中国组织工程研究

中国组织工程研究 ›› 2021, Vol. 25 ›› Issue (13): 1982-1987.doi: 10.3969/j.issn.2095-4344.3514

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

动态压力对负载胰岛素样生长因子1基因兔脂肪间充质干细胞增殖能力和机械性能的影响

张传辉1，李建军2，杨  军2   

  1. 1朝阳市中心医院骨外科，辽宁省朝阳市   122000；2中国医科大学附属盛京医院创伤骨科，辽宁省沈阳市   110004
  • 收稿日期:2020-04-16 修回日期:2020-04-21 接受日期:2020-06-12 出版日期:2021-05-08 发布日期:2020-12-28
  • 通讯作者: 张传辉，朝阳市中心医院骨外科，辽宁省朝阳市 122000
  • 作者简介:张传辉，男，1982年生，辽宁省庄河市人，汉族，2010年中国医科大学毕业，硕士，副主任医师，主要从事关节外科、创伤骨科、手外科专业疾病的诊疗研究。
  • 基金资助:
    辽宁省自然科学基金项目(20102264)，项目负责人：李建军

Effects of dynamic pressure on the proliferation and mechanical properties of rabbit adipose mesenchymal stem cells transfected with insulin-like growth factor-1

Zhang Chuanhui1, Li Jianjun2, Yang Jun2   

  1. 1Department of Orthopedics, Chaoyang Central Hospital, Chaoyang 122000, Liaoning Province, China; 2Department of Traumatology and Orthopedics, Shengjing Hospital, China Medical University, Shenyang 110004, Liaoning Province, China
  • Received:2020-04-16 Revised:2020-04-21 Accepted:2020-06-12 Online:2021-05-08 Published:2020-12-28
  • Contact: Zhang Chuanhui, Department of Orthopedics, Chaoyang Central Hospital, Chaoyang 122000, Liaoning Province, China
  • About author:Zhang Chuanhui, Master, Associate chief physician, Department of Orthopedics, Chaoyang Central Hospital, Chaoyang 122000, Liaoning Province, China
  • Supported by:
    the Natural Science Foundation of Liaoning Province, No. 20102264 (to LJJ)

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摘要：

文题释义：
脂肪间充质干细胞/壳聚糖支架复合物：将脂肪间充质干细胞制成细胞浓度为5×1010 L-1的细胞悬液，均匀置于壳聚糖支架中，每个支架滴加200 μL，于6孔培养板中温箱孵育4 h，待细胞附着贴壁后，每孔加5 mL完全培养基(DMED-HG)于37 ℃、体积分数为5% CO2培养箱中培养2 d。
胰岛素样生长因子1：是一种丝裂素生长因子，与前胰岛素在氨基酸序列上有同源性，是一种既有促进细胞增殖活性，又具有胰岛素样作用的生长激素依赖性多肽，它通过生长激素-胰岛素样生长因子轴来实现其强有力的刺激合成代谢的作用。

