中国组织工程研究 ›› 2018, Vol. 22 ›› Issue (12): 1829-1834.doi: 10.3969/j.issn.2095-4344.0199

• 软骨组织构建 cartilage tissue construction • 上一篇    下一篇

骨软骨前体细胞分离鉴定及转化生长因子β3对其成软骨分化的影响

左  伟,程文俊,焦  竞,黄玉成,肖  飞,王俊文   

  1. 武汉市第四医院 华中科技大学同济医学院附属普爱医院骨科,湖北省武汉市  430033
  • 收稿日期:2017-11-27 出版日期:2018-04-28 发布日期:2018-04-28
  • 通讯作者: 王俊文,硕士,主任医师,武汉市第四医院 华中科技大学同济医学院附属普爱医院骨科,湖北省武汉市 430033
  • 作者简介:左伟,男,1984年生,湖北省武汉市人,汉族,2013年北京协和医学院毕业,博士,主治医师,主要从事骨关节退行性疾病的病因与防治研究。
  • 基金资助:

    湖北省自然科学基金项目(2017CFB255);武汉市卫计委基金项目(WX15C23)

Osteochondroprogenitor cells: isolation, identification and chondrogenesis under the induction of transforming growth factor beta3

Zuo Wei, Cheng Wen-jun, Jiao Jing, Huang Yu-cheng, Xiao Fei, Wang Jun-wen   

  1. Department of Orthopedics, Wuhan Forth Hospital & Pu’ai Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430033, Hubei Province, China
  • Received:2017-11-27 Online:2018-04-28 Published:2018-04-28
  • Contact: Wang Jun-wen, Master, Chief physician, Department of Orthopedics, Wuhan Forth Hospital & Pu’ai Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430033, Hubei Province, China
  • About author:Zuo Wei, M.D., Attending physician, Department of Orthopedics, Wuhan Forth Hospital & Pu’ai Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430033, Hubei Province, China
  • Supported by:

    the Natural Science Foundation of Hubei Province, No. 2017CFB255; the Project of Health and Family Planning Commission of Wuhan, No. WX15C23

摘要:

文章快速阅读:

文题释义:
骨软骨前体细胞:分布在不同组织中的间充质干细胞可以视为组织特异性干细胞,一定条件下可向相应组织的成熟细胞分化,骨软骨前体细胞也称为骨软骨祖细胞,是指在特定条件下能够分化为骨细胞及软骨细胞的间充质干细胞。
转化生长因子β:是属于一组新近发现的调节细胞生长和分化的转化生长因子β超家族。这一家族除转化生长因子β外,还有活化素(activins)、抑制素(inhibins)、缪勒氏管抑制质(Mullerian inhibitor substance,MIS)和骨形成蛋白。转化生长因子β的命名是根据这种细胞因子能使正常的成纤维细胞的表型发生转化,即在表皮生长因子同时存在的条件下,改变成纤维细胞贴壁生长特性而获得在琼脂中生长的能力,并失去生长中密度信赖的抑制作用。转化生长因子β与早先报道的从非洲绿猴肾上皮细胞BSC-1所分泌的生长抑制因子是同一物。
摘要
背景:
关节软骨中存在着前软骨干细胞,可能成为软骨组织工程潜在的种子细胞,转化生长因子β3对前软骨干细胞的增殖及成软骨分化具有正向调节作用。
目的:分析转化生长因子β3对骨软骨前体细胞成软骨的分化作用。
方法:分离筛选出晚期骨关节炎患者软骨组织CD146+软骨细胞并鉴定。培养CD146+软骨细胞团块分为4组:为普通培养基组,转化生长因子β3-诱导组,转化生长因子β3+诱导组(含2.5 μg/L重组人转化生长因子β3)和转化生长因子β3++诱导组(含10 μg/L重组人转化生长因子β3)。培养4周后行组织块Ⅱ型胶原、Aggrecan免疫组织化学及相关基因实时荧光定量PCR检测。
结果与结论:①成软骨诱导培养时,转化生长因子β3++诱导组所形成软骨块>转化生长因子β3+诱导组>转化生长因子β3-诱导组;②免疫组织化学结果显示,转化生长因子β3++诱导组Ⅱ型胶原和Aggrecan的表达明显高于转化生长因子β3+诱导组(P < 0.05),转化生长因子β3+诱导组表达明显高于转化生长因子β3-诱导组(P < 0.05);③实时荧光定量PCR检测显示,转化生长因子β3++诱导组Ⅱ型胶原和Aggrecan mRNA的表达明显强于转化生长因子β3+诱导组(P < 0.05),转化生长因子β3+诱导组2指标表达明显强于转化生长因子β3-诱导组(P < 0.05);转化生长因子β3++诱导组和转化生长因子β3+诱导组性别决定区Y框蛋白-9的表达均明显强于转化生长因子β3-诱导组(P < 0.05);④结果表明,晚期骨关节炎患者残余关节软骨中存在着具有干细胞特性的骨软骨前体细胞;转化生长因子β3具有较强促骨软骨前体细胞成软骨分化的能力,其有可能成为软骨组织工程中理想的细胞因子。

