中国组织工程研究

中国组织工程研究 ›› 2017, Vol. 21 ›› Issue (21): 3312-3319.doi: 10.3969/j.issn.2095-4344.2017.21.006

• 脂肪干细胞 adipose-derived stem cells • 上一篇    下一篇

残耳软骨细胞诱导脂肪来源干细胞体内软骨的形成

蔡 震1，蒋海越2   

  1. 1四川省医学科学院•四川省人民医院整形外科，四川省成都市 610072；2中国医学科学院，北京协和医学院整形外科医院整形七科，北京市 100144
  • 修回日期:2017-03-22 出版日期:2017-07-28 发布日期:2017-08-02
  • 通讯作者: 蒋海越，博士生导师，教授，主任医师，中国医学科学院，北京协和医学院整形外科医院整形七科，北京市 100144
  • 作者简介:蔡震，男，1976年生，天津市人，汉族，博士，副主任医师，主要从事组织移植与外耳再造研究。

Chondrogenesis of adipose tissue-derived stem cells induced by auricular chondrocytes from microtia in vivo

Cai Zhen1, Jiang Hai-yue2   

  1. 1Department of Plastic Surgery, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, Chengdu 610072, Sichuan Province, China; 2Plastic Surgery Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100144, China
  • Revised:2017-03-22 Online:2017-07-28 Published:2017-08-02
  • Contact: Jiang Hai-yue, Doctoral supervisor, Professor, Chief physician, Doctoral supervisor, Professor, Chief physician, Plastic Surgery Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100144, China
  • About author:Cai Zhen, M.D., Associate chief physician, Department of Plastic Surgery, Sichuan Academy of Medical Sciences & Sichuan Provincial People's Hospital, Chengdu 610072, Sichuan Province, China

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文题释义：
先天性小耳畸形：耳郭先天性发育不良，常伴有外耳道闭锁、中耳畸形和颌面部畸形。其发生率因种族不同而有区别。一般按照耳郭发育情况分为3度。Ⅰ度：耳郭各部分尚可辨认，有小耳甲腔及耳道口，轮廓较小，耳道内常为盲端；Ⅱ度耳郭多数结构无法辨认，残耳不规则，呈花生状、舟状或者腊肠状，外耳道常闭锁；Ⅲ度，残耳仅为小的皮赘呈小丘状或者仅有异位的耳垂。极少情况耳郭完全没有发育，局部无任何痕迹为无耳症。致病原因及发病机制不明确，治疗方法为耳郭再造手术。
组织工程技术：应用工程学及生命科学的原理，产生一种可以恢复、维持或改善受损组织和器官功能的新的组织和器官。具有3个优点：以少量的组织和器官形成大块的组织和器官，达到真正意义的无创伤修复创伤；利用有功能的活的组织和器官修复；可按照损伤组织和器官的形态进行修复。

 

摘要
背景：先天性小耳畸形残耳软骨细胞从细胞数量和质量上难以作为种子细胞构建出正常人耳郭大小的同质耳软骨支架。
目的：探讨残耳软骨细胞能否在裸鼠体内非软骨环境下模拟软骨诱导微环境，促进脂肪来源的干细胞向软骨分化并形成组织工程软骨，从而为解决组织工程化人耳郭软骨支架制备做基础准备工作。 
方法：①实验分为4组：第2代残耳软骨细胞与第3代脂肪来源的干细胞以3∶7比例混合作为共移植组，单纯残耳软骨细胞作为阳性对照组(残耳软骨细胞组)，单纯脂肪来源的干细胞作为阴性对照组(脂肪干细胞组)，以上3组接种细胞终浓度为5.0×1010 L-1，低浓度残耳软骨细胞对照组细胞终浓度为1.5×1010 L-1；②按照实验分组将0.2 mL细胞-Pluronic-F127复合物注射到裸鼠背部皮下，体内培养8周后对新生组织进行大体观察、湿质量测量、糖胺多糖含量测定、组织学及免疫组化染色检测。

结果与结论：①共移植组平均湿质量达到残耳软骨细胞组的80%以上；低浓度残耳软骨细胞对照组平均湿质量低于残耳软骨细胞组的30%；②共移植组和残耳软骨细胞组的平均湿质量和糖胺多糖平均含量均显著高脂肪干细胞组和低浓度残耳软骨细胞对照组(P < 0.05)；③组织学染色：共移植组、残耳软骨细胞组与低浓度残耳软骨细胞对照组标本均有成熟的软骨陷窝形成，低浓度残耳软骨细胞对照组软骨陷窝松散排列不均，胞外基质着色淡；脂肪干细胞组为纤维样组织，未见软骨陷窝形成；④Ⅱ型胶原免疫组化染色：共移植组、残耳软骨细胞组与低浓度残耳软骨细胞对照组可见成熟软骨陷窝周围有不同程度的棕黄色沉淀即Ⅱ型胶原表达；脂肪干细胞组未见Ⅱ型胶原表达；⑤结果提示，残耳软骨细胞能够在裸鼠体内非软骨环境下模拟软骨诱导微环境，促进脂肪干细胞向软骨分化并生成组织工程软骨。

 

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
ORCID: 0000-0001-6647-0310(蔡震)

关键词: 干细胞, 脂肪干细胞, 残耳软骨细胞, 脂肪来源干细胞, 软骨形成, 共移植, 诱导, 体内

Abstract:

BACKGROUND: Due to quantity and quality deficiencies, chondrocytes from microtia are difficult to act as seed cells to construct an ear cartilage scaffold with the normal human auricle size.
OBJECTIVE: To test the hypothesis that auricular chondrocytes from microtia can promote chondrogenic differentiation and chondrogenesis of human adipose tissue-derived stem cells (ADSCs) at non-chondrogensis site in vivo, which is the preparatory work for preparation of human tissue-engineered auricle cartilage scaffold. 
METHODS: Human ADSCs at passage 3 and auricular chondrocytes at passage 2 were mixed at a ratio of 7:3 and 5.0×1010/L mixed cells were suspended in 0.2 mL of 30% Pluronic F-127, and then the mixture was injected subcutaneously into Balb/c nude mice as experimental group. Auricular chondrocytes or ADSCs at the concentration of 5.0×1010/L were mixed with 0.2 mL of Pluronic F-127 and injected respectively as positive and negative control groups. 1.5×1010/L auricular chondrocytes were mixed and injected as low-concentration chondrocyte control group. All specimens were collected at the 8th week post-injection. Newborn tissues in nude mice were taken out for morphological examination, wet weight measurement, determination of glycosaminoglycans, histological and immunohistochemical staining. 
RESULTS AND CONCLUSION: The wet weight of specimens in the experimental group was over 80% of that in the positive control group, and the wet weight of specimens in the low-concentration chondrocyte control group was less than 30% of that in the positive control group. The average wet weight and glycosaminoglycan content were significantly higher in the experimental and positive control group than in the negative control and low-concentration chondrocyte control groups (P < 0.05). In all the groups except for the negative control group, mature cartilage lacunas could be observed by histological staining and collagen type II could be detected for expression by immunohistochemistry to different extents. In the low concentration chondrocyte control group, cartilage lacunas were incompact and inhomogeneous, and the extracellular matrix was slightly stained. In the negative control group, mature cartilage lacunae and collagen type II could not be detective. To conclude, auricular chondrocytes from microtia can promote chondrogenic differentiation and chondrogenesis of ADSCs at the non-chondrogenesis site in vivo.

 

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

Key words: Chondrocytes, Fats, Stem Cells, Tissue Engineering

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