中国组织工程研究

中国组织工程研究 ›› 2018, Vol. 22 ›› Issue (17): 2661-2668.doi: 10.3969/j.issn.2095-4344.0537

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

人脂肪间充质干细胞和滑膜间充质干细胞协同抑制炎性软骨细胞的退变

宋卓悦，王 洋，连晓磊，丁 康，李广恒   

  1. 郑州大学第一附属医院骨科，河南省郑州市 450052
  • 修回日期:2018-03-22 出版日期:2018-06-18 发布日期:2018-06-18
  • 通讯作者: 李广恒，博士，教授，主任医师，博士生导师，郑州大学第一附属医院骨科，河南省郑州市 450052
  • 作者简介:宋卓悦，男，1986年生，河南省郑州市人，汉族，郑州大学在读硕士，主要从事关节外科基础及临床研究。
  • 基金资助:

    国家自然科学基金(81472136)

Human adipose-derived mesenchymal stem cells and synovial-derived mesenchymal stem cells synergistically inhibit the degeneration of inflammatory chondrocytes

Song Zhuo-yue, Wang Yang, Lian Xiao-lei, Ding Kang, Li Guang-heng   

  1. Department of Orthopedics, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan Province, China
  • Revised:2018-03-22 Online:2018-06-18 Published:2018-06-18
  • Contact: Li Guang-heng, M.D., Professor, Doctoral supervisor, Department of Orthopedics, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan Province, China
  • About author:Song Zhuo-yue, Master candidate, Department of Orthopedics, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan Province, China
  • Supported by:

    the National Natural Science Foundation of China, No. 81472136

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摘要：

文章快速阅读：

文题释义：
三维立体培养: 利用磁性微球载体和磁悬浮技术使贴有细胞的微球载体悬浮在培养液中，确保了高质量、高密度的细胞繁殖，突破了传统有盖培养皿、培养瓶或微孔板细胞培养耗时繁琐，细胞产量微小等局限性。
联合应用脂肪来源间充质干细胞和滑膜来源间充质干细胞协同抑制炎性软骨细胞退变：一方面通过脂肪来源间充质干细胞来抑制炎性软骨细胞的炎症反应，另外一方面通过滑膜来源间充质干细胞促进再生软骨细胞及炎性软骨的修复。通过这两方面的协同作用可以使软骨细胞的修复最大化。

 

摘要
背景：将具有目的功能的细胞通过注射的方法注入关节腔内来改变病变的微环境，作用于早期骨性关节炎的炎性软骨细胞，通过发挥植入细胞本身的特性来影响疾病的进程。
目的：探讨联合应用人膝关节脂肪来源间充质干细胞及滑膜来源间充质干细胞对炎性软骨细胞的退变是否具有协同抑制作用。
方法：原代培养脂肪来源间充质干细胞、滑膜来源间充质干细胞和炎性软骨细胞。体外二维培养条件下，采用MTS细胞增殖实验检测3种细胞的增殖情况，定量PCR及免疫荧光分析3种贴壁细胞成软骨标志物基因水平及蛋白水平的差异。体外3D混合培养条件下分3组：脂肪来源间充质干细胞+炎性软骨细胞组(A+C组)，滑膜来源间充质干细胞+炎性软骨细胞组(S+C组)，脂肪来源间充质干细胞+滑膜来源间充质干细胞+炎性软骨细胞组(A+S+C组)。收集3组混合培养的细胞团块，进行阿尔新蓝染色、番红O染色及Ⅱ型胶原免疫组化染色，并做定量分析。采用定量PCR检测成软骨分化标志物的基因水平差异。收集培养液上清通过酶联免疫吸附技术检测促炎因子及抗炎因子的分泌情况。
结果与结论：①体外二维培养条件下，脂肪来源间充质干细胞的增殖速率高于炎性软骨细胞和滑膜来源间充质干细胞，差异有显著性意义(P < 0.05)；炎性软骨细胞的成软骨标志物Ⅱ型胶原、蛋白聚糖 mRNA表达量以及Ⅱ型胶原蛋白表达量明显高于脂肪来源间充质干细胞和滑膜来源间充质干细胞(P < 0.01)；②体外3D混合培养条件下，A+S+C组的成软骨分化特异性染色阳性区域面积百分比均明显高于S+C组和A+C组(P < 0.05)；A+S+C组成软骨分化标志物蛋白聚糖及Ⅱ型胶原表达量明显高于S+C组和A+C组(P < 0.05)；S+C组促炎因子白细胞介素1、白细胞介素6、肿瘤坏死因子α水平明显高于A+C组及A+S+C组(P < 0.05)，A+C组及A+S+C组抗炎因子白细胞介素10水平明显高于S+C组(P < 0.05)；③结果表明，联合应用脂肪来源间充质干细胞及滑膜来源间充质干细胞对炎性软骨细胞退变具有协同抑制作用。

