切换至 "中华医学电子期刊资源库"

中华关节外科杂志(电子版) ›› 2019, Vol. 13 ›› Issue (06) : 693 -698. doi: 10.3877/cma.j.issn.1674-134X.2019.06.008

所属专题: 文献

基础论著

三维共培养体系促进去分化的软骨细胞再分化的实验研究
张廷帅1, 邹健宇1, 陈汉政1, 刘日许1, 郑仕聪1, 陈艺1, 张姝江1, 姚咏嫦1,()   
  1. 1. 510120 广州医科大学附属第一医院关节外科;510120 广州,广东省骨科矫形技术及植入材料重点实验室
  • 收稿日期:2019-09-06 出版日期:2019-12-01
  • 通信作者: 姚咏嫦
  • 基金资助:
    广州市科技计划项目一般项目(201804010479和201904010174); 广州市属高校科研项目重点项目(1201610097)

Experimental study of three-dimensional co-culture system promoting dedifferentiated chondrocyte redifferentiation

Tingshuai Zhang1, Jianyu Zou1, Hanzheng Chen1, Rixu Liu1, Shicong Zheng1, Yi Chen1, Shujiang Zhang1, Yongchang Yao1,()   

  1. 1. Department of Joint Surgery, 1st Affiliated Hospital of Guangzhou Medical University; Guangdong Key Laboratory of Orthopaedic Technology and Implant Materials, Guangzhou 510120, China
  • Received:2019-09-06 Published:2019-12-01
  • Corresponding author: Yongchang Yao
  • About author:
    Corresponding author: Yao Yongchang, Email:
引用本文:

张廷帅, 邹健宇, 陈汉政, 刘日许, 郑仕聪, 陈艺, 张姝江, 姚咏嫦. 三维共培养体系促进去分化的软骨细胞再分化的实验研究[J]. 中华关节外科杂志(电子版), 2019, 13(06): 693-698.

Tingshuai Zhang, Jianyu Zou, Hanzheng Chen, Rixu Liu, Shicong Zheng, Yi Chen, Shujiang Zhang, Yongchang Yao. Experimental study of three-dimensional co-culture system promoting dedifferentiated chondrocyte redifferentiation[J]. Chinese Journal of Joint Surgery(Electronic Edition), 2019, 13(06): 693-698.

目的

探讨海藻酸钠水凝胶三维共培养体系对去分化的软骨细胞再分化的影响。

方法

将第1代(P1)软骨细胞及第4代(P4)软骨细胞分别与ATDC5按3 ∶1的比例在海藻酸钠水凝胶内进行三维共培养,即AP1组及AP4组。另建立单纯P1、P4及ATDC5的三维培养体系,即P1、P4及ATDC组,全部以软骨诱导液为培养基,提供外源性转化生长因子(TGF-β3)。培养28 d后,通过Q-PCR检测Ⅰ型胶原、Ⅱ型胶原、蛋白聚糖基因的表达情况,组织学染色观察等手段,比较各组的Ⅱ型胶原及蛋白聚糖基因的表达水平,结果以Bonferroni检验法进行统计学分析。

结果

AP4组中Ⅱ型胶原及蛋白聚糖基因的表达明显上调,且与P1、P4组的差异有统计学意义(Ⅱ型胶原:F=38.41,P<0.01;蛋白聚糖:F=5,P<0.01),且在组织学染色上也能观察到相关蛋白产物明显沉积。

结论

三维共培养体系能让去分化的软骨细胞重新出现其特有的表型,其机制涉及细胞因子及细胞直接接触等方面。

Objective

To investigate the effect of alginate hydrogel 3D co-culture on redifferentiation of dedifferentiated chondrocytes.

