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中华细胞与干细胞杂志(电子版)

中华细胞与干细胞杂志(电子版) ›› 2019, Vol. 09 ›› Issue (05) : 267 -275. doi: 10.3877/cma.j.issn.2095-1221.2019.05.003

所属专题： 文献；

论著

人体皮肤成纤维细胞的快速分离及多向分化能力鉴定
魏传飞1, 王伟1, 刘延明1, 段婧1, 芦现杰1, 王琰1, 张男1, 李孟鹏1, 陈超1, 章阳1, 佟加贝2, 韩发彬3,†()   
  1. 1. 252000　聊城，聊城大学/聊城市人民医院组织工程与再生医学研究所
    2. 250000　济南，山东美加赛培生物科技有限公司
    3. 252000　聊城，聊城大学/聊城市人民医院组织工程与再生医学研究所；250000　济南，山东大学第二医院转化医学研究所
  • 收稿日期:2019-08-03 出版日期:2019-10-01
  • 通信作者: 韩发彬
  • 基金资助:
    国家自然科学基金面上项目(81571241); 山东省重点研发计划(公益类专项2017GSF18104)

Isolation and identification of multidirectional differentiation of human skin fibroblasts

Chuanfei Wei1, Wei Wang1, Yanming Liu1, Jing Duan1, Xianjie Lu1, Yan Wang1, Nan Zhang1, Mengpeng Li1, Chao Chen1, Yang Zhang1, Jiabei Tong2, Fabin Han3,†()   

  1. 1. The Institute for Tissue Engineering and Regenerative Medicine, Liaocheng University/The Liaocheng People's Hospital, Liaocheng 252000, China
    2. Shandong Meijia Saipei Biotechnology Co., Ltd. Jinan 250000, China
    3. The Institute for Tissue Engineering and Regenerative Medicine, Liaocheng University/The Liaocheng People's Hospital, Liaocheng 252000, China; Institute of Translational Medicine, The Second Hospital of Shandong University, Jinan 250000, China
  • Received:2019-08-03 Published:2019-10-01
  • Corresponding author: Fabin Han
  • About author:
    Corresponding author: Han Fabin, Email:
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引用本文：

魏传飞, 王伟, 刘延明, 段婧, 芦现杰, 王琰, 张男, 李孟鹏, 陈超, 章阳, 佟加贝, 韩发彬. 人体皮肤成纤维细胞的快速分离及多向分化能力鉴定[J]. 中华细胞与干细胞杂志(电子版), 2019, 09(05): 267-275.

Chuanfei Wei, Wei Wang, Yanming Liu, Jing Duan, Xianjie Lu, Yan Wang, Nan Zhang, Mengpeng Li, Chao Chen, Yang Zhang, Jiabei Tong, Fabin Han. Isolation and identification of multidirectional differentiation of human skin fibroblasts[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2019, 09(05): 267-275.

目的

建立快速分离人皮肤成纤维细胞的方法，并探讨成纤维细胞在成脂、成骨、成软骨和成神经的多向分化潜能。

方法

利用组织块培养法分离人体皮肤成纤维细胞，通过形态学观察、流式分析、Vimentin蛋白染色鉴定成纤维细胞；再利用生长曲线、核型分析、线粒体染色分析不同传代细胞的增殖速度，线粒体及染色体形态的改变；最后进行成纤维细胞成脂、成骨、成软骨和成神经的诱导分化实验，鉴定其多向分化潜能。两代细胞增殖速度及线料体相对量的比较采用t检验统计分析。

结果

分离的皮肤成纤维细胞呈典型梭状及多角形；高表达细胞表面标记物CD90 （NCF1，NCF2占比分别为99.9%，98.7%）和CD73 （NCF1，NCF2占比分别为98.2%，85.6%），但极少表达造血干细胞标记物CD34 （NCF1，NCF2占比分别为1.8%，2.6%）；细胞Vimentin蛋白表达呈阳性，阳性率为100%；对细胞生长曲线进行分析，表明分离后不同代次细胞增殖差异无统计学意义（t?= 1.586，P?= 0.1567）；线粒体相对含量统计分析，同一株细胞系第5代（相对荧光强度值：6876±577.8）与第10代（相对荧光强度值：7371±471.9）之间的差异无统计学意义（t?= 0.664，P?= 0.543）；核型分析分别显示传代后保持染色体数目为正常46条且形态无明显异常；经诱导后成纤维细胞可向成脂、成骨、成软骨和类神经分化。

结论

利用组织块培养法分离出的人体皮肤成纤维细胞状态稳定，增殖能力强，具有成脂、成骨、成软骨和成神经多向分化诱导潜能，为细胞移植修复骨损伤、软骨损伤和神经损伤性疾病的临床研究提供细胞来源及实验依据。

关键词: 皮肤成纤维细胞, 组织块培养法, 流式分析, 多向分化
Objective

To efficiently isolate human skin fibroblast cells with rapid proliferation and to investigate the multi-lineage differentiation potential of fibroblasts into adipogenic, osteogenic, chondrogenic and neurogenic directions.

Methods

The human primary fibroblast cells were isolated and cultured from the skin tissues. Morphological structures, flow cytometry and vimentin staining were used to identify the fibroblasts. Growth curves analysis, mitochondrial staining and karyotype analysis were used to identify the cell state of different generations. And finally fibroblasts were induced to adipogenic, osteogenic, chondrogenic and neurogenic directions. The difference between cell proliferation rate and the relative amount of mitochondrial was compared by t-test.

Results

The isolated fibroblasts showed a typical spindle-like morphology. Flow cytometry analysis showed that the isolated cells were highly express fibroblast surface markers CD90 (NCF1, NCF2 accounted for 99.9%, 98.7%, respectively) and CD73 (NCF1, NCF2 accounted for 98.2%, 85.6%, respectively) , rarely express hematopoietic stem cell marker CD34 (NCF1, NCF2 accounted for 1.8%, 2.6%, respectively) . All the fibroblasts positively express vimentin, the positive rate was 100%. The results of growth curves analyse showed that there was no significant difference in cell proliferation rate among multiple passages (t = 1.586, P = 0.1567) . The relative content of mitochondria showed no significant difference (t = 0.664, P = 0.543) between the fifth generation (relative fluorescence intensity: 6876±577.8) and the 10th generation (relative fluorescence intensity: 7371 ± 471.9) of the same cell line. Karyotype analysis showed that the number of chromosomes was 46 and the morphology of chromosomes were similar during passage. The multi-lineage differentiation assay showed that the fibroblasts could express adipogenic, osteogenic, cartilaginous and neurogenic differentiation markers.

Conclusion

Isolated fibroblasts have adipogenic, osteogenic, chondrogenic, neurogenic multi-directional differentiation potential, which can meet the needs of preclinical studies and provide cell sources for bone trauma, cartilage injury and neurodegenerative diseases.

Key words: Skin fibroblasts, Explant culture, Flow cytometry analysis, Multidirectional differentiation
图1 光学显微镜下分离皮肤成纤维细胞的形态（×100）
图2 分离皮肤成纤维细胞的流式分析
图3 荧光显微镜下分离皮肤成纤维细胞鉴定（Vimentin荧光染色，×100）
图4 分离皮肤成纤维细胞生长曲线分析
图5 荧光显微镜下皮肤成纤维细胞线粒分析（线粒体活体染色，×100）
图6 同一株人体皮肤成纤维细胞不同代次P5，P10代细胞线粒体相对荧光强度值
图7 分离皮肤成纤维细胞核型分析（×200）
图8 荧光显微镜下分离皮肤成纤维细胞多向诱导分化后免疫荧光染色鉴定（×200）
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