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
ORCID: 0000-0001-9952-4346(左伟)

关键词: 骨软骨前体细胞, 转化生长因子β3, 软骨组织工程, 成软骨分化, 骨关节炎, 组织构建

Abstract:

BACKGROUND: Precartilaginous stem cells exist in articular cartilage, which may become potential seed cells for cartilage tissue engineering. Transforming growth factor-β3 (TGF-β3) has positive regulation effect on the proliferation and chondrogenic differentiation of precartilagious stem cells.
OBJECTIVE: To investigate the effect of TGF-β3 on the chondrogenesis of osteochondroprogenitor cells.
METHODS: CD146+ chondrocytes were isolated from the patients with advanced osteoarthritis, and were then identified. CD146+ chondrocytes were cultured in the normal medium (blank control group), chondrogenic induced medium (control group), chondrogenic induced medium containing 2.5 and 10 μg/L recombinant human TGF-β3, respectively. The immunohistochemistry of collagen II and aggrecan was performed, and the related gene expression was tested by real-time quantitative PCR after 4 weeks of culture.
RESULTS AND CONCLUSION: When chondrogenic differentiation was performed, the number of cell pellets in the 10 μg/L TGF-β3 group was greater than that in the 2.5 μg/L TGF-β3 group, and the number of cell pellets in the 2.5 μg/L TGF-β3 group was greater than that in the control group. The expression levels of collagen II and aggrecan in the 10 μg/L TGF-β3 group was significantly higher than that in the 2.5 μg/L TGF-β3 group (P < 0.05). The expression levels of collagen II and aggrecan in the 2.5 μg/L TGF-β3 group were significantly higher than those in the control group (P < 0.05). Real-time quantitative PCR results showed that the expression levels of collagen II and aggrecan mRNA in the 10 μg/L TGF-β3 group were significantly higher than those in the 2.5 μg/L TGF-β3 group (P < 0.05), but the expression level of SOX-9 showed insignificant difference between two groups (P > 0.05), and the expression level of SOX-9 in the 10 and 2.5 μg/L TGF-β3 groups was significantly higher than that in the control group (P < 0.05); the expression levels of collagen II and aggrecan mRNA in the 2.5 μg/L TGF-β3 group were higher than those in the control group (P < 0.05). Our findings suggest that osteochondroprogenitor cells with stem cell characteristics exist in the residual articular cartilage of the patients with advanced osteoarthritis. TGF-β3 has the ability of promoting chondrogenic differentiation of osteochondroprogenitor cells, which may be an ideal cytokine for cartilage tissue engineering.

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: Osteoarthritis, Chondrocytes, Tissue Engineering

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