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: 0000-0002-1568-5941(宋卓悦)

关键词: 骨性关节炎, 脂肪间充质干细胞, 滑膜间充质干细胞, 炎性软骨细胞, 三维培养, 软骨退变, 软骨再生, 炎症反应, 成软骨分化, 干细胞, 国家自然科学基金

Abstract:

BACKGROUND: Intra-articular injection of cells targeting to change the microenvironment in lesions can act on early osteoarthritis of inflammatory chondrocytes. Implanted cells affect the progress of the disease by the cell characteristics.
OBJECTIVE: To explore the synergistic effect of mesenchymal stem cells from human knee adipose (ADMSCs) and synovial tissues (SDMSCs) to inhibit the degeneration of inflammatory chondrocytes.
METHODS: ADMSCs, SDMSCs and inflammatory chondrocytes were primary cultured. Under in vitro two-dimensional culture conditions, cell proliferation assay (MTS) was performed to detect the proliferation of three kinds of cells. Differences in chondrogenic markers at mRNA and protein levels between three kinds of adherent cells were detected by quantitative PCR and immunofluorescence. Under in vitro three-dimensional mixed culture conditions, three groups were set up: (1) ADMSCs+inflammatory chondrocytes (A+C group), (2) SDMSCs+inflammatory chondrocytes (S+C group), and (3) ADMSCs-SDMSCs+inflammatory chondrocytes (A+S+C group). Alcian blue staining, safranin O staining and type II collagen immunohistochemistry staining were performed on the mixed-cultured cell mass paraffin sections followed by quantitative analysis. Chondrogenic differentiation in each group was detected by quantitative PCR. Culture supernatants were collected to detect the secretion of pro-inflammatory and anti-inflammatory factors by enzyme-linked immunosorbent assay.
RESULTS AND CONCLUSION: Under the two-dimensional culture, the proliferative rate of ADMSCs was significantly higher than that of inflammatory chondrocytes and SDMSCs (P < 0.05). The expression of type II collagen mRNA and protein and proteoglycan protein in inflammatory chondrocytes was significantly higher than that in the other two kinds of cells (P < 0.01). Under the three-dimensional culture, the percentage of chondrogenic area per total area was significantly higher in the A+S+C group than the S+C and A+C groups (P < 0.05). The expression of type II collagen and proteoglycan was significantly higher in the A+S+C group than the S+C and A+C groups (P < 0.05). Compared with the other two groups, the S+C group showed higher levels of interleukin 1, interleukin 6, and tumor necrosis factor α, but lower level of interleukin 10 (P < 0.05). To conclude, the combined use of ADMSCs and SDMSCs synergistically inhibits the degeneration of inflammatory chondrocytes.

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: Adipose Tissue, Synovial Membrane, Mesenchymal Stem Cells, Chondrocytes, Coculture Techniques, Osteoarthritis, Tissue Engineering

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