Methods

The first generation (P1) chondrocytes and the fourth generation (P4) chondrocytes were co-cultured with ATDC5 at the ratio of 3: 1, and wee named as AP1 and AP4 respectively. Another 3D culture system consisting of single P1, P4 and ATDC5 was established, and was named as P1, P4 and ATDC, respectively. All the groups were cultured in the chondrocyte-inducing medium with the supply of TGF-β3. After 28 days, the expression of collagen typeⅠ (ColⅠ), collagen typeⅡ (ColⅡ) and aggrecan (Acan) genes was assayed by Q-PCR. Histological staining was performed. The cartilage-related genes (Col Ⅱ and Acan) of each group were compared. The results were analyzed by Bonferroni test.

Results

The expression of ColⅡ and Acan in AP4 group was significantly up-regulated compared to P1 and P4 groups (ColⅡ: F=38.41, P<0.01, Acan: F=5, P<0.01 ), and the deposition of relative protein products could also be clearly observed on histological staining.

Conclusion

The 3D co-culture system can make the dedifferentiated chondrocytes regain their unique phenotype, the mechanism of which involves cellular factors and the cell-cell connection.

表1 Q-PCR所用引物
图1 软骨细胞在单层培养下的细胞形态。图A 为P1(第1代)平面培养的软骨细胞,可见其细胞形态为多边形(比例尺100 μm);图B 为P4(第4代)软骨细胞,其多边形态基本消失(比例尺100 μm)
图2 P1(第1代)及P4(第4代)细胞的软骨标志性基因及肥大化基因的表达情况。蛋白聚糖、II型胶原及I型胶原基因的表达对比中,P1组和与P4组两两比较均有统计学差异(P<0.05)
图3 各组细胞增殖活性对比。图中可见P4共培养组的细胞增殖活性高于其余各组,差异具有统计学意义(*-P<0.05,**-P <0.01 vs P4 co-culture); ATDC(单纯ATDC5三维培养组),P1(单纯第1代软骨细胞三维培养组),P1 co-culture(ATDC5与第1代软骨细胞三维共培养组);P4(单纯第4代软骨细胞三维培养组);P4 co-culture(ATDC5与第4代软骨细胞三维共培养组)
图4 共培养体系及单层培养的软骨标志性基因表达情况。图A 为4组中I型胶原基因表达情况,*-与AP4组差异有统计学意义(P <0.05);图B 为4组中Ⅱ型胶原基因表达情况,**-与AP4组的差异有明显统计学(P <0.01);图C 为4组中蛋白聚糖基因表达情况,**-与AP4组的差异有明显统计学(P <0.01); plate P4(第4代软骨细胞平面培养组);alginate P4(第4代软骨细胞三维培养组);AP4(ATDC5与第4代软骨细胞三维共培养组);AP1(ATDC5与第1代软骨细胞三维共培养组)
图5 各组微球的番红O染色结果。图A 为AP1(ATDC5与第1代软骨细胞三维培养组)组,图中可见细胞成团聚集,其周围有较多的红色深染区,表明有GAG沉积;图B 为AP4 (ATDC5与第4代软骨细胞三维培养组),图中也可见细胞团周围有较多的红染区,但较AP1组少;图C 为P4组(第4代软骨细胞三维培养组),图中可见细胞周围有少量的红染区,较AP1及AP4明显要少;图D 为ATDC组(单纯ATDC5三维培养组),图中所示细胞周围无乎没有红染区,细胞分散
[1]
Lin Z, Willers C, Xu JK, et al. The chondrocyte: Biology and clinical application[J]. Tissue Eng, 2006, 12(7): 1971-1984.
[2]
Aigner T, Stove J. Collagens-major component of the physiological cartilage matrix, major target of cartilage degeneration, major tool in cartilage repair[J]. Adv Drug Deliv Rev, 2003, 55(12): 1569-1593.
[3]