背景：脂肪间充质干细胞是目前公认的较为优秀的组织工程软骨种子细胞，基因转染技术可以有效诱导其成软骨分化，应用生物反应器模拟体内力学环境，是目前广大学者探索体外构建组织工程软骨的新思路。
目的：探讨周期性动态压应力联合胰岛素样生长因子1基因转染对种植于壳聚糖/明胶支架中的兔脂肪间充质干细胞增殖能力和弹性模量的影响。
方法：在脂质体介导下应用pcDNA3.1-IGF-1基因转染兔脂肪间充质干细胞，G418筛选，获得稳定转染的细胞株；将负载或不负载胰岛素样生长因子1基因的脂肪间充质干细胞以5×1010 L-1的密度接种于壳聚糖/明胶支架中培养2 d，然后分别于动态压力(2%形变，1 Hz，4 h/d)环境中或静态培养条件下培养7 d；扫描电镜、Masson三色染色及阿尔新蓝染色观察细胞/支架复合物的形态学变化，MTT法绘制细胞增殖曲线，CM-Dil荧光染料标记细胞计数评估细胞增殖效率，DMMB定量测定软骨特异性细胞外基质糖胺聚糖含量，Real time PCR法检测软骨特异性基因Ⅱ型胶原的相对表达，应用BioDynamicTM mechanical tester测量各组细胞/支架复合物不同形变时的轴线压应力，计算对应的弹性模量。
结果与结论：动态压力联合胰岛素样生长因子1转染可显著提高脂肪间充质干细胞的增殖能力，细胞分布更为均匀，软骨特异性细胞外基质糖胺聚糖及胶原分泌增多，上调Ⅱ型胶原基因的表达，机械性能显著改善。单纯胰岛素样生长因子1组的细胞增殖能力和弹性模量均优于单独动态压力组，但动态压力条件下细胞在支架中分布更为均匀。结果表明，动态压力与胰岛素样生长因子1基因转染均可显著提升兔脂肪间充质干细胞的增殖能力和机械性能，二者呈协同作用。

https://orcid.org/0000-0002-4742-2150(张传辉) 

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



关键词: 干细胞, 脂肪间充质干细胞, 压力, 软骨细胞, 胰岛素样生长因子1, 弹性模量, 组织工程

Abstract: BACKGROUND: Adipose mesenchymal stem cells are currently recognized as excellent seed cells for tissue engineering cartilage. Gene transfection technology can effectively induce them to differentiate into cartilage. The bioreactor is used to simulate the mechanical environment in vivo. It is a new idea for the majority of scholars to explore the construction of tissue engineering cartilage in vitro.  
OBJECTIVE: To investigate the effects of cyclic dynamic compressive stress combined with insulin-like growth factor-1 gene transfection on the proliferation and elastic modulus of rabbit adipose mesenchymal stem cells implanted in chitosan/gelatin scaffold.
METHODS:  Rabbit adipose mesenchymal stem cells were transfected with pcDNA3.1-IGF-1 gene mediated by liposome. The stable transfected cell lines were screened by G418. The adipose mesenchymal stem cells transfected with or without insulin-like growth factor-1 gene were inoculated in chitosan/gelatin scaffold at the density of 5×1010 L-1 for 2 days, and cultured under dynamic pressure (2% at 1 Hz, 4 hours per day) or static culture conditions for 7 days, respectively. The morphological changes of the cell/scaffold complex were observed by scanning electron microscope, Masson trichrome staining and alcian blue staining. The cell proliferation curve was drawn by MTT assay. The cell proliferation efficiency and distribution were evaluated by CM-Dil fluorescence-labeling method, and the content of total glycosaminoglycan was quantitatively determined by DMMB. The differences of type II collagen among different groups were compared with real time PCR. Compressive mechanical properties of the cell/scaffold constructs were assessed using a BioDynamicTM mechanical tester, and the corresponding elastic modulus was calculated.
RESULTS AND CONCLUSION: Dynamic pressure combined with insulin-like growth factor-1 transfection could significantly improve the cell proliferation ability of the cell/scaffold complex; the cell distribution was more uniform; glycosaminoglycan and collagen secretion in the cartilage-specific extracellular matrix were increased; the expression levels of type II collagen were up-regulated; and the mechanical properties were significantly improved. The cell proliferation and elastic modulus of insulin-like growth factor-1 group were better than those of single pressure group, but the distribution of cells in scaffolds was more uniform under dynamic pressure. The results indicate that both dynamic pressure and insulin-like growth factor-1 gene transfection can significantly improve the proliferation and mechanical properties of rabbit adipose mesenchymal stem cells; the two have synergistic effect. 


Key words: stem cells, adipose mesenchymal stem cells, pressure, chondrocytes, insulin-like growth factor-1, elasticity modulus, tissue engineering

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