblasts.  Results Primary ADSCs grew in long spindles,showed strong positive expression of alkaline phosphatase (ALP) after osteogenesis induction,and alizarin red staining showed red mineralized nodules;Aggregation of intracellular red-stained lipid droplets after adipogenic induction were found;Flow cytometry showed positive expression of mesenchymal stem cell-related marker CD90,and hematopoietic stem cell marker CD45 was negative. Morphology of ADSCs started to change on day 2 after induction into fibroblasts. On the 4th day after induction, the cells were in the shape of water droplets or short rods. On the 6th day after induction, the cells were protruded polygonal or triangular. Cells crowded and covered the bottom of the bottle on day 8 after induction,becoming slender fibrous. MTT test showed that the cell viability was significantly lower on the second day after induction than in the control group. There were no significant differences in cell viability on the 4th, 6th, and 8th days after induction compared with the control group. Scanning electron microscopy showed that the cells were triangular on the 6th day after induction, and the surface had more cilia. On the 8th day after induction, the cells were slender and fibrous, with small protrusions, and the surface cilia were dense. Vimentin was positively expressed in most cells on the 8th day after induction.  Conclusion ADSCs can have the morphological characteristics of fibroblasts after induced differentiation in vitro;that can express fibroblasts marker protein.

Key words: Adipose-derived stem cell, Induced differentiation, Tissue Engineering, Flow cytometry, Scanning electron microscopy, Rat

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