Carnes J, Stannus O, Cicuttini FM, et al. Knee cartilage defects in a sample of older adults: natural history, clinical significance and factors influencing change over 2.9 years[J]. Osteoarthritis Cartilage, 2012, 20(12): 1541-1547.
[4]
Zhu Y, Yuan M, Meng HY, et al. Basic science and clinical application of platelet-rich plasma for cartilage defects and osteoarthritis: a review[J]. Osteoarthritis Cartilage,2013, 21(11): 1627-1637.
[5]
Mastbergen SC, Saris DBF, Lafeber FPJG. Functional articular cartilage repair: here, near, or is the best approach not yet clear?[J]. Nat Rev Rheumatol, 2013, 9(5) : 277-290.
[6]
Richter DL, Schenck RC, Wascher DC, et al. Knee articular cartilage repair and restoration techniques: a review of the literature[J]. Sports Health, 8(2): 153-160.
[7]
Makris EA, Gomoll AH, Malizos KN, et al. Repair and tissue engineering techniques for articular cartilage[J]. Nat Rev Rheumatol, 2015, 11(1): 21-34.
[8]
Dai WD, Kawazoe N, Lin XT, et al. The influence of structural design of PLGA/collagen hybrid scaffolds in cartilage tissue engineering[J]. Biomaterials, 2010, 31(8): 2141-2152.
[9]
邹健宇,刘日许,郑仕聪,共培养体系在关节软骨组织工程中的应用研究[J/CD]. 中华关节外科杂志(电子版), 2018, 12(6): 835-841.
[10]
Qing C, Wei-Ding C, Wei-Min F. Co-culture of chondrocytes and bone marrow mesenchymal stem cells in vitro enhances the expression of cartilaginous extracellular matrix components[J]. Braz J Med Biol Res, 2011, 44(4): 303-310.
[11]
Bekkers JE, Tsuchida AI, van Rijen MH, et al. Single-stage cell-based cartilage regeneration using a combination of chondrons and mesenchymal stromal cells comparison with microfracture[J]. Am J Sports Med, 2013, 41(9): 2158-2166.
[12]
Giovannini S, Diaz-Romero J, Aigner T, et al. Micromass co-culture of human articular chondrocytes and human bone marrow mesenchymal stem cells to investigate stable neocartilage tissue formation in vitro[J]. Eur Cell Mater, 2010, 20, (5): 245-259.
[13]
Gomez-Leduc T, Desance M, Hervieu MA, et al. Hypoxia is a critical parameter for chondrogenic differentiation of human umbilical cord blood mesenchymal stem cells in type I/III collagen sponges[J/OL]. Int J Mol Sci, 2017, 18(9):pii: E1933. doi: 10.3390/ijms18091933.
[14]
徐磊,叶朝阳,周燕,等.体外传代培养兔关节软骨细胞的去分化现象[J].中国组织工程研究,2013,17(20):3626-3634.
[15]
Callahan LA, Ganios AM, Mcburney DL, et al. ECM production of primary human and bovine chondrocytes in hybrid PEG hydrogels containing type I collagen and hyaluronic acid[J]. Biomacromolecules, 2012, 13(5): 1625-1631.
[16]
Zeng L, Chen XF, Zhang Q, et al. Redifferentiation of dedifferentiated chondrocytes in a novel three-dimensional microcavitary hydrogel[J]. J Biomed Mater Res A, 2015, 103(5): 1693-1702.
[17]
Schuh E, Hofmann S, Stok K, et al. Chondrocyte redifferentiation in 3D: the effect of adhesion site density and substrate elasticity[J]. J Biomed Mater Res A, 2012, 100A(1): 38-47.
[18]
Sailor LZ, Hewick RM, Morris EA. Recombinant human bone morphogenetic protein-2 maintains the articular chondrocyte phenotype in long-term culture[J]. J Orthop Res, 1996, 14(6): 937-945.
[19]
Yamamoto Y, Mochida J, Sakai D, et al. Upregulation of the viability of nucleus pulposus cells by bone marrow-derived stromal cells: significance of direct cell-to-cell contact in coculture system[J]. Spine (Phila Pa 1976), 2004, 29(14): 1508-1514.
[20]
孙明林,朱雷,吕丹.Ⅱ型胶原蛋白对兔去分化软骨细胞再分化的作用[J].中国修复重建外科杂志,2010,24(10):1244-1248.
[21]
Zuo Q, Cui W, Liu F, et al. Co-cultivated mesenchymal stem cells support chondrocytic differentiation of articular chondrocytes[J]. Int Orthop, 2013, 37(4): 747-752.
[1] 罗璠, 饶志涛. 机械敏感蛋白Piezo1介导创伤后骨关节炎的作用及机制[J]. 中华关节外科杂志(电子版), 2023, 17(04): 528-533.
[2] 傅子财, 黄勇, 陈斐, 刘澍雨, 朱伟民. 间充质干细胞来源外泌体在骨关节炎治疗中的研究进展[J]. 中华关节外科杂志(电子版), 2022, 16(02): 196-201.
[3] 柴浩卜, 王俏杰, 张先龙. 具有骨免疫调节性能的骨科生物材料研究进展[J]. 中华关节外科杂志(电子版), 2022, 16(01): 37-43.
[4] 于承浩, 张益, 陈进利, 戚超, 李海峰, 于腾波. 肩袖补片在巨大肩袖损伤治疗中的研究进展[J]. 中华关节外科杂志(电子版), 2021, 15(02): 225-230.
[5] 戴佑任, 张悦, 李扬, 王聪, 陈婷, 程文俊, 罗成燕. 未分化/去分化子宫内膜癌的临床病理学特征及治疗研究[J]. 中华妇幼临床医学杂志(电子版), 2022, 18(06): 660-668.
[6] 黄晓罡, 牛东升, 闫香果, 张克松, 何军民, 王晓军, 刘媛媛. 局部应用重组人Ⅲ型胶原蛋白水凝胶对糖尿病患者创面愈合的影响[J]. 中华损伤与修复杂志(电子版), 2022, 17(05): 430-434.
[7] 王晟, 许卓然, 夏德萌, 李磊, 许硕贵. 穿皮骨整合截肢假体与上皮细胞生长迁移的研究进展[J]. 中华损伤与修复杂志(电子版), 2021, 16(02): 166-169.
[8] 王智忠, 刘利华, 曹强, 查天建, 张娜, 禹学彬, 刘小龙. 水凝胶联合藻酸盐银敷料在面部深Ⅱ度烧伤创面治疗中的应用研究[J]. 中华损伤与修复杂志(电子版), 2021, 16(01): 50-54.
[9] 邢超, 徐灵巧, 廖文婷, 孙养鹏, 叶钟泰, 张志光. 骨髓间充质干细胞来源的外泌体促进髁突软骨细胞再生的研究[J]. 中华口腔医学研究杂志(电子版), 2021, 15(04): 207-214.
[10] 郑泽坤, 刘卓恒, 邹浩, 胡会元, 李妲, 吴巍. 扩大根治性手术切除复发性巨大腹膜后去分化脂肪肉瘤1例[J]. 中华普外科手术学杂志(电子版), 2023, 17(05): 588-590.
[11] 刘阳阳, 王梁, 戴志红, 郝建戈, 张力仁, 刘志宇. 腹膜后去分化脂肪肉瘤合并左肾透明细胞癌一例报告[J]. 中华腔镜泌尿外科杂志(电子版), 2023, 17(04): 412-414.
[12] 杜凯玥, 袁博伟, 洪晶. 水凝胶在角膜修复中的应用研究进展[J]. 中华眼科医学杂志(电子版), 2022, 12(05): 298-304.
[13] 李欣达. 同轴水凝胶制造技术在神经再生中的应用进展[J]. 中华脑科疾病与康复杂志(电子版), 2021, 11(03): 180-182.
[14] 葛校永, 李亚华, 李宗明, 周子鹤, 吴昆鹏, 李一帆, 韩新巍, 任克伟. 新型温敏性水凝胶在经导管动脉化疗栓塞中的应用进展[J]. 中华介入放射学电子杂志, 2023, 11(03): 268-274.
[15] 徐高丽, 张建兴, 周健, 沈文俊, 谷志远, 徐国超. 静压力下缺氧诱导因子-1α信号通路对髁突软骨细胞增殖与凋亡的调控作用[J]. 中华老年病研究电子杂志, 2021, 08(03): 24-29.
阅读次数
全文